CN109055550B - Target protein PCBP1 of lncRNA H19 and application thereof - Google Patents

Abstract

The invention discloses an application of a target protein PCBP1 of lncRNA H19 in preparation and/or screening of a medicament for treating tumors; also discloses application of the combination of IncRNA H19 and PCBP1 in preparation and/or screening of a medicament for treating tumors. After the targeted inhibitor of the PCBP1 is adopted, the colony forming capability of K562 cells is weakened, which shows that the proliferation of tumors can be effectively inhibited through targeted inhibition of the expression level of the PCBP 1; meanwhile, the reagent capable of promoting the expression level of IncRNA H19 has the effect of inhibiting the tumor by improving the expression level of IncRNA H19 to inhibit the growth of the tumor and prolong the survival time of a mouse with the tumor.

Description

Target protein PCBP1 of lncRNA H19 and application thereof

Technical Field

The invention relates to a method for treating malignant tumors, in particular to a target protein PCBP1 of lncRNA H19 and application thereof.

Background

Chronic Myelocytic Leukemia (CML) is a malignant myeloproliferative tumor that occurs on pluripotent hematopoietic stem cells. CML patients often develop characteristic chromosomal ectopic t (9:22) (q34: q11) (i.e., Ph chromosome) with translocation of the C-ABL proto-oncogene on chromosome 9, 2, to the Breakpoint Cluster Region (BCR) of the long arm of chromosome 22, formation of a BCR-ABL fusion gene, abnormal expression of the BCR-ABL protein, resulting in sustained activation of tyrosine kinase and activation of downstream signal transduction pathways, thereby regulating cytokine expression, ultimately resulting in massive proliferation of bone marrow stem and progenitor cells, decreased apoptosis and decreased adhesion of bone marrow stromal cells, resulting in release of large numbers of immature marrow cells into peripheral blood, leading to CML development. Clinically, the progression is divided into three stages, namely, Chronic Phase (CP), Accelerated Phase (AP) and acute phase (BP), according to the overall progression of the disease. The chronic disease symptoms are slight and nonspecific, and are mainly manifested by abdominal distension, abdominal mass, hypodynamia, low fever and splenomegaly, which are seen in 90 percent of patients and have different disease degrees.

With the rapid development of genome sequencing technology, it was found that RNA can be transcribed from 60% to 70% of genome sequences, but less than 2% of the sequences encoding proteins, the remainder being called non-coding RNA. In fact, many years ago, many non-coding RNAs were found to have important effects on vital activities, such as ribosomal RNAs (rrnas), transfer RNAs (trnas), and small nuclear RNAs (snrnas) involved in protein synthesis, and these non-coding RNAs were commonly referred to as housekeeping non-coding RNAs. Non-coding RNAs, which are of widespread interest in recent years, are primarily non-coding RNAs with life-regulating effects, including micrornas and RNAs studied herein (lncrnas). It has been found that LncRNA refers to RNA greater than 200 nucleotides in length and transcribed by RNA polymerase ii and lacking or lacking an open reading frame, and it can be derived from intergenic, enhancer elements, antisense strands of genes, other locations in the genome of intron regions.

With the continuous and deep research on long-chain non-coding RNA, LncRNA is found to have a plurality of important functions and participate in important biological processes of organisms, including cell differentiation, aging, proliferation, apoptosis, occurrence and development of tumors and the like. Various LncRNAs such as H19, X chromosome-specific inactivation transcript (Xist), etc. are involved in gene imprinting.

Pachnis et al, 1984, have found that the H19 gene, although transcribed by RNA polymerase II, can be cleaved and tailed, does not translate into biologically functional proteins, but rather functions in the form of noncoding RNA. Since the length of the H19 product exceeded 200nt, it was defined as LncRNA. H19 and IGF2 are present on chromosome seven in mice and at 11p15.5 in humans, which are 90kb apart, which belongs to the first pair of genes that were confirmed to have imprinting properties. H19 is embodied as paternal and maternal expression, while IGF2 is embodied as paternal and maternal imprinting. Both imprinted properties are regulated by a methylation Difference Modification Region (DMR) or imprinted regulatory region (ICR) 4kb upstream of the H19 promoter. In species evolution, H19 has a highly conserved characteristic, highly expressed in three phases, endoderm, mesoderm and tissues derived therefrom at embryonic development, but after birth, it is hardly expressed in other tissues than heart and skeletal muscle, unless expression is reactivated in three cases: tissue damage repair and stress status and tumorigenesis.

H19 is 2.3kb in length, consists of 35 small open reading frames, and has a 5 'end cap structure and a 3' poly A tail structure similar to mRNA, and theoretically can encode a protein comprising 256 amino acid molecules, but LncRNA only functions at mRNA level and cannot encode any protein molecule. The miRNA-675 molecule encoded by the highly conserved region of the first promoter of H19 controls the growth of the placenta during late gestation by regulating the expression of the IGFLR gene. The study found that H19 was highly concentrated in human placental peptide and some embryonic tissues, indicating that H19 may play a significant role in embryogenesis and growth development. After birth, H19 expression was down-regulated, but basal expression levels were maintained in breast, adrenal and uterine tissues. It has been reported that H19 acts as an oncogene inhibitor in wilms' tumor and embryonal rhabdomyosarcoma, whereas H19 is an oncogene in many solid tumors (including breast cancer, liver cancer, bladder cancer, etc.). This suggests that H19 participates in the formation and progression of body tumors through different mechanisms in humans. Many unknown links to these different mechanisms remain to be discovered in further research.

Disclosure of Invention

Based on the above problems, the inventors of the present application, in the course of studying a specific mechanism of H19 involved in the formation and progression of a tumor in a body, unexpectedly found that the proliferation of tumor cells can be effectively inhibited by increasing the expression level of incrna H19 or inhibiting PCBP1, a target protein of incrna H19. Specifically, the technical scheme of the invention comprises the following aspects:

in a first aspect, the invention provides an application of a target protein PCBP1 of IncRNA H19 in preparation and/or screening of a medicament for treating tumors.

Preferably, the tumor is a leukemia.

In a second aspect, the invention provides the use of the combination of lncRNA H19 and PCBP1 in the preparation and/or screening of a medicament for the treatment of tumours. Among them, the over-expression of lncRNA H19 could inhibit the proliferation of tumor cells, and the low expression of PCBP1 could inhibit the colony forming ability (colony) of tumor cells.

Preferably, the tumor is a malignant tumor.

In a third aspect, the invention provides a medicament for the treatment of tumours, which comprises an inhibitor of PCBP 1.

Preferably, the inhibitor is PCBP 1-targeted SiRNA.

Preferably, the medicament also contains an agent or a vector capable of promoting the expression level of IncRNA H19.

Preferably, the base sequence of the inhibitor is shown as PCBP1-SiRNA-03 (i.e., SEQ ID NO. 8).

More preferably, the tumor is a blood cancer.

In conclusion, the beneficial effects of the invention are as follows:

after the targeted inhibitor of the PCBP1 is adopted, the colony forming capacity of K562 cells is weakened, and the targeted inhibition of the expression level of the PCBP1 is shown to effectively inhibit the proliferation of tumors; meanwhile, the reagent capable of promoting the expression level of IncRNA H19 has the effect of inhibiting the tumor by improving the expression level of IncRNA H19 to inhibit the growth of the tumor and prolong the survival time of a mouse with the tumor.

Drawings

FIG. 1 is a schematic diagram of a lentiviral vector;

FIG. 2 is a graph showing the results of measurement of expression levels of H19 in K562 and KCL-22 cells in a normal control group;

FIG. 3 is a diagram showing the results of the Real-time PCR screening for effective sequences of H19-SiRNA;

FIG. 4 is a graph showing the results of measuring the expression level of H19 in K562-H19LentiV and K562empty LentiV;

FIG. 5 is a graph showing the results of detection of colony numbers in the K562-H19Lenti and K562-empty LentiV groups, wherein A is a colony graph (40 ×); b is relative colony number;

FIG. 6 is a graph showing the effect of H19 overexpression on BALB/c nude mice; wherein,

a shows the size of the mouse tumor after injection of P210 cells overexpressing H19;

b shows the effect on mouse survival after injection of P210 cells overexpressing H19;

c shows the statistical results of mouse tumor volume after injection of P210 cells overexpressing H19;

FIG. 7 is a graph showing the results of NOD/SCID mouse tail vein transplantation K562-NEG-LentiV and K562-H19Lenti V, observation of mouse incidence rate by in vivo imaging, and recording of mouse survival rate.

FIG. 8 shows the results of PCR product identification; wherein A is an agarose gel electrophoresis picture after PCR products. B is the result of PCR product sequencing (the sequence is already provided with a T7 promoter);

FIG. 9 is a photograph of an in vitro transcribed IncRNA H19 agarose gel electrophoresis;

FIG. 10 is a graph showing the results of RNA pull-down of K562 cell lysate incubated with RNA, in which the proteins in the complex were separated by 10% SDS-PAGE, stained with Coomassie Brilliant blue;

FIG. 11 is a graph showing the results predicted by cataPID software and startbase v2.0 software; wherein it is shown that in the long non-coding RNAH19pull-down product PCBP1 and FUS proteins are present, and the catapid software and the startbase v2.0 software predict that long non-coding RNA H19 interacts with PCPB1 and FUS proteins;

FIG. 12 is a graph showing the results of verifying the expression of two proteins in the RNA pull-down product using the PCBP1 primary antibody and the FUS primary antibody;

FIG. 13 shows the results of screening PCBP1-SiRNA and FUS-SiRNA for effective sequences (P <0.01, P <0.05, PCBP1-siRNA and FUS-siRNA transfection groups compared to blank group and SCR control group);

FIG. 14 is a graph showing the effect of different concentrations of Imatinib on the relative activity of K562 cells after transfection with SCR, PCBP1-siRNA and FUS-siRNA (P <0.01, P <0.05, PCBP1-siRNA and FUS-siRNA transfection groups compared to blank group and SCR control group);

FIG. 15 shows the colony results for the K562, SCR, PCPB1-siRNA, and FUS-siRNA groups; where a is the colony pattern (40 ×), B is the relative colony count (. about.p <0.01, PCPB1-siRNA and FUS-siRNA groups compared to K562, SCR groups).

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. Embodiments of the present invention generally include three major components, wherein reagents, consumables, and other test items are available from the market or other public sources.

A first part: functional characterization of lncRNA H19

H19-SiRNA sequence and H19 lentiviral expression vector

1.1H19SiRNA sequence

1.2H19 Lentiviral expression vector

The H19 lentivirus expression vector was constructed from Guangzhou pluripotent Gene, Inc.

H19 lentiviral expression vector: CS-GS3160-LV 201;

empty lentiviral expression vectors: CS-NEG-LV-201

2. Cell line

K562 cell line, human Chronic myeloid leukemia line, purchased from Shanghai cell Bank of Chinese academy of sciences

3. Preparation of main reagent

3.1 cell culture Medium containing 10% serum

Placing Australian fetal calf serum in an autoclave for 56 deg.C, extinguishing fire for 30min, subpackaging in 10mL kangning centrifuge tube, and storing the subpackaged serum in-20 deg.C refrigerator. The RPMI-1640 medium was dispensed in 200mL wide-mouth bottles (180 mL each), and stored in a 4 ℃ refrigerator. Each 180mL of the culture medium was supplemented with 20mL of Australian fetal bovine serum, and the serum content in the RPMI-1640 culture medium was 10%.

32pH72 Phosphate Buffer (PBS)

Weighing the above components respectively, dissolving the weighed components with double distilled water, diluting to a constant volume of 1000mL, sterilizing with high pressure steam for 30min, taking out, and storing at 4 deg.C.

3.3 preparation of 2.7% semisolid Medium for methylcellulose

Weighing 2.7g of methylcellulose powder into a wide-mouth bottle, adding 50mL of boiling water, dissolving under stirring for 30min, taking out, cooling, adding 50mL of sterile 2X 1640 culture medium into a clean bench, stirring until dissolving, and storing in a refrigerator at 4 ℃ for later use.

Example 1K 562 cell culture

Inoculating CML cell K562 to RPMI-1640 culture medium with serum content of 10%, placing the inoculated K562 cell at 37 deg.C and CO₂The culture was carried out in a cell culture chamber with a volume fraction of 5%.

Example 2 screening of effective sequences of H19-SiRNA

2.2.1 transfection of K562 cells with H19-SiRNA

(1) The experimental groups are: BLANK control group (BLANK), SCR, H19-SiRNA-01, H19-SiRNA-02, H19-SiRNA-03 group, SCR, H19-SiRNA-01, H19-SiRNA-02, H19-SiRNA-03 concentration is 100nM.

(2) Taking logarithmic growth phase cells, adjusting the cell suspension concentration to 30 x 10 by using serum-free and antibiotic-free RPMI-1640 culture medium⁴Per mL, 1.5mL of cell suspension per well was seeded in 6-well plates.

(3) Preparation of Lipofectamine^TM2000 complex with RNA:according to Lipofectamine^TM2000 instructions, respectively taking the required siRNA amount and Lipofectamine^TM2000, diluted with optimized medium Opti-MEM, respectively.

(4)Lipofectamine^TMAfter the dilution was completed at 2000 deg.C, it was allowed to stand at room temperature for 5 min.

(5) Lipofectamine diluted and left to stand for 5min^TM2000 and diluted SiRNA are mixed according to the proportion of 1:1, and the mixture is stood for 20min at room temperature to obtain Lipofectamine^TM2000-RNA complex.

(6) The Lipofectamine required by each group was added^TM2000-RNA complexes were slowly added dropwise to the cell suspension in the well plate to give a final volume of 2mL per well. Placing the 6-hole plate at 37 ℃ and CO ₂5% volume fraction in a cell culture chamber.

2.2.2Real-time PCR detection of the relative expression level of H19mRNA in various groups of cells

2.2.2.1 Total RNA extraction

Grouping experiments: BLANK control group (BLANK), SCR, H19-SiRNA-01, H19-SiRNA-02, H19-SiRNA-03

(1) The cells of each group in the logarithmic growth phase were centrifuged at 1500rpm for 5min, and the supernatant was discarded.

(2) The cell pellet was washed once with PBS, centrifuged at 1500rpm for 5min, and the supernatant was discarded.

(3) 1mL of trizol was added to the cell pellet and lysed on ice for 5 min.

(4) To the cell lysate, 200. mu.L of chloroform was added, shaken for 45s, and allowed to stand on ice for 10 min.

(5) Centrifuge at 12000rpm, 4 ℃ for 15 min. Carefully pipette the supernatant into a clean EP tube and leave at minus 20 ℃ for 20 min.

(6) Centrifuge at 12000rpm, 4 ℃ for 10 min. The supernatant was carefully discarded.

(7) The RNA precipitate was washed with 70% alcohol at 12000rpm at 4 ℃ for 5min and repeated three times.

(8) The precipitate was air dried in a fume hood for 2-3 min. To the pellet was added 30. mu.L of RNA enzyme-free water. The RNA was stored at minus 80 ℃.

2.2.2.2 Reverse Transcription (RT)

(1) Taking 1 mu g of RNA sample with A260/A280 of 1.8-2.0 and a PCR tube without nuclease pollution, and placing each group of RNA on ice for later use.

(2) Reverse transcription was performed by adding the following components to a nucleic acid-free tube (ice-on procedure) according to the instructions of the All-in-One cDNA Synthesis SuperMix of bimake:

(3) after the components are added, the mixture is instantaneously centrifuged for 10 s.

(4) The PCR tube was placed in a PCR instrument and reverse transcription was performed according to the standard reaction procedure provided with the kit: 15min at 42 ℃; at 85 ℃ for 2 min.

2.2.2.3 Real-time PCR

(1) The relative expression level of H19mRNA in each group was detected by 2 XSSYBR Green qPCR Master Mix kit from bimake, with GAPDH as internal reference, and 1. mu.L of cDNA was taken for Real-time PCR amplification.

(2) The reaction system is as follows:

relative expression levels of H19 and GAPDH 2^-△△CTAnd (4) calculating.

EXAMPLE 3 establishment of K562 cell line stably expressing H19

3.3.1H 19 Lentiviral expression vector and empty Lentiviral expression vector transfected K562 cells

(1) Taking logarithmic growth phase cells, adjusting the cell suspension concentration to 30 x 10 by using serum-free and antibiotic-free RPMI-1640 culture medium⁴Per mL, 1.75mL of cell suspension per well was seeded in 6-well plates.

(2) Preparation of Lipofectamine^TM3000 complex in DNA: according to Lipofectamine^TM3000 SpecificationPreparation, 2.5. mu.g of DNA and 3.75. mu.L of Lipofectamine per well^TM3000 amounts, diluted with 250. mu.L of Opti-MEM, respectively.

(3) DNA and Lipofectamine^TM3000, uniformly mixing the diluted solution according to the proportion of 1:1, standing at room temperature for 5min to obtain Lipofectamine^TM3000-DNA complex.

(4) Mixing Lipofectamine^TMThe 3000-DNA complex was slowly added dropwise to the cell suspension in the well plate to a final volume of 2mL per well. Placing the six-hole plate at 37 deg.C and CO₂The cells were cultured in a 5% volume fraction cell culture chamber, and after 6 hours, 2mL of RPMI-1640 medium containing 20% serum was added.

3.3.2 puromycin screening of K562 cell lines stably expressing H19

After transfection of K562 cells with H19 lentiviral vector, puromycin was added to the culture system to give a final concentration of 1mg/mL puromycin, and the culture medium was changed every 2 days.

Example 4 Real-time PCR detection of the relative expression level of H19mRNA in cells

4.4.1 the extraction of total RNA,

according to 2.2.2.1

4.4.2 Reverse Transcription (RT)

According to 2.2.2.2

4.4.3 Real-time PCR

According to 2.2.2.3

Example 5 Effect of H19 on Colony formation in K562 cells

5.5.1 Experimental groups

Blank control group K562

Group K562-H19LentiV (K562 cells stably expressing H19)

Group K562-empty LentiV (K562 cells with empty lentiviral vector)

5.5.2 COLONY experiment

The experiments were divided into two groups: k562 cells stably expressing H19 and K562 cells that have been transfected with an empty lentiviral expression vector. The cells of each experimental group in the logarithmic growth phase were taken and the K562 cells were diluted in serum-free medium so that the density was 1X 10³mL, 450. mu.L of K562Adding the cell suspension into 550 μ L semisolid culture medium containing 20% fetal calf serum, 5 μmol/L beta-mercaptoethanol, 2 mmol/L-glutamine and 0.9% methylcellulose, mixing the cell suspension and semisolid culture medium, inoculating 1mL of the cell suspension and semisolid culture medium into a 24-well plate, repeating three wells for each experiment, placing the 24-well plate at 37 deg.C and CO₂Culturing in 5% cell culture box for 7-14 days. The COLONY size and morphology were observed under an inverted microscope with a cell mass of approximately greater than 40 cells as 1 COLONY, stored by photography, and the experiment was repeated three times.

Example 6 functional study of H19 in a CML mouse model

6.6.1 Balb/c nude mice subcutaneous tumor formation experiment

Transfecting H19 lentivirus vector and empty lentivirus vector in P210 cell to construct P210 cell stably over-expressing H19, and introducing the P210 cell into a cell body with density of 1 × 10⁷The tumors were observed in BALB/c nude mice by inoculating 200. mu.L of P210-H19 and P210-NEG cells to the left lower limb axilla of the mice at a/mL ratio, and the change in tumor size of the mice was measured daily after the mice had tumors.

6.6.2 NOD/SCID mouse Tail vein experiment

Transfecting H19 lentivirus vector and empty lentivirus vector in K562 cell to construct K562 cell stably over-expressing H19, and introducing the K562 cell into a culture medium at a density of 1 × 10⁷The mice were observed and recorded in vivo imaging by transplanting 200. mu.L of K562-H19 and K562-NEG cells into/mL cells, and injecting K562-H19 and K562-NEG cells into tail vein.

The experimental results are as follows:

effect of H19 on K562 cell progression

1.1 detection of expression level of H19

The results of taking normal human blood, extracting RNA from K562 cells, KCL-22 cells and 293T cells, and detecting the expression level of H19 in each group by fluorescence quantitative PCR are shown in FIG. 2, and compared with the normal control group, the expression level of H19 in K562 and KCL-22 is lower.

1.2 screening of effective sequences of H19-SiRNA

SCR (serum creatinine) with the concentration of 100nm and H19-siRNA transfect K562 cells, after normal culture for 48H, extracting total RNA of each group of cells, detecting the relative expression level of H19mRNA of each group by Real-time PCR, and detecting the cell activity of each group of K562 cells after H19-SiRNA transfection by MTT. The results are shown in FIG. 3.

1.3 testing the efficiency of transfection of K562 cells with the Lentiviral vector H19

The H19 lentiviral vector (CS-GS3160-LV201) and the empty lentiviral vector (CS-NEG-LV-201) were used as supplied by Guangzhou pluripotent Gene Co., Ltd. After the H19 lentiviral vector transfects K562 cells, the K562 cells stably expressing H19 are obtained by puromycin screening. Groups of K562 cells in the logarithmic growth phase were collected and total RNA was extracted to measure the relative expression level of H19 mRNA. Relative expression levels of H19 in K562 cells of each group As shown in FIG. 4, the expression level of H19mRNA was significantly increased in the H19 lentiviral vector group (H19LentiV) relative to the Empty lentiviral vector (Empty LentiV).

1.4 Effect of H19 overexpression on Colony formation in K562 cells

The colony formation method using methylcellulose as a carrier can be used for detecting the proliferation capability of single tumor cells. The colony experiments were performed using K562, K562-empty LentiV and K562-H19LentiV at the logarithmic growth phase, and the results are shown in FIG. 5, in which the number and size of colonies in K562-H19LentiV were reduced (P <0.05) relative to those in K562 and K562-empty LentiV groups.

2. Effect of over-expression of H19 on Balb/c nude mice of CML model mice

The influence of H19 overexpression on the tumor size of a BALB/c nude mouse and the survival of the BALB/c nude mouse are explored by subcutaneous tumor formation of a nude mouse, in order to explore the influence of H19 on the P210 tumor size of the BALB/c nude mouse, an H19 lentiviral vector and an empty lentiviral vector are transfected in a P210 cell to construct a P210 cell stably overexpressing H19, and the density is 1 multiplied by 10⁷The tumors were observed in BALB/c nude mice by inoculating 200. mu.L of P210-H19 and P210-NEG cells to the left lower limb axilla of the mice at a/mL ratio, and the change in tumor size of the mice was measured daily after the mice had tumors. It can be seen from fig. 6 that the H19-overexpressing group had smaller tumors than the control group.

3. Effect of overexpression of H19 on the CML model mouse NOD/SCID

Mice were injected intravenously with K562-H19 cells and K562-empty cells and the incidence and survival of the mice were recorded by in vivo imaging. The results are shown in FIG. 7.

And (4) analyzing results:

the invention firstly detects the expression quantity of H19, takes the blood of normal people, K562 cells and KCL-22 cells, extracts RNA, and detects the expression quantity of H19. As shown in FIG. 2, it can be seen that the expression amount of H19 was relatively low in K562 and KCL-22 compared with that of the normal control group. Next, SiRNA of H19 was screened for effective sequences, and H19-siRNA and SCR were synthesized by Ruibo Bio Inc., Guangzhou. Through Real-time PCR screening, two sequences of H19-SiRNA-02 and H19-SiRNA-03 are effective, as shown in figure 3, but after the target inhibition of H19, the influence on the activity of K562 cells is not obvious. Therefore, the inventor utilizes H19 lentiviral vector to transfect K562 cells, puromycin screens positive cells to ensure that H19 is over-expressed, a stable strain is named as K562-H19LentiV, the effect of H19 over-expression on the malignant progression of the K562 cells is researched, and as shown in figure 4, H19 over-expression inhibits the clonogenic of the K562 cells. Lentiviral expression vectors CS-GS3160-LV201 and CS-NEG-LV-201 construction and technical support were provided by Guangzhou pluripotent Gene.

CS-GS3160-LV201 contains H19 and eGFP target gene fragment, and CS-NEG-LV-201 only contains eGFP, so that after transfection is completed, positive cells of GFP protein are observed, transfection efficiency can be preliminarily judged, and puromycin is used for preliminary screening according to a resistance marker gene on a vector. RNA is extracted from the K562 stable strain after lentivirus transfection, and the relative expression level of H19 is detected by Real-time PCR. FIG. 5 shows that the expression level of H19mRNA in K562-H19LentiV cells is obviously increased compared with that of the K562-empty LentiV cell group, and the results show that the K562 cells stably expressing H19 are successfully established, so that an experimental model is provided for researching the function of H19 in CML.

Clonal proliferation is an important feature of the CML K562 cell line, and therefore, studies on the clonogenic capacity of K562 cells can prove the influence of H19 on the progression of CML cells. The colony formation experiment refers to the growth condition of single cells, especially tumor cells, in the culture condition of a methylcellulose semisolid culture medium, and the proliferation capacity of the single cells can be evaluated by observing the cell number and the colony form through an inverted microscope. It was found from the colony formation experiment that the size and number of colony cells decreased in K562 cells after H19 overexpression, and the expression level of H19 was closely related to the proliferative capacity of K562 cells.

Chronic myeloid leukemia has characteristic BCR/ABL fusion gene and its product BCR/ABL fusion protein (P210), which has strong tyrosine kinase activity and can abnormally activate multiple signal pathways, resulting in malignant transformation of blood cells. BaF-P210 cells, P210 for short, are cell lines stably expressing BCR/ABL. Thus, this section was an animal level investigation of the effect of H19 overexpression on BALB/c nude mouse neoplasia and survival. The inventor constructs P210 cells stably over-expressing H19 at a density of 1 × 10⁷Mice were observed for neoplasia and survival from 200. mu.L/mL of P210-H19 and P210-NEG cells inoculated to the date of axillary injection of the left lower limb of BALB/c nude mice. As can be seen in fig. 6, overexpression of H19 inhibited tumor growth and prolonged survival of mice compared to the control group. Meanwhile, the inventor constructs K562 cells stably over-expressing H19 at a density of 1 × 10⁷The cells were transplanted 200. mu.L into NOD/SCID mice by tail vein injection of K562-H19 and K562-NEG cells per mL, and the mice were observed for morbidity and survival by in vivo imaging, as can be seen from FIG. 7, overexpression of H19 inhibited tumor growth and prolonged survival compared to the control group.

Functional study of target protein PCBP1 of second part lncRNA H19

1. The sequences of primers for the H19 gene having the T7 promoter sequence are shown in the following Table, and the primers were designed and synthesized by Biotechnology (Shanghai) Co., Ltd.

2. Cell line

The K562 cell line, human Chronic myeloid leukemia line, was purchased from Shanghai cell Bank, Chinese academy of sciences.

3. Preparation of main reagent

3.1 preparation of 10 XTAE electrophoresis buffer

Weighing the components in a beaker, adding 800mL double distilled water into the beaker, and stirring to fully dissolve the components; adding 11.4mL of glacial acetic acid, and fully stirring; and (5) adding double distilled water to a constant volume of 1L, and storing at room temperature for later use.

3.2 preparing 0.8% agarose gel

Weighing 0.4g of agarose, dissolving in 50mL of double distilled water, stirring uniformly, heating by a microwave oven until the agarose is boiled, taking out, cooling to about 60 ℃, adding 3 mu L of GeneRed, stirring fully, pouring into a gel preparation tank, inserting a comb, and standing until the gel is solidified for later use.

3.3 preparation of LB Medium

Weighing the above components in a beaker, adding distilled water, stirring and fixing volume to 1L, placing in a high pressure steam sterilization pot for sterilization, and keeping for later use.

3.4 preparation of LB solid Medium

Adding 15g of agar in addition to the components in the liquid culture medium, adding double distilled water to a constant volume of 1L, sterilizing for about 30min, taking out, cooling to about 60 ℃, pouring into a culture dish added with antibiotics, shaking uniformly, solidifying, and storing at 4 ℃ for later use.

3.5 preparation of 5M sodium chloride

292.5g of sodium chloride is weighed, added with double distilled water and stirred until dissolved, and the volume is up to 1L. And (5) standby.

3.610 XPTBS buffer (pH 7.6)

Weighing the above components, dissolving with appropriate amount of double distilled water, adjusting pH to 7.6 with HCl, adding double distilled water to constant volume of 1L, and storing at room temperature. When used, 10 × TBS was diluted to 1 × TBS.

3.7 preparation of 1 XTSST buffer

100mL of 1 xTBS solution is measured, 1mL of Tween-20 is added, and a proper amount of double distilled water is added to the solution until the volume is 1L, namely the 1 xTBST buffer solution.

3.8 preparation of 1 Xtransmembrane buffer (pH 8.5)

Weighing the above components, adding appropriate amount of double distilled water to dissolve, adjusting pH to 8.5 with HCL, adding double distilled water to constant volume of 1L, and storing at room temperature.

3.9 preparation of sealing liquid

5g of skimmed milk powder is weighed and dissolved in 100ml of LTBST, and the skimmed milk powder is prepared in situ.

3.10 preparing antibody diluent

5g of skim milk is dissolved in 50ml of LTBST to prepare 5 percent skim milk which is prepared in situ.

3.115 XSDS-PAGE electrophoresis buffer

Adding appropriate amount of double distilled water, stirring to dissolve, and adding double distilled water to constant volume of 1L. Storing at room temperature. When used, the mixture was diluted with double distilled water to 1 XSDS-PAGE.

3.121.5 mol/L Tris-HCl buffer (pH 8.8)

Adding a proper amount of double distilled water to dissolve, and fixing the volume to 100 mL. Storing at 4 ℃.

31330% acrylamide

The components are weighed, dissolved in double distilled water and subjected to constant volume of 100 mL. Storing at 4 ℃.

3.1410% separating gel

3.155% concentrated gum

Example 7 obtaining templates for in vitro transcription

1) Preparation of competent Escherichia coli

(1) Taking a strain preservation bacterium solution escherichia coli DH5 alpha frozen and stored at minus 70 ℃, sucking 5 mu L of bacterium solution and 5mL of LB liquid culture medium, and activating by a shaking table at 37 ℃ and 250rpm overnight.

(2) Taking 50 mu L of the bacterial liquid in the step (1), inoculating the bacterial liquid into 5mL of fresh LB liquid culture medium, culturing the bacterial liquid in a shaking table at 37 ℃ and 250rpm for 3h, taking a proper amount of the bacterial liquid and measuring an OD value (0.3-0.4OD value) in a 1.5mL EP tube, and carrying out an experiment.

(3) 1mL of the suspension was taken out from a 1.5mL sterile EP tube, centrifuged at 5000rpm for 5min, and the supernatant was removed. 1mL of precooled 0.1mol/l CaCl was added₂Resuspend the bacterial solution, and place on ice for 30 min.

(4) Centrifuging at 5000rpm and 4 deg.C for 5min, and removing supernatant.

(5) 50 μ L of pre-cooled 0.1mol/L CaCl was added₂Resuspending the bacterial solution, i.e., competent cells (stored in a refrigerator at minus 80 ℃ C.)

2) H19 lentivirus expression vector transformed escherichia coli

(1) And (3) thawing the competent cells on ice, adding 1 mu L of the H19 lentivirus overexpression vector DNA into the competent cell suspension after the competent cells are thawed, gently blowing and beating the mixture by using a pipette, and uniformly mixing the mixture for ice bath for 30 min.

(2) The tube was quickly transferred to an ice bath by hot shock at 42 ℃ for 45s and allowed to stand on ice for 3 min.

(3) Adding 450 μ L of sterile LB medium without antibiotic into the centrifuge tube, mixing, placing in a 37 deg.C shaking table, and shaking and culturing at 150rpm for 45min to allow the thallus to recover.

(4) 100 μ L of the bacterial liquid was spread on LB plates containing ampicillin, spread evenly using a sterile spreading bar, the plates were placed at 37 ℃ until the liquid was absorbed, cultured upside down, and cultured overnight at 37 ℃.

(5) The next morning, a 10mL centrifuge tube was prepared, 5mLLB medium was added, 2.5. mu.L ampicillin was added, and a single colony was inoculated into LB medium containing ampicillin and cultured with shaking at 37 ℃ for about 10 hours.

3) Extraction of plasmid DNA

The reagents used for the extraction are shown in the following table:

(1) 5mL of overnight-cultured bacterial liquid was taken, added to a centrifuge tube, centrifuged at 13000rpm for 1min to collect the bacterial pellet, and the supernatant was carefully aspirated.

(2) 250. mu.L of Buffer P1 was added to the centrifuge tube containing the precipitate, mixed well using a pipette, and the precipitate was suspended.

(3) Add 250. mu.L of Buffer P2 into the centrifuge tube, mix 10 times by gently turning upside down to fully lyse the thallus, and leave it at room temperature for 5 min. At this point the solution became clear and viscous.

(4) Adding 250 μ L of Buffer E3 into a centrifuge tube, immediately turning upside down and mixing for 10 times, wherein white flocculent precipitate appears, standing at room temperature for 5min, centrifuging at 13000rpm for 5min, sucking supernatant, adding the supernatant into a filter aid (Endo-Remover FM), centrifuging at 13000rpm for 1min, filtering, and collecting filtrate in the centrifuge tube.

(5) To the filtrate was added 225. mu.L of isopropyl alcohol, and the mixture was inverted upside down and mixed.

(6) Column balancing: 200. mu.L of Buffer PS was added to the adsorption column (Spin Col. mu. mns DM) loaded into the collection tube, centrifuged at 13000rpm for 1min, the waste liquid in the collection tube was decanted, and the adsorption column was replaced into the collection tube.

(7) And (4) transferring the mixed solution of the filtrate and the isopropanol in the step (5) to an adsorption column (filled into a collecting pipe) with good balance.

(8) Centrifuging at 13000rpm for 1min, pouring off waste liquid in the collecting tube, and replacing the adsorption column in the collecting tube again. (maximum volume of adsorption column 750. mu.L, if sample volume is more than 750. mu.L can be added in batches.)

(9) 750. mu.L of Buffer PW to which absolute ethanol had been added in advance was added to the adsorption column, and centrifuged at 13000rpm for min, and the waste liquid in the collection tube was decanted.

(10) The adsorption column was replaced into the collection tube and centrifuged at 13000rpm for 1 min. (Note: the purpose of this step is to remove residual ethanol from the column, which could affect the subsequent enzymatic reaction.)

(11) The adsorption column was placed in a new collection tube, 50-100. mu.L Edno-Free Buffer EB was added to the middle of the adsorption membrane, left at room temperature for 5min, centrifuged at 13000rpm for 2min, and the plasmid solution was collected in a centrifuge tube. The plasmid was stored at minus 20 ℃.

4) Plasmid DNA quantitation

And (3) plasmid DNA quantification, namely quantifying by using a nucleic acid quantifier, and averaging the quantification for three times.

5) PCR amplification

PCR amplification was performed according to Thermo Scientific usage High-Fidelity DNA Polymerase of Thermo Scientific Co

(1) A200 μ L EP tube from EXYGEN was taken.

(2) The amplification system is shown in the following table:

(3) after fully mixing, the mixture is put on a machine for PCR amplification.

(4) Amplification was performed according to the following procedure

6) Identification of PCR amplification product by agarose gel electrophoresis

(1) Take 10. mu.L of PCR product in a new 200. mu.L EP tube

(2) 2 μ L of 5-by-Loading buffer was added to the EP tube and mixed with a microsyringe.

(3) Add 1 XTAE fresh running buffer to the running bath and remove the comb to allow the running solution to go through the gel.

(4) The sample is sequentially loaded with 5. mu.L of a mixture of DNA Ladder, PCR product and Loading buffer. The PCR product of interest is plated in 14 duplicate wells, so that the amount of DNA recovered is sufficient for subsequent experiments.

(5) Setting conditions, regulating the voltage to 110 volts and the time to 30 min. Agarose gel electrophoresis was performed.

(6) And after electrophoresis, observing whether the position of the strip is correct under an ultraviolet lamp, and photographing and storing.

7) Purification and recovery of gel DNA

The reagents and other components used to recover the DNA are shown in the following table:

(1) the single target DNA band was excised from the agarose gel (excess was removed as much as possible) and placed in a clean centrifuge tube and weighed.

(2) 3 volumes of Buffer NJ were added: if the weight of the gel is 0.1g, the volume of the gel is 0.1mL in a water bath at the temperature of 55-65 ℃ until the gel is completely melted. (Note: after the gel was completely dissolved, the color of the mixture was observed, and if it was pink, 5. mu.L of 3M sodium acetate at pH 5.2 was added to lower the pH value, and the color of the mixture was adjusted to be light yellow.)

(3) The solution obtained in the previous step was applied to a GBC adsorption column (note: the column volume was 800. mu.L; if the sample volume was more than 800. mu.L, it was added after batch centrifugation). After standing at room temperature for 2min, centrifuge at 1000 Xg for 60 s. Discard the tube filtrate and loop the column back to the 2mL collection tube.

(4) Discard the tube filtrate and loop the column back to the 2mL collection tube. Add 600. mu.L of the LDNA Wash Buffer to the column. Centrifuging at room temperature at 10000 Xg for 30-60 s.

(5) The column was washed repeatedly with DNA Wash Buffer. Centrifuging at room temperature at 10000 Xg for 30-60 s.

(6) Discard the tube filtrate and loop back to the 2mL collection tube. At room temperature, 13000 Xg centrifugation for 2min to spin dry the column matrix residual liquid.

(7) The column was mounted in a clean 1.5mL centrifuge tube, 15-25. mu.L (depending on the final product concentration) of Elution Buffer was added to the column matrix, and the column was left at room temperature for 1min and centrifuged at 12000 Xg for 1min to elute the DNA.

Example 8 obtaining of desthiobiotin-labeled IncRNA H19

1) Purification of recovered PCR product as template for in vitro transcription

(1) Thawing each reagent: placing the RNA Polymerase Enzyme Mix on ice;

(2) 10 × Reaction Buffer and 4 Ribonucleotide solutions (ATP, CTP, GTP and UTP) were vortexed gently until dissolved. After dissolution, 10 × Reaction Buffer was placed at normal temperature, and four ribonucletide solvents were placed on ice.

(3) The transcription reaction was performed at room temperature.

First, 200. mu.L of RNase-free EP tube was taken

mu.L of LATP solution, 2. mu.L of CTP solution, 2. mu.L of LGTP solution, 2. mu.L of LUTP solution, 2. mu.L of 10 × Reaction Buffer, and 1. mu.g of template DNA were sequentially added using a microsyringe, 2. mu.L of Enzyme Mix was added, and 20. mu.L of nucleic acid-free water was used for the completion.

② lightly mixing up and down by using a trace sample injector.

③ incubation at 37 ℃ overnight.

And fourthly, after the incubation is finished, taking out, adding 1 mu L of Dnase into the reaction system, mixing uniformly, and incubating for 15min at 37 ℃. The purpose of Dnase is to remove template DNA inside the system.

Fifthly, terminating the reaction and precipitating RNA. mu.L of nucleic-free Water and 30. mu.L of lithium chloride precipitant were added to the reaction system.

Sixthly, completely mixing the materials evenly, and precipitating the mixture for 30min and more than 30min at the temperature of minus 20 ℃.

Seventhly, carrying out centrifugation for 15min at the maximum rotating speed at 4 ℃ to precipitate RNA.

Eighthly, carefully removing the supernatant. The precipitate was washed once with 1mL of 70% ethanol. The aim is to remove the impurities to the maximum extent. Centrifuging at 4 deg.C for 15 min.

Ninthly, carefully removing 70% of ethanol, resuspending RNA by using nucleic acid enzyme-free water, quantifying the RNA, and storing at minus 70 ℃.

2) Desulfurbiotin labeling of RNA after in vitro transcription

(1) Placing DMSO on ice for thawing; 30% PEG (polyethylene glycol) was heated at 37 ℃ until it became a flowable liquid. The remaining reagents were thawed on ice.

(2) A heater was prepared and adjusted to 85 ℃.

(3) A200. mu.L EP tube was taken, and 5. mu.L of unlabeled RNA (50pmol) was added, followed by 1.25. mu.L of DMSO. The DMSO function is:

(4) the reaction system was added as follows:

(5) incubation at 16 ℃ overnight, overnight incubation can increase reaction efficiency.

(6) After the reaction, 70. mu.L of nucleic acid-free water was added to the reaction system. Mixing well.

(7) mu.L of chloroform: alcohol (24:1) (from which RNA Ligase was removed) was added to the reaction system, mixed gently, and centrifuged at the maximum speed for 3 min. The supernatant was carefully aspirated and transferred to a new nuclease-free EP tube.

(8) To the supernatant in the seventh step were added 10. mu.L of NaCl at a concentration of 5M, 1. mu.L of glycogen and 300. mu.L of 100% cold ethanol, respectively. Placing at least 1hour at minus 20 ℃.

(9) Centrifuge at 13000 Xg for 15min at 4 ℃ and carefully remove the supernatant taking care not to discard the pellet.

(10) Adding 300 μ L of 70% cold ethanol, mixing gently, and washing the precipitate. Centrifuge at 13000 Xg for 15min at 4 ℃, carefully remove supernatant and air dry the pellet (not more than 5 min).

(11) The pellet was resuspended in 20. mu.L of core-free water and stored at minus 80 ℃.

Example 9 labeling of completed RNAs RNA-protease Pull Down experiments

1) Preparation of cell lysate:

(1) collecting cells in logarithmic phase, collecting cell suspension in a 15mL centrifuge tube, centrifuging at 1500rmp for 5min, collecting cells, and discarding supernatant.

(2) The cells were washed once with PBS, blown and mixed well, 1500rmp, centrifuged for 5min to collect the cells.

(3) Adding appropriate amount of cell lysis solution according to cell precipitation amount, mixing, and performing ice lysis for 30min to completely lyse cells.

(4) Centrifuge at 12000rmp for 30min at 4 ℃.

(5) The supernatant was pipetted into a new and clean 1.5mL EP tube and the protein concentration was determined according to the instructions of the Coomassie Brilliant blue protein concentration determination kit. The protein concentration in the cell lysate needs to be greater than 20 mg/mL.

(6) The prepared cell lysate was placed on ice for use.

2) Binding the labeled RNA to streptavidin magnetic beads

The amount of bound RNA was 50pmol, as follows:

(1) a1.5 mL EP tube was loaded with 50. mu.L of streptavidin magnetic beads.

(2) The EP tube is placed on a magnetic frame, magnetic beads are collected at one end of the EP tube, and the supernatant is removed.

(3) An equal volume (50. mu.L) of 20mM Tris (pH7.5) was added for washing and the magnetic beads were resuspended using a tip.

(4) And (4) repeating the second step and the third step.

(5) And (3) placing the EP tube in a magnetic frame, collecting magnetic beads at the other end of the EP tube, and removing the supernatant.

(6) An equal volume (50. mu.L) of 1 × RNA Capture Buffer was added and the beads were resuspended using a pipette tip.

(7) 50pmol of labeled RNA was added to the magnetic beads, and the mixture was gently mixed.

(8) Mixing at room temperature, and incubating for 30 min.

3) RNA-Binding Protein Binding to labeled lncRNA

(1) And (3) taking down the EP tube incubated in the step (8) to complete the magnetic bead, placing the EP tube on a magnetic frame, and collecting the magnetic bead at one end of the EP tube. The supernatant was removed.

(2) An equal volume (50. mu.L) of 20mM Tris (pH7.5) was added for washing, and the beads were resuspended by gentle pipetting using a pipette tip.

(3) And (3) repeating the steps (1) and (2).

(4) And (3) placing the EP tube in a magnetic frame, collecting magnetic beads at the other end of the EP tube, and removing the supernatant.

(5) 10 XProtein-RNA Binding Buffer was diluted to 1X (for each reaction, 1 o. mu.L of 10 XProtein-RNA Binding Buffer was taken, 90. mu.L of RNA enzyme-free water was added, and mixed) for use.

(6) Add 100. mu.L of the diluted 1 XProtein-RNA Binding Buffer to the magnetic beads and mix well.

(7) Preparing an RNA-Protein Binding Reaction system, wherein each component is as follows:

(8) and (4) placing the EP tube in the step (6) on a magnetic frame, collecting magnetic beads at one end of the EP tube, and removing the supernatant.

(9) Adding 100 mu L of the prepared Master Mix into the magnetic beads, and gently mixing.

(10) The cells were incubated at 4 ℃ for 60min with rotation.

4) Elution of RNA-Binding Protein complexes

(1) And (3) placing the EP tube in a magnetic frame, collecting magnetic beads at the other end of the EP tube, and removing the supernatant.

(2) Complex washes were performed with an equal volume (100. mu.L) of 1 × wash buffer.

(3) Repeating the steps (1) and (2) twice.

(4) And (3) placing the EP tube in a magnetic frame, collecting magnetic beads at the other end of the EP tube, and removing the supernatant.

(5) Add 50. mu.L of Elution Buffer to the beads, mix well, incubate at 37 ℃ for 30 min.

(6) And (3) placing the EP tube on a magnetic frame, collecting magnetic beads at the other end of the EP tube, and collecting supernatant. For subsequent analysis.

(7) Add 1 × loading buffer to the complex. Boiling in boiling water for 10min, and storing at minus 20 deg.C.

Example 10 detection of RNA-Binding Protein Complex

1) Western Blot electrophoresis detection and Coomassie brilliant blue staining detection are carried out on the eluted RNA-Binding Protein complex

(1) Electrophoresis: preparing SDS-PAGE gel, loading 50 μ L of the complex, performing 80V electrophoresis for 30min, and performing 120V electrophoresis for 1 h.

(2) Dyeing: after the electrophoresis is finished, the gel is taken out, put into a 10cm dish, added with 100mL of distilled water, shaken on a shaking table for 5min, poured off, repeated twice and washed three times in total. (the purpose of this step is to wash away interfering substances such as SDS from the gel)

(3) Distilled water was discarded and an appropriate volume of Coomassie Brilliant blue fast staining solution was added to allow the staining solution to cover the gel and stain on a horizontal shaker.

(4) According to the molecular weight of the protein, a band with the molecular weight of the protein appears for about 30min, and most of the band appears in about 2 h.

(5) And (3) finishing dyeing, and decoloring: adding a proper amount of distilled water, decoloring on a shaking table, adding distilled water for multiple times for washing until a clear strip is seen, and photographing for recording.

(6) The gel was stored at 4 ℃.

2) The gel after electrophoresis is subjected to mass spectrometry analysis and identification

And (3) performing mass spectrum identification on the gel which is decolorized and stored, wherein the mass spectrum identification is completed by Guangzhou Jun organisms.

Example 11 Western Blot to verify the protein of interest

1) Obtaining RNA-Protein biding complexes

(1) In vitro transcription and RNA pulldown experiments were performed as described above to obtain RNA-Protein biding complexes.

(2) Adding the sample buffer solution according to the proportion, and boiling in a boiling water bath for 10 min.

2) Western Blot for verifying target protein

(1) And (4) electrophoresis. SDS-PAGE gel is prepared, wherein the concentration of the separation gel is 10 percent, and the concentration of the concentrated gel is 5 percent. And (4) after sample loading, carrying out electrophoresis at 80V for about 40min until the protein enters the separation gel and clear bands appear in MARKER, carrying out electrophoresis at 120V for 1h to enable the MARKER to run out sufficiently, and stopping electrophoresis.

(2) And (5) after electrophoresis is finished, transferring the membrane. Firstly, putting two pieces of filter paper special for the membrane transfer with the same size into membrane transfer liquid for full soaking, cutting out separation gel containing target protein according to the position of MARKER and the molecular weight of the target protein, putting the cut gel into the membrane transfer liquid for full soaking, putting the cut PVDF membrane into methanol for activation for 30s, taking out the PVDF membrane, and putting the PVDF membrane into the membrane transfer liquid for soaking. The PVDF membrane and the gum were placed in the wet transfer device in the following order: the positive electrode (white board), the spongy cushion, the filter paper, the film (smooth surface up), the glue, the filter paper, the spongy cushion and the negative electrode (blackboard) are placed into the film rotating groove, the glass rod is used for gently removing bubbles between the glue and the film, and finally the device is closed for 100V and 1 h.

(3) And (5) sealing. And (3) immediately placing the membrane in TBST after the membrane is completely transferred, washing the membrane in a decoloring shaking table for three times, repeating the washing for 5min each time, and after the membrane is completely transferred, placing the membrane in a sealing solution of 5% skimmed milk, and sealing the membrane for 2h at room temperature.

(4) Primary antibody incubation. After blocking, the membrane was placed in TBST and washed three times in a decolorizing shaker for 10min each. The antibody was diluted to the appropriate concentration according to the antibody instructions. The membrane was placed in the diluted primary antibody incubation chamber and incubated overnight on a shaker at 4 ℃.

(5) The membrane was placed in TBST and washed three times for 10min each.

(6) And (5) incubating a secondary antibody. Secondary antibodies were diluted according to secondary antibody instructions. The membrane was placed in an incubation box with diluted secondary antibody, incubated for 2h at room temperature on a shaker.

(7) The membrane was placed in TBST and washed three times for 10min each.

(8) And (4) emitting light. After washing, sucking the excess water on the membrane with filter paper, preparing ECL luminescent solution, mixing solution A and solution B according to a ratio of 1:1, and storing in dark place. And (3) dripping an appropriate amount of ECL luminescent liquid onto the film, putting the film into a dark box, and taking a picture for storage by using a gel imaging system.

The bioinformatics software involved in the present invention includes the following:

1) RNA protein interaction prediction software: catRAPID http:// service. tartaglalab. com/update _ submission/108643/f560944664

2) RNA protein interaction prediction software: http:// starbase

The experimental results are as follows:

1. the in vitro transcribed template with the T7 promoter was amplified using the H19 lentiviral vector as a template, and the results are shown in FIG. 8.

2. Identification of the in vitro transcript IncRNA H19

The DNA with the T7 promoter is used as a template, in vitro transcription is carried out according to the kit instruction, the transcribed RNA is subjected to agarose gel electrophoresis, the relative molecular mass and the size of the marker are compared, and the electrophoresis result is shown in FIG. 9.

Identification of RNA binding protein after RNA pull-down

After in vitro transcription is finished, biotin labeling is carried out on RNA, the labeled RNA and lysate of K562 cells are incubated together to obtain a compound of RNA protein and streptavidin magnetic beads, and the compound of RNA and protein is obtained by elution. And (3) performing polyacrylamide gel electrophoresis on the compound, performing Coomassie brilliant blue dyeing and decoloring with a decoloring solution on the electrophoresis gel, and then sending a sample to mass spectrometry for identification. SDS-PAGE gels were subjected to full lane protein mass spectrometry, resulting in the identification of multiple specific proteins by the long non-RNAH 19 and protein complex lane.

Protein mass spectrometric identification of RNA pull-down products

To verify which proteins interact with the long non-coding RNAH19, the inventors transcribed RNA in vitro and labeled it with biotin, incubated the biotin-labeled RNA with K562 cell lysate for RNA pull-down, and the resulting complex product was electrophoresed and stained with coomassie brilliant blue, as shown in fig. 10.

Mass spectrometric detection and software prediction of coexisting protein molecules in lncRNA H19pull-down

The catapid software and the startbase v2.0 software predicted proteins that interact with long-chain non-coding RNAH19, and as a result, as shown in fig. 11, it was found that two of the predicted and mass-identified proteins were common, PCBP1 and FUS.

Western Blot to verify target proteins PCBP1 and FUS of long-chain non-coding RNA H19

In order to further verify the interaction between long-chain non-coding RNA H19 and PCBP1 and FUS, the inventor carried out Western Blot verification on the product of RNA pull-down, and the result is shown in FIG. 12; from FIG. 9, it can be seen that PCBP1 and FUS can be detected in the RNA pull-down product.

And (4) analyzing results:

type of interaction of LncRNA with protein: first, lncRNA acts as a guide molecule for proteins. In the study of p53 signal path by Huart et al, it was found that long-chain intergenic non-coding RNA p21(long endogenous ncRNA p21, lincRNA-p21) induced by p53 can be used as a guide molecule of heterogeneous nuclear ribonucleoprotein-K (hnRNP-K), and the guide molecule is positioned in the promoter region of p21 gene to activate p21 transcription expression. Secondly, the method comprises the following steps: lncRNA as a protein scaffold molecule. The LncRNA can also serve as a scaffold molecule for two or more proteins, and these proteins are combined into a protein complex map. Hox antisense intergenic RNA (HOTAIR) is located in the Homebox C (HOXC) gene cluster and is expressed in cells distal to the posterior extremity of the limb. HOTAIR can inhibit transcription of homeobox D (HOXD) gene cluster by trans-action, and inhibit HOTAIR can activate expression of HOXD gene cluster. Thirdly, the method comprises the following steps: lncRNA acts as a decoy molecule for proteins. IncRNA-DNA damage activation P21-associated non-coding RNA (P21associated ncRNA DNA damageactivated, PANDA) is often involved in gene expression regulation as a transcription factor decoy molecule. PANDA is located 5kb upstream of cyclin-dependent kinase inhibitor1A (cyclin-dependent kinase inhibitor1A, CDKN1A, i.e., p21) gene, and has 5 'cap and 3' poly A tail structure without cleavage modification. When DNA damage occurs, PANDA is transcribed, and the interaction with a target gene is prevented by combining a nuclear transcription factor Y alpha subunit (NF-YA), so that the survival time of cells is regulated.

In this experiment, the inventors obtained lncRNA H19 in vitro by using an in vitro transcription method, and then obtained a complex of lncRNA H19 and its binding protein and a complex of lncRNA H19 and RNA by using biotin-labeled RNA and an RNA pull-down technique. Protein detection using Mass Spectrometry (MS), a variety of proteins were identified.

catapid is an online algorithm that estimates the tendency of interaction between RNA and protein mainly based on their secondary structure, hydrogen bond and intermolecular forces, and then predicts the interaction between RNA and protein. Starbase V2.0 software can predict miRNA-lncRNA interactions, miRNA-circRNA interactions, miRNA-sncRNA interactions, miRNA-mRNA interactions, Protein-lncRNA interactions, Protein-mRNA interactions, Protein-pseudo-RNA interactions, and Protein-sncRNA interactions. After RNA pull-down, mass spectrum identification shows that a plurality of protein molecules are detected, the cat rapid online algorithm and Starbase V2.0 software predict that 9 proteins interact with lncRNA H19, and two proteins identified by western blot are PCBP1 and FUS.

Functional study of third-part lncRNA H19 target protein

1. The PCPB1-SiRNA sequence was designed and synthesized by Ruibo, Guangzhou

2. Cell lines:

the K562 cell line, human Chronic myeloid leukemia line, was purchased from Shanghai cell Bank, Chinese academy of sciences.

EXAMPLE 12 screening of PCBP1-SiRNA effective sequences

See example 2 for the test methods.

Example 13 Targeted inhibition of the Effect of PCBP1 on Colony formation in K562 cells

The specific method is shown in 5.5.2 in example 5.

Example 14 Targeted inhibition of the Effect of PCPB1 on Imatinib drug sensitivity in K562 cells

1) Transfection of K562 cells with PCBP1-siRNA and SCR

(1) The experimental groups are: BLANK control group (BLANK), random control group, PCBP1-siRNA, each group set up 3 multiple wells, PCBP1-siRNA and SCR concentration is 100nM.

(2) Cells in logarithmic growth phase were taken and cell suspension concentration was adjusted to 30X 104/mL using serum-free, antibiotic-free RPMI-1640 medium, 50. mu.L of cell suspension per well was seeded in 96-well plates and triplicate wells per experimental group.

(3) Preparation of Lipofectamine^TM2000 complex with RNA: according to Lipofectamine^TM2000 instructions, respectively taking the required siRNA amount and Lipofectamine^TM2000, diluted with optimized medium Opti-MEM, respectively.

(4)Lipofectamine^TMAfter the dilution was completed at 2000 deg.C, it was allowed to stand at room temperature for 5 min.

(5) Lipofectamine diluted and left to stand for 5min^TM2000 and diluted siRNA are mixed according to the proportion of 1:1, and the mixture is stood for 20min at room temperature to obtain Lipofectamine^TM2000-RNA complex.

(6) The Lipofectamine required by each group was added^TMThe 2000-RN complex was slowly added dropwise to the cell suspension in the well plate such that the final volume per well was 100. mu.L. Placing 96-well plate at 37 deg.C and CO ₂5% volume fraction in a cell culture chamber.

2) Imatinib action

Grouping experiments:

blank control group: (K562) (ii) a

Imatinib-acting SCR, PCBP1-siRNA group.

Preparing an Imatinib working solution: imatinib with a mother liquor concentration of 10mM was diluted with RPMI-1640 containing 20% fetal bovine serum to working concentrations which were, in order: 0.05, 0.1, 0.15,0.2,0.25 μm. Taking out the 96-well plate transfected with SCR and PCBP1-siRNA for 6h, slowly adding diluted Imatinib working solution into corresponding wells with a volume of 100 μ L per well to make the final volume of each well 200 μ L, adding 100 μ L of RPMI-1640 medium containing 20% serum into blank control group, mixing, placing 96-well plate at 37 deg.C and CO₂Culturing in a cell culture box with the volume fraction of 5%.

3) CCK8 cell activity detection kit for detection

48h later, the detection of the Biotool Vita-Blue cell activity detection kit is carried out

(1) mu.L (1/10 vol.) of Vita-Blue cell activity assay reagent was added to the cell culture medium and mixed well without air bubbles.

(2) The cells were cultured for 1-4h, but direct exposure to light was avoided. Description of the drawings: the sensitivity of the detection increases with the incubation time. For a smaller number of cells, it can be detected after 24h of culture.

(3) When the broth changed from pure blue to light pink, fluorescence readings were recorded using fluorescence microscopy or fluorometer (excitation light 530nm, emission light 590-620 nm). Since the experiment is a live cell experiment, multiple time points should be chosen to determine the best results. However, complete disappearance of the blue color indicates too long incubation. Description of the drawings: there was slight autofluorescence at 590nm using Vita-Blue cell activity detection reagent. This fluorescence varies with the medium, pH, incubation time, exposure time to light, and other factors. To correct for background, one or more cell-free media control wells must be prepared, the mean fluorescence calculated, and then subtracted from the air in the assay.

(4) And (3) calculating the cell viability:

relative cellular activity (%) ═ a (drug +) -a (blank) ]/[ a (drug-) -a (blank) ] × 100%

A (drug +), absorbance of the wells with cells, Vita-Blue reagent and drug solution;

a (drug-): absorbance of wells with cells, Vita-Blue reagent and no drug solution A (blank): absorbance of wells with medium, Vita-Blue reagent and no cells.

The bioinformatics software involved in the present invention includes:

RNA protein interaction prediction software: catRAPID http:// service. tartaglalab. com/update _ submission/108643/f560944664

RNA protein interaction prediction software: http:// starbase

Signal path integration website: http:// kobas. cbi. pku. edu. cn:// kobas

The data processing in the invention uses SPSS statistical software, the data is expressed by mean plus or minus standard deviation, the significance of the difference between each group of data is analyzed by adopting a one-way ANOVA method, and the difference P <0.05 is taken as the significant difference.

The experimental results are as follows:

screening of effective sequences of PCPB1-SiRNA

SCR and PCBP1-siRNA with the concentration of 100nm transfect K562 cells, after the cells are normally cultured for 48h, total RNA of each group of cells is extracted, and Real-time PCR (polymerase chain reaction) is carried out to detect the relative expression level of PCBP1mRNA of each group (as shown in figure 13), so that PCBP1-SiRNA-01 and PCBP1-SiRNA-03 are screened out as effective sequences. The sequences used in the subsequent functional experiments were: PCBP 1-SiRNA-03.

Study of the malignant progression of PCPB1 in K562 cells

2.1 Effect of Targeted inhibition of PCPB1 on malignant progression of K562 cells

2.1.1 Targeted inhibition of PCBP1 Effect on K562 cell Imatinib drug sensitivity

After K562 cells are transfected by SCR and PCBP1-siRNA with the concentration of 100nm, the transfected cells are treated by different concentrations of imatinib, the relative activity of the cells is detected after the cells are cultured for 48h normally, and as a result, as shown in FIG. 14, the drug sensitivity of the cells of the PCBP1-siRNA transfection group to imatinib is obviously improved (the significant difference P is less than 0.05 compared with a blank group and an SCR group), and the relative activity of the cells is reduced along with the increase of the drug concentration of the imatinib.

2.1.2 Effect of Targeted inhibition of PCPB1 on Colony formation in K562 cells

After SCR-siRNA and PCBP1-siRNA with the concentration of 100nm were transfected in K562 cells, the colony formation experiment was performed, and the results are shown in FIG. 15, and compared with the blank group and the SCR group, the cells of the PCBP1-siRNA transfected group were found to have reduced colony formation ability and to have statistical significance (P < 0.05).

And (4) analyzing results:

the experiment of the part is mainly divided into two parts, wherein in the first part, in order to research the function of the target protein of lncRNA, the inventor designs and synthesizes PCPB1-siRNA, firstly, after the low expression of the PCPB1-siRNA is realized, the influence of PCBP1 on K562 cells is researched. PCBP1-siRNA and SCR were synthesized in Ruibo Bio Inc., Guangzhou.

PCBP1, also known as poly (rC) binding protein 1, is located in the 3-band 3 sub-band (2p13.3) of the short arm 1 region of human chromosome 2 in humans. Despite the numerous approaches to the treatment of CML, the first generation protein tyrosine kinase inhibitor Imatinib remains widely used as the first line drug for the treatment of CML. Firstly, SCR and PCBP1-siRNA are transfected in K562 cells, an effective SiRNA sequence is selected, PCBP1-SiRNA-03 has the best effect (figure 13), then the effective sequence is transfected into the K562 cells, and the change of drug sensitivity of each group of cells to imatinib is detected by using a Biotool Vita-Blue cell activity detection reagent, as shown in figure 14, after PCBP1 is targeted and inhibited, the drug sensitivity of the K562 cells to imatinib is enhanced. As shown in FIG. 15, the colony experiment was performed on K562 cells transfected with SCR and PCBP1-siRNA, and it was found that the targeted inhibition of PCBP1 reduced the colony forming ability of K562 cells.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (1)

1. A medicament for treating chronic myeloid leukemia, which comprises an inhibitor of PCBP 1; the inhibitor is targeted SiRNA of PCBP 1; the base sequence of the inhibitor is shown as SEQ ID NO. 8.

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