CN109091492A - The target miRNA miR-106b-5p of lncRNA H19 and its application - Google Patents

Abstract

The invention discloses application of the target miRNA miR-106b-5p of lncRNA H19 in the drug for being used in combination in preparation and/or screening treatment tumour of application and lncRNA H19 and miR-106b-5p in the drug for preparing and/or screening treatment tumour.The overexpression of H19 inhibits the growth of tumour and extends the survival of mouse；Meanwhile the target miRNA that the present invention also passes through software prediction lncRNA H19 has miR-106b-5p, and by being verified；Finally, inventor has found targeted inhibition miR-106b-5p, it is suppressed that the proliferation of K562 cell reduces the colony Forming ability of K562 cell.

Description

The target miRNA miR-106b-5p of lncRNA H19 and its application

Technical field

The present invention relates to the target miRNA miR-106b-5p of the treatment method of malignant tumour, especially lncRNA H19 and It is applied.

Background technique

Chronic myelocytic leukemia (chronic myelocytic leukemia, CML) is a kind of occurs in multipotency hematopoiesis Pernicious bone marrow proliferative tumour on stem cell.CML patient often will appear characteristic chromosome translocation t (9:22) (q34: Q11) (i.e. Ph chromosome), on No. 9 chromosomes 2 transposition of C-ABL proto-oncogene to No. 22 chromosome long arms breaking point gathering Area (BCR) forms BCR-ABL fusion, and unconventionality expression BCR-ABL protein leads to the sustained activation of tyrosine kinases, and Cause the signal transduction pathway in downstream to be activated, to adjust the expression of cell factor, it is a large amount of to eventually lead to marrow stem/progenitor cells Hyperplasia, apoptosis reduce and the decline of the adhesiveness of marrow stromal cell, and a large amount of still immature myelocytes is caused to be discharged into peripheral blood, Lead to CML.According to the overall development process of disease, clinically its development process is divided into three periods, respectively slowly Property phase (Chronic phase, CP), accelerated period (Accelerated phase, AP) and rapid change period (Blastic phase, BP).Chronic phase disease symptoms slightly without specificity, are mainly shown as abdominal distension, abdominal mass, out of strength, low-heat, splenomegaly, see 90% patient, illness degree are different.

With the rapid development of genomic sequencing technique, it has been found that RNA can by 60%~70% genome sequence Transcribe, but wherein only less than 2% coding protein sequence, remaining is referred to as non-coding RNA.In fact, very Many years ago, many non-coding RNAs are just found to have a major impact vital movement, such as are related to the ribosomes of protein synthesis RNA (rRNAs), transfer RNA (tRNA) and small nuclear rna (snRNA), and these non-coding RNAs are commonly known as non-volume of running one's home Code RNA.Non-coding RNA attracts extensive attention in recent years, these RNA are primarily referred to as the non-coding RNA with life adjustment effect, The RNA (LncRNA) studied including microRNA and herein.Research has been found that LncRNA refers to that length is greater than 200 nucleotide And transcribed by RNA polymerase II and lacked or the RNA without open reading frame, it can between gene, enhancer original part, The antisense strand of gene, the other positions for including sub-district genome.

With deepening continuously to long-chain non-coding RNA research, it is found that it is many important LncRNA has the function of, takes part in The important biological process of body, the occurrence and development etc. including cell differentiation, aging, increment, apoptosis and tumour.A variety of LncRNA Such as H19, X chromosome specificity inactive transcription object (Xist) participate in Genomic Imprinting.

Pachnis etc., although can be transcribed by RNA polymerase II, can be sheared simultaneously tailing in discovery h19 genes in 1984, But its product for transcribing out can not translate into the protein of biological function, but be sent out in the form of non-coding RNA The effect of waving.Since H19 product length has been more than 200nt, therefore it is defined as LncRNA.H19 and IGF2 exists in Mice Body In No. seven chromosome, then it is located at 11p15.5 in human body, at a distance of 90kb, this belongs to first pair and is proved to be with the marking the two The gene of characteristic.H19 is presented as the paternal marking and maternal expression, and IGF2 is then presented as paternal expression and the maternal marking.The two Marking characteristic all by the methylation differential modified region (DMR) of H19 promoter upstream 4kb or marking regulatory region (ICR) Regulation.On spore, H19 has highly conserved feature, the high expression within three periods, when being embryonic development respectively It is several in hetero-organization other than heart and skeletal muscle in entoderm, mesoderm and its tissue that differentiates, but after being born It does not express, unless expression can be reactivated under three circumstances: when tissue damage reparation and stress situation and tumour occur.

H19 overall length 2.3kb by 35 small open reading frame, and has 5 ' end cap minor structures and the 3 ' ends of similar mRNA Poly-A tail fawns on structure, theoretically a kind of protein comprising 256 amino acid moleculars of codified, but LncRNA is only It plays a role in mRNA level in-site, and any protein molecule can not be encoded.It compiles in highly conserved region in first promoter of H19 The miRNA-675 molecule of code can control the growth of placenta in latter half of gestation by adjusting the expression of IGFLR gene.The study found that H19 largely assembles in human body placenta element and some embryonic tissues, indicates that H19 may rise very in embry ogenesis and growth and development stage Important role.After fetal birth, H19 expression is lowered, but maintenance basal expression is thrown away in mammary gland, adrenal gland and uterine tissue Amount.It has been reported that and shows in the nephroblastoma, embryonal rhabdomyosarcoma at present, H19 plays tumor suppressor gene, however many In solid tumor (including breast cancer, liver cancer, bladder cancer etc.), H19 is an oncogene.This shows that H19 passes through difference in human body Mechanism participate in body tumour formation and progress.Need further to be ground there are many more unknown link in these different mechanism Study carefully discovery.

Summary of the invention

Based on the above issues, present inventor participates in the formation of body tumour and the specific machine of progress in research H19 During system, it has unexpectedly been found that by promoting the expression of lncRNA H19 or inhibiting the target miRNA of lncRNA H19 MiR-106b-5p (its base sequence is as shown in SEQ ID NO.6), can effectively inhibit the proliferation of tumour cell.Specifically, originally The technical solution of invention includes the following aspects:

In the first aspect, the present invention provides the target miRNA miR-106b-5p of lncRNA H19 to prepare and/or sieve Application in the drug of choosing treatment tumour.

Preferably, the tumour is leukaemia.

In the second aspect, the present invention provides lncRNA H19 and miR-106b-5p be used in combination preparation and/or Application in the drug of screening treatment tumour.Wherein, the overexpression of lncRNA H19 can inhibit the proliferation of tumour cell, targeting Inhibit miR-106b-5p that can inhibit the Colony forming ability (colony) of tumour cell and the proliferation of tumour cell.

Preferably, the tumour is malignant tumour.

In the third aspect, the present invention provides a kind of drug for treating tumour, contain miR-19a-3p in the drug Inhibitor.

Preferably, the inhibitor is the antisense nucleic acid of miR-106b-5p.

Preferably, also containing the reagent or carrier that can promote lncRNA H19 expression in the drug.

Preferably, the base sequence of the antisense nucleic acid of the miR-106b-5p such as t-anti-miR-106b-5p (i.e. SEQ ID NO.7) shown in.

It is highly preferred that the tumour is leukemia.

In conclusion the invention has the benefit that

It is proposed that the overexpression of lncRNA H19 inhibits the growth of tumour and extends the survival of mouse in the present invention；Together When, the target miRNA that the present invention also passes through software prediction lncRNA H19 has miR-106b-5p, and by being verified； Finally, inventor has found targeted inhibition miR-106b-5p, it is suppressed that the proliferation of K562 cell reduces K562 cell Colony Forming ability.

Detailed description of the invention

Fig. 1 is slow virus carrier structure chart；

Fig. 2 is the testing result figure of Normal group and the expression of K562 and KCL-22 cell H19；

Fig. 3 is the selection result figure of the Real-time PCR to H19-SiRNA ordered sequence；

Fig. 4 is the testing result figure of the expression of the H19 in K562-H19LentiV and K562empty LentiV；

Fig. 5 is the colony number testing result figure of K562-H19Lenti and K562-empty LentiV group, and wherein A is Colony figure (40 ×)；B is opposite colony number；

Fig. 6 is influence result figure of the H19 overexpression to BALB/c nude mouse；Wherein,

After A shows that injection is overexpressed the P210 cell of H19, the size of mouse tumor；

B shows that injection is overexpressed influence to mouse survival after the P210 cell of H19；

After C shows that injection is overexpressed the P210 cell of H19, the statistical result of mouse tumor volume；

Fig. 7 is that NOD/SCID mouse tail vein transplants K562-NEG-LentiV and K562-H19Lenti V, living imaging Mouse invasion rate is observed, the result figure of mouse survival rate is recorded；

Fig. 8 is the flow chart of tiny RNA sample handling procedure；

Fig. 9 is the flow chart of tiny RNA data analysis step；

Figure 10 is the qualification result figure of PCR product；Wherein, A is the agarose gel electrophoresis figure after PCR product, B PCR (sequence has taken T7 promoter) result figure is sequenced in product；

Figure 11 is the lncRNA H19 agarose gel electrophoresis figure after being transcribed in vitro；

The result schematic diagram of 2.0 software prediction of Figure 12 StarBase v, wherein display and LncRNA H19 interaction In the microRNA that microRNA and RNA-seq are detected, 5 be it is common, they are respectively: hsa-miR-19a-3p, hsa-miR-106b-5p,hsa-miR-148a-3p,hsa-miR-148b-3p,hsa-miR-454-3p；

Figure 13 is the result figure of real-time quantitative PCR；It demonstrates miR-19a-3p and miR- in RNA pull-down product 106b-5p；

Figure 14 is lncRNA-miRNA interaction model；

Figure 15 be transfect t-antimiR-106b-5p after to K562 cell activity influence result figure (* * P < 0.01, * P < 0.05, t-antimiR-106b-5p compared with blank group and SCR group)；

Figure 16 is K562, the colony result figure of SCR and t-antimiR-106b-5p group, wherein A is colony figure (40 ×), B is opposite colony number (* P < 0.05, t-antimiR-19a-3p and t-antimiR-106b-5p group and K562, SCR group phase Than).

Specific embodiment

To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair The present invention is described further.The embodiment of the present invention mainly includes three parts, reagent, consumptive material, the Yi Jiqi being directed to Its test article can be obtained from market or other public's channels.

First part: the Function Identification of lncRNA H19

1.H19-SiRNA sequence and H19 Lentiviral

1.1H19SiRNA sequence

1.2 H19 Lentivirals

H19 Lentiviral is implemented in Guangzhou FulenGen Co., Ltd..

H19 Lentiviral: CS-GS3160-LV201；

Empty Lentiviral: CS-NEG-LV-201

2. cell strain

K562 cell strain, human chronic myelogenous leukemia system, is purchased from Shanghai Cell Bank of the Chinese Academy of Sciences

3. main agents are prepared

3.1 contain the cell culture medium of 10% serum

Australia fetal calf serum is placed in high-pressure sterilizing pot 56 DEG C, is sub-packed in the healthy and free from worry centrifuge tube of 10mL after 30min fire extinguishing, Serum after packing saves in -20 DEG C of refrigerators.RPMI-1640 culture medium is sub-packed in 200mL wide-mouth bottle, and every bottle of 180mL is put in It is saved in 4 DEG C of refrigerators.Fetal calf serum 20mL in Australia is added in every 180mL culture medium, prepares so that blood in RPMI-1640 culture medium Clear content is 10%.

3.2 pH7.2 phosphate buffers (PBS)

Said components are weighed respectively, dissolve the above-mentioned component weighed up using distilled water and are settled to 1000mL, and high pressure is steamed Vapour sterilizing 30min, takes out, 4 DEG C of preservations.

3.3 prepare 2.7% methylcellulose semisolid culturemedium

2.7g methylcellulose powder is weighed in wide-mouth bottle, 50mL boiling water is added, dissolves while stirring, 30min takes Out, to its cooling, sterile 2 × 1640 culture medium of 50mL is added in superclean bench, stirring is stored in 4 DEG C until dissolution Refrigerator is stand-by.

1 K562 cell culture of embodiment

CML cell K562 is inoculated in RPMI-1640 culture medium, the content of serum is 10% in RPMI-1640, will be inoculated with It is 37 DEG C, CO that good K562 cell, which is placed in temperature,₂It is cultivated in the cell incubator that volume fraction is 5%.

The screening of 2 H19-SiRNA ordered sequence of embodiment

2.2.1 H19-SiRNA transfection K 562 cell

(1) experimental group are as follows: blank control group (BLANK), SCR, H19-SiRNA-01, H19-SiRNA-02, H19- SiRNA-03 group, the concentration of SCR, H19-SiRNA-01, H19-SiRNA-02, H19-SiRNA-03 are 100nM.

(2) logarithmic growth phase cell, with serum-free, antibiotic-free RPMI-1640 culture medium by concentration of cell suspension It is adjusted to 30 × 10⁴/ mL, the cell suspension inoculation of every hole 1.5ml is in 6 orifice plates.

(3) Lipofectamine is prepared^TMThe compound of 2000 and RNA: according to Lipofectamine^TM2000 specifications, The siRNA amount and Lipofectamine that each takes what he needs^TM2000 amounts are diluted with Optimal Medium Opti-MEM respectively.

(4)Lipofectamine^TMAfter the completion of 2000 dilutions, it is stored at room temperature 5min.

(5) dilute and stand the Lipofectamine of 5min^TM2000 with diluted SiRNA and mixed according to the ratio of 1:1, It is stored at room temperature after 20min to get Lipofectamine is arrived^TM2000-RNA compound.

(6) by Lipofectamine needed for each group^TM2000-RNA compound is slowly added drop-wise to the cell suspension in orifice plate In, so that the final volume in every hole is 2mL.6 orifice plates are placed in 37 DEG C, CO₂In the cell incubator that volume fraction is 5%.

2.2.2 Real-time PCR detects the relative expression levels of H19mRNA in group of cells

2.2.2.1 Total RNAs extraction

Experimental group: blank control group (BLANK), SCR, H19-SiRNA-01, H19-SiRNA-02, H19-SiRNA-03

(1) group of cells of logarithmic growth phase, 1500rpm are centrifuged 5min, abandon supernatant.

(2) PBS washes cell precipitation one time, and 1500rpm is centrifuged 5min, abandons supernatant.

(3) trizol of 1mL is added into cell precipitation, cracks 5min on ice.

(4) chloroform of 200 μ L is added into cell pyrolysis liquid, vibrates 45s, stands 10min on ice.

(5) 12000rpm, is centrifuged 15min by 4 DEG C.Careful supernatant of drawing is in a clean EP pipe, minus 20 DEG C of placements 20min。

(6) 12000rpm, is centrifuged 10min by 4 DEG C.Carefully discard supernatant.

(7) 70% alcohol washes RNA precipitate, and 12000rpm, is centrifuged 5min, in triplicate by 4 DEG C.

(8) precipitating is dried in draught cupboard, 2-3min.The RNA of 30 μ L is added in precipitating without enzyme water.RNA is placed in minus 80 DEG C save.

2.2.2.2 reverse transcription reaction (RT)

(1) PCR pipe for taking RNA sample 1 μ g of the A260/A280 between 1.8-2.0 and nuclease free to pollute, each group RNA It is placed in spare on ice.

(2) according to the specification of the All-in-One cDNA Synthesis SuperMix of bimake company, in nucleic acid Reverse transcription reaction (operating on ice) is carried out without following components in enzyme pipe, is added:

(3) after adding well according to said components, brief centrifugation 10s.

(4) PCR pipe is put into PCR instrument, the standard reaction program progress reverse transcription reaction provided according to kit: 42 DEG C 15min；85℃2min.

2.2.2.3 Real-time PCR

(1) each group is detected according to 2 × SYBR Green qPCR Master Mix kit of bimake company The relative expression levels of H19mRNA, GAPDH are internal reference, and 1 μ L cDNA is taken to carry out Real-time PCR amplification.

(2) reaction system is as follows:

The relative expression levels of H19 and GAPDH are with 2^-△△CTIt calculates.

Embodiment 3 establishes the K562 cell strain for stablizing expression H19

3.3.1 H19 Lentiviral and empty Lentiviral transfection K 562 cell

(1) logarithmic growth phase cell, with serum-free, antibiotic-free RPMI-1640 culture medium by concentration of cell suspension It is adjusted to 30 × 10⁴/ mL, every hole 1.75mL cell suspension inoculation is in 6 orifice plates.

(2) Lipofectamine is prepared^TM3000 in the compound of DNA: according to Lipofectamine^TM3000 specifications It prepares, every hole takes the amount of DNA of 2.5 μ g and the Lipofectamine of 3.75 μ L^TM3000 amounts, respectively with the Opti-MEM of 250 μ L Dilution.

(3) DNA and Lipofectamine^TM3000 dilutions are completed, and dilution are uniformly mixed according to the ratio of 1:1, room temperature After standing 5min, Lipofectamine is obtained^TM3000-DNA compound.

(4) by Lipofectamine^TM3000-DNA compound is slowly dropped in the cell suspension in orifice plate, and every hole is most Final volume is 2mL.It is 37 DEG C, CO that six orifice plates, which are placed in temperature,₂It cultivates in the cell incubator that volume fraction is 5%, after 6h, adds Enter the RPMI-1640 culture medium that 2mL is 20% containing serum.

3.3.2 the K562 cell strain of expression H19 is stablized in puromycin screening

After H19 slow virus carrier transfection K 562 cell, puromycin is added in cultivating system, keeps the end of puromycin dense Degree was 1mg/mL, every 2 days replacement culture solutions.

4 Real-time PCR of embodiment detects the relative expression levels of intracellular H19mRNA

4.4.1 Total RNAs extraction,

According to 2.2.2.1

4.4.2 reverse transcription reaction (RT)

According to 2.2.2.2

4.4.3Real-time PCR

According to 2.2.2.3

The influence that 5 H19 of embodiment forms K562 cell colony

5.5.1 experimental group

1.K562 blank control group

2.K562-H19LentiV group (the K562 cell for stablizing expression H19)

3.K562-empty LentiV group (containing the K562 cell for being free slow virus carrier)

5.5.2 COLONY is tested

Experiment is divided into two groups: stablizing the K562 cell of expression H19 and has transfected the K562 cell of empty Lentiviral. The cell of each experimental group of logarithmic growth phase dilutes K562 cell with serum free medium, so that its density is 1 × 10³/ ML takes the K562 cell suspension of 450 μ L, is added to 550 μ L containing 20% fetal calf serum, 5 μm of ol/L beta -mercaptoethanols, 2mmol/L L-Glutamine and 0.9% methylcellulose semisolid culturemedium in, cell suspension and semisolid culturemedium is sufficiently mixed After even, every hole 1mL is inoculated in 24 orifice plates, and each experiment repeats three multiple holes, and it is 37 DEG C, CO that 24 orifice plates, which are placed in temperature,₂Body It is cultivated 7-14 days in the cell incubator that fraction is 5%.It is placed under inverted microscope and observes COLONY size and form, with The cell mass of approximately greater than 40 cells is 1 COLONY, and preservation of taking pictures, experiment is in triplicate.

Functional study of 6 H19 of embodiment in CML mouse model

6.6.1 the tumor formation of Balb/c nude mice by subcutaneous is tested

H19 slow virus carrier and empty slow virus carrier are transfected in P210 cell, and it is thin to construct the stable P210 for being overexpressed H19 Density is 1 × 10 by born of the same parents⁷200 μ L to the BALB/c nude mouse left lower extremity of P210-H19 and P210-NEG cell inoculation of/mL Its tumor formation situation is observed in oxter, after mouse tumor formation, the daily variation for measuring mouse tumor size.

6.6.2 NOD/SCID mouse tail vein is tested

H19 slow virus carrier and empty slow virus carrier are transfected in K562 cell, and it is thin to construct the stable K562 for being overexpressed H19 Density is 1 × 10 by born of the same parents⁷The 200 μ L of K562-H19 and K562-NEG cell transplantation of/mL, tail vein injection K562-H19 cell With K562-NEG cell, living imaging observes and records the morbidity and survival condition of mouse.

Experimental result:

Influence of the 1.H19 to K562 cell progression

The detection of 1.1 H19 expression quantity

The blood of normal person is taken, RNA is extracted, while to K562 cell, KCL-22 cell, 293T cell extraction RNA, fluorescence is fixed The expression quantity for measuring PCR detection each group H19, as a result as shown in Fig. 2, compared with Normal group, the table of H19 in K562, KCL-22 It is lower up to measuring.

The screening of 1.2 H19-SiRNA ordered sequences

Concentration is SCR (serum creatinine), the H19-siRNA transfection K 562 cell of 100nm, after normal culture 48h, extracts each group The total serum IgE of cell, Real-time PCR detect the relative expression levels of each group H19mRNA, MTT detection transfection H19-SiRNA Afterwards, the cell viability of each group K562 cell.As a result as shown in Figure 3.

The Efficiency testing of 1.3 H19 slow virus carrier transfection K 562 cells

H19 slow virus carrier (CS-GS3160-LV201) used and empty slow virus carrier (CS-NEG-LV-201) be by Provided by Guangzhou FulenGen Co., Ltd..H19 slow virus carrier screens after transfection K 562 cell through puromycin, obtains To the K562 cell for stablizing expression H19.The each group K562 cell of logarithmic growth phase is collected, the phase of total serum IgE detection H19mRNA is extracted To expression.The relative expression levels of the intracellular H19 of each group K562 are as shown in figure 4, relative to empty slow virus carrier (Empty LentiV), the expression of the H19mRNA of H19 slow virus carrier group (H19LentiV) is significantly raised.

1.4 H19 are overexpressed the influence formed to K562 cell colony

The competence for added value that method can be used to detect single tumor cell is formed by the colony of carrier of methylcellulose.It takes K562, K562-empty LentiV and K562-H19LentiV of logarithmic growth phase carry out colony experiment, as a result such as Fig. 5 institute Show, relative to K562 group and K562-empty LentiV group, the colony number and size of K562-H19 LentiV group are reduced (P<0.05)。

2. being overexpressed influence of the H19 to CML model mouse Balb/c nude mice

Influence of the H19 overexpression to BALB/c nude mouse tumor size is probed into nude mice by subcutaneous tumor formation, to BALB/c The influence of nude mouse survival, in order to probe into influence of the H19 to BALB/c nude mouse P210 tumor size, in P210 cell Middle transfection H19 slow virus carrier and empty slow virus carrier, construct the P210 cell stablized and be overexpressed H19, are 1 × 10 by density⁷/ 200 μ L to the BALB/c nude mouse left lower extremity oxter of P210-H19 and P210-NEG cell inoculation of mL, observes its tumor formation feelings Condition, after mouse tumor formation, the daily variation for measuring mouse tumor size.As can be seen from Figure 6 the tumour ratio of H19 overexpression group Control group it is small.

3. being overexpressed influence of the H19 to CML model mice NOD/SCID

Mouse tail vein injection K562-H19 cell and K562-empty cell, living imaging observe and record the morbidity of mouse And survival condition.As a result as shown in Figure 7.

Interpretation of result:

The present invention first detects the expression quantity of H19, takes the blood and K562 cell and KCL-22 cell of normal person, RNA is extracted, H19 expression quantity is carried out and is detected.Such as Fig. 2, it can be seen that compared with Normal group, in K562 and KCL-22 The expression quantity of H19 is relatively low.Secondly, having carried out the screening of ordered sequence, H19-siRNA and SCR synthesis to the SiRNA of H19 In the sharp rich biological Co., Ltd in Guangzhou.It is screened through Real-time PCR, H19-SiRNA-02 and H19-SiRNA-03 two sequences Effectively, unobvious to the effect of vigor of K562 cell such as Fig. 3, but after targeted inhibition H19.Therefore, inventor is sick slowly using H19 Poisonous carrier transfection K 562 cell, puromycin screens positive cell, so that H19 overexpression, stablizes strain and be named as K562- H19LentiV, research H19 are overexpressed the effect being in progress to K562 malignant, and such as Fig. 4, H19 overexpression inhibit K562 cell Clone formation.Lentiviral CS-GS3160-LV201 and CS-NEG-LV-201 building and technical support are answered by Guangzhou It can genome company's offer.

CS-GS3160-LV201 contains H19 and eGFP target gene fragment, and CS-NEG-LV-201 contains only eGFP, institute To observe the positive cell of GFP albumen after transfection is completed, transfection efficiency can be tentatively judged, it is anti-according to what is had on carrier Property marker gene using puromycin carry out preliminary screening.Strain is stablized to the K562 after transfection slow virus and extracts RNA, Real- The relative expression levels of time PCR detection H19.Fig. 5 shows compared with K562-empty LentiV groups of cells, K562- The expression of H19LentiV intracellular H19mRNA is significantly raised, the above result shows that, stablize the K562 cell of expression H19 It is successfully established, this provides experimental model for function of the research H19 in CML.

Clonal hyperplasia is one important feature of CML K562 cell strain, therefore, to K562 cell clonal formation energy Power, which carries out research, can prove influence of the H19 to CML cell progression.Colony forms experiment and refers to individual cells especially tumour Upgrowth situation of the cell under methylcellulose semisolid culturemedium condition of culture, by inverted microscope observe cell number with And colony form, it can be used to evaluate the proliferative capacity of individual cells.Experiment is formed by colony to learn, is overexpressed H19 The colony size and number of K562 cell are reduced afterwards, and the proliferative capacity of the expression and K562 cell of H19 is closely related.

Chronic myelocytic leukemia has characteristic BCR/ABL fusion and its product BCR/ABL fusion protein (P210), the albumen have strong tyrosine kinase activity, can abnormal activation many A signal pathways, cause haemocyte pernicious Conversion.BaF-P210 cell, abbreviation P210 are the cell strains for stablizing expression BCR/ABL.Therefore, this part is in animal water The flat influence for having visited influence and survival that H19 is overexpressed to BALB/c nude mouse tumor formation.Inventor stablized constructing Density is 1 × 10 by the P210 cell for expressing H19⁷200 μ L to the BALB/c of P210-H19 and P210-NEG cell inoculation of/mL From the mouse left lower extremity oxter nude is injected, the tumor formation situation and survival condition of mouse are observed.Fig. 6 can be seen that with it is right It is compared according to group, the overexpression of H19 inhibits the growth of tumour and extends the survival of mouse.Meanwhile inventor is constructing Stablize the K562 cell for being overexpressed H19, is 1 × 10 by density⁷K562-H19 the and K562-NEG cell of/mL is infused by tail vein It penetrates, by 200 μ L to NOD/SCID Mice Body of cell transplantation, living imaging observes the incidence and survival condition of mouse, From figure 7 it can be seen that compared with the control group, the overexpression of H19 inhibits the growth of tumour and extends the survival of mouse.

Second part: the identification of lncRNA H19 target miRNA

1. with T7 promoter sequence h19 gene primer sequence (as shown in the table), by give birth to work bioengineering (on Sea) limited liability company designs and synthesizes.

2.K562 cell strain, human chronic myelogenous leukemia system, is purchased from Shanghai Cell Bank of the Chinese Academy of Sciences.

3. main agents are prepared

3.1 prepare 10 × TAE electrophoretic buffer

Said components are weighed in beaker, 800mL distilled water, stirring, so that it is sufficiently dissolved are added into beaker；It is added 11.4mL glacial acetic acid, is sufficiently stirred；1L is settled to distilled water, room temperature preservation, for use.

3.2 prepare 0.8% agarose gel

It weighs 0.4g agarose and is dissolved in 50mL distilled water, stir evenly, and microwave stove heating, until boiling, takes out, to Temperature drops to 60 DEG C or so, and the GeneRed of 3 μ L is added, is sufficiently stirred, pours into stick mixing tank, is inserted into comb, standby to its solidification With.

3.3 LB culture mediums are prepared

Above-mentioned each component is weighed in beaker, distilled water is added to stir and is settled to 1L, is placed in high-pressure steam sterilizing pan sterilizing, It is spare.

3.4 LB solid mediums are prepared

Other than ingredient each in aforesaid liquid culture medium, then plus 15g agar, equally plus distilled water constant volume arrive 1L, sterilizing greatly It after about 30min, takes out, drops to 60 DEG C or so to temperature, be poured into the culture dish for having added antibiotic, shake up, to solidifying Gu after, 4 DEG C of preservations are spare.

3.5 prepare the sodium chloride of 5M

292.5g sodium chloride is weighed, distilled water is added to stir, until dissolution, and is settled to 1L.It is spare.

3.6 10 × TBS buffer (pH=7.6)

Said components are weighed, appropriate distilled water dissolution, being adjusted to pH with HCL is 7.6, and distilled water is added to be settled to 1L, room temperature It saves.10 × TBS is diluted to 1 × TBS when use.

3.7 prepare 1 × TBST buffer

100mL1 × TBS solution is measured, 1mLTween-20 is added, appropriate distilled water is added and is settled to 1L, as 1 × TBST buffer.

3.8 prepare 1 × transferring film buffer (pH=8.5)

Said components are weighed, appropriate distilled water dissolution is added, is 8.5 with HCL adjustment pH, is settled to 1L, room with distilled water Temperature saves.

3.9 prepare confining liquid

5g skimmed milk power is weighed, is dissolved in 100mLTBST, matching while using.

3.10 preparing antibody diluent

5g skim milk is dissolved in 50mLTBST, 5% skim milk is made into, first with now matching.

3.11 5 × SDS-PAGE electrophoretic buffer

Add appropriate distilled water, stirring and dissolving adds distilled water to be settled to 1L.Room temperature preservation.Distilled water is added to be diluted to 1 when use ×SDS-PAGE。

3.12 1.5mol/L pH=8.8Tris-HCL buffers

Add appropriate distilled water to dissolve, and is settled to 100mL.4 DEG C of preservations.

3.13 30% acrylamide

Weigh said components, distilled water dissolution and constant volume value 100mL.4 DEG C of preservations.

3.14 10% separation gel

3.15 5% concentration glue

Embodiment 7 obtains the template being transcribed in vitro

1) preparation of competent E.coli

(1) the one minus 70 DEG C conservation bacterium solution bacillus coli DH 5 alphas frozen are taken, 5 μ L bacterium solutions and 5mLLB Liquid Culture are drawn In base, 37 DEG C, the activation of 250rpm shaking table is overnight.

(2) 50 μ L are taken from the bacterium solution of step (1), are inoculated into the fresh LB liquid medium of 5mL, 37 DEG C, 250rpm Shaking table culture 3h takes survey OD value in the EP pipe of appropriate bacterium solution and 1.5mL (0.3-0.4OD value can be tested).

(3) take the bacterium solution of 1mL in the sterile EP tube of 1.5mL in super-clean bench, 5000rpm is centrifuged 5min, removes supernatant.It is added The CaCl of the 0.1mol/l of 1mL pre-cooling₂Resuspended bacterium solution places 30min on ice.

(4) 5000rpm, is centrifuged 5min, removes supernatant by 4 DEG C.

(5) CaCl of the 0.1mol/l of 50 μ L pre-cooling is added₂Resuspended bacterium solution, as competent cell (are placed in minus 80 DEG C of ice Case saves)

2) H19 Lentiviral converts Escherichia coli

(1) competent cell is placed on ice to melt, after it melts, it is sick slowly that H19 is added into competent cell suspension Malicious over-express vector DNA1 μ L, mixing, ice bath 30min are gently blown and beaten with pipettor.

Centrifuge tube, is transferred in ice bath rapidly, stands 3min on ice by (2) 42 DEG C of thermal shock 45s.

(3) the sterile not antibiotic LB culture medium of 450 μ L is added into centrifuge tube, mixes, is placed in 37 DEG C of shaking tables, 150rpm shaken cultivation 45min, so that thallus is recovered.

(4) 100 μ L bacterium solutions are taken, are coated on the LB plate containing ampicillin, will uniformly be applied using sterile spreading rod It opens, plate is placed in 37 DEG C and is absorbed to liquid, be inverted culture, 37 DEG C of overnight incubations.

(5) prepare the centrifuge tube of a 10mL the next morning, add 5mLLB culture solution, then plus the concentration of 2.5 μ L be ammonia benzyl Penicillin, picking single bacterium colony are inoculated in the LB culture medium for having added ampicillin, 37 DEG C of shaken cultivations, 10 left sides h It is right.

3) extraction of Plasmid DNA

Extracting the reagent used see the table below:

(1) bacterium solution for taking 5mL to be incubated overnight is added in centrifuge tube, and 13000rpm is centrifuged 1min and collects bacterial sediment, carefully Suck supernatant.

(2) 250 μ L Buffer P1 are added into the centrifuge tube containing precipitating, are mixed well using pipettor, it is heavy to suspend It forms sediment.

(3) 250 μ L Buffer P2 are added into centrifuge tube, mixing 10 times of mildly turning upside down cracks thallus sufficiently, It is placed at room temperature for 5min.Solution should become limpid sticky at this time.

(4) 250 μ L Buffer E3 are added into centrifuge tube, white wadding occurs at this time in mixing 10 times of turning upside down immediately Shape precipitating is placed at room temperature for 5min, and 13000rpm is centrifuged 5min, draws supernatant, and filtering note (Endo-Remover is added in supernatant FM in), 13000rpm is centrifuged 1min filtering, and filtrate is collected in centrifuge tube.

(5) 225 μ L isopropanols, mixing of turning upside down are added into filtrate.

(6) 200 μ L Buffer column equilibration: are added into the adsorption column (Spin Col μm ns DM) for having been charged into collecting pipe PS, 13000rpm are centrifuged 1min, outwell the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.

(7) mixed liquor of filtrate and isopropanol in step (5) is transferred to the adsorption column balanced and (has been charged into collection Pipe) in.

(8) 13000rpm is centrifuged 1min, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.(absorption The maximum volume of column is 750 μ L, if sample volume is greater than 750 μ L and can be added portionwise.)

(9) Buffer PW, the 13000rpm centrifugation that 750 μ L have been added to dehydrated alcohol in advance is added into adsorption column Min outwells the waste liquid in collecting pipe.

(10) adsorption column is placed back in collecting pipe, 13000rpm is centrifuged 1min.(note: the purpose of this step be by Remaining ethyl alcohol removes in adsorption column, and ethyl alcohol residual will affect subsequent enzymatic reaction.)

(11) adsorption column is placed in a new collecting pipe, 50-100 μ L Edno- is added to the intermediate position of adsorbed film Free Buffer EB, is placed at room temperature for 5min, and 13000rpm is centrifuged 2min, plasmid solution is collected into centrifuge tube.Minus 20 DEG C Save plasmid.

4) Plasmid DNA is quantitative

Plasmid DNA is quantitative: being quantified using nucleic acid quantification instrument, is quantitatively averaged three times.

5) PCR amplification

According to the Thermo Scientific Phusion High-Fidelity of thermo scientific company DNA Polymerase carries out PCR amplification

(1) EP of the EXYGEN of one 200 μ L is taken to manage.

(2) amplification system see the table below:

(3) after mixing well, upper machine carries out PCR amplification.

(4) it is expanded according to following procedure

6) agarose gel electrophoresis carries out the identification of pcr amplification product

(1) take the PCR product of 10 μ L in the EP pipe of a 200 new μ L.

(2) 5 × Loading buffer of 2 μ L is added into EP pipe, is mixed with micro sample adding appliance.

(3) the fresh electrophoretic buffer of 1 × TAE is added into electrophoresis tank, comb is pulled out, so that electrophoresis liquid there was not gel.

(4) successively loading: the mixed liquor of DNA Ladder of 5 μ L, PCR product and Loading buffer.Purpose PCR is produced 14 multiple holes on object, so that the enough subsequent experimentals of amount of DNA of recycling.

(5) condition is set, and the electricity is at 110 volts of pressure for adjusting, time 30min.Carry out agarose gel electrophoresis.

(6) electrophoresis terminate under ultraviolet lamp observe pillar location it is whether correct, preservation of taking pictures.

6.1.7 purification and recovery gel DNA

The reagent and other compositions that recycling DNA is used see the table below:

(1) single goal DNA band is cut into (excision redundance as far as possible) from Ago-Gel and is put into clean centrifugation Guan Zhong weighs weight.

(2) the Buffer NJ of 3 times of volumes is added: if gel weight is 0.1g, then its volume is 0.1mL in 55-65 DEG C of water Melt completely in bath to gel.(note: after gel is completely dissolved, observation mixture color is then wanted if it is pink It is 3M, the sodium acetate that pH is 5.2, to turn down its pH value that 5 μ L concentration, which are added,.Through overregulating, the color of mixture should be pale yellow Color.)

(3) the resulting solution of previous step is added on GBC adsorption column to (note: absorption column volume is 800 μ L, if sample Volume is greater than 800 μ L.It is added can be centrifuged in batches after).After placing 2min at room temperature, 1000 × g is centrifuged 60s.It abandons in collecting pipe and filters Pillar is recovered 2mL collecting pipe by liquid.

(4) filtrate in collecting pipe is abandoned, pillar is recovered into 2mL collecting pipe.600 μ LDNA Wash Buffer are added to pillar In.10000 × g is centrifuged 30-60s at room temperature.

(5) it repeats to wash pillar with DNA Wash Buffer.10000 × g is centrifuged 30-60s at room temperature.

(6) filtrate in collecting pipe is abandoned, pillar recovers 2mL collecting pipe.At room temperature, 13000 × g is centrifuged 2min to dry pillar Matrix residual liquid.

(7) pillar on the centrifuge tube of a clean 1.5mL, 15-25 μ L is added and (it is dense to be specifically dependent upon final product Degree) on Elution Buffer to base for post matter, it is placed at room temperature for 1min, 12000 × g is centrifuged 1min with eluted dna.

Embodiment 8 obtains the lncRNA H19 of desthiobiotin label

1) PCR product of purification and recovery is transcribed in vitro as template

(1) thaw each reagent: RNA Polymerase Enzyme Mix is placed on ice；

(2) by 10 × Reaction Buffer and 4 kinds of Ribonucleotide solutions (ATP, CTP, GTP and UTP) vortex gently vibrates, until dissolution.After dissolution, 10 × Reaction Buffer is placed in room temperature, by four kinds Ribonucleotide solutions is placed on ice.

(3) responsive transcription is carried out in room temperature.

1. taking the EP pipe without RNA enzyme of a 200 μ L

2 μ LATP solution, 2 μ L CTP solution, 2 μ LGTP are sequentially added using micro sample adding appliance Solution, 2 μ LUTP solution, 2 μ L 10 × Reaction Buffer, 1 μ g of template DNA, add the Enzyme of 2 μ L Mix, finally using nucleic acid without 20 μ L of enzyme water polishing.

2. being mixed gently up and down using micro sample adding appliance.

3. 37 DEG C of overnight incubations.

It completes, takes out 4. being incubated for, the Dnase of 1 μ L is added inside reaction system, mix, 37 DEG C of incubation 15min.Dnase's Purpose is the template DNA inside removal system.

5. terminating reaction, RNA is precipitated.In the reaction system plus the chlorine of the Nuclease-free Water of 30 μ L and 30 μ L Change lithium precipitating reagent.

6. thoroughly mixing, -20 DEG C of precipitating 30min and 30min or more.

7. 4 DEG C, maximum (top) speed is centrifuged 15min to precipitate RNA.

8. carefully removal supernatant.It is primary that precipitating is washed with the ethyl alcohol of 1mL70%.Purpose is farthest to remove impurity.4 DEG C, maximum (top) speed is centrifuged 15min.

9. the carefully ethyl alcohol of removal 70% is resuspended RNA without enzyme water with nucleic acid, quantifies to RNA, minus 70 DEG C of preservations.

2) RNA completed is transcribed in vitro and carries out desthiobiotin label

(1) DMSO is placed in and is thawed on ice；By 30% PEG (polyethylene glycol) in 37 DEG C of heating until it becomes flowable Liquid.Remaining reagent thaws on ice.

(2) prepare heater, be adjusted to 85 DEG C.

(3) it takes the EP of a 200 μ L to manage, the unmarked RNA (50pmol) of 5 μ L is added, adds the DMSO of 1.25 μ L. The effect of DMSO is:

(4) add reaction system according to following table:

(5) 16 DEG C of overnight incubations, reaction efficiency can be increased by being incubated overnight.

(6) reaction terminates, and in the reaction system plus the nucleic acid of 70 μ L is without enzyme water.It mixes well.

(7) chloroform:alcohol (24:1) of 100 μ L is added (to remove RNA therein in the reaction system Ligase), mix gently, maximum (top) speed is centrifuged 3min.It is careful to draw supernatant, it is transferred to the EP pipe of a new nuclease free In.

(8) concentration that 10 μ L are separately added into the supernatant into the 7th step is the glycogen and 300 μ of the Nacl of 5M, 1 μ L The cold ethyl alcohol of the 100% of L.Minus 20 DEG C of placements at least 1hour.

(9) 4 DEG C, 13000 × g is centrifuged 15min, carefully removes supernatant, is careful not to abandon precipitating.

(10) the cold ethyl alcohol pipette tips that 300 μ L 70% are added mix gently up and down, wash precipitating.4 DEG C, 13000 × g centrifugation 15min carefully removes supernatant, and precipitating is dried (no more than 5min).

(11) precipitating, minus 80 DEG C of preservations is resuspended with (nucleic acid is without enzyme water) the nuclease-free water of 20 μ L.

The combination of long-chain non-coding RNA H19 and RNA that embodiment 9 has marked

1) preparation of RNA:

(1) take the K562 cell of a certain amount of logarithmic growth phase in 15mL centrifuge tube, 1500rpm is centrifuged 5min.

(2) supernatant is abandoned, is washed cell one time, 1500rpm with the PBS of 2mL, 5min is centrifuged.

(3) supernatant is abandoned, extra supernatant is sopped up, the Trizol of 1mL is added into centrifuge tube, gently piping and druming mixes, and places on ice 5min。

(4) cell pyrolysis liquid for having added Trizol clean and the 1.5mL without RNA enzyme a EP is sucked to manage, And the chloroform of 200 μ L is added, 30s is vibrated, stands 10min on ice.

(5) 12000rpm, is centrifuged 15min by 4 DEG C.

(6) supernatant is carefully drawn in a new EP pipe without RNA enzyme, and isometric isopropanol, minus 20 DEG C of placements is added At least 20min.

(7) 12000rpm, is centrifuged 10min by 4 DEG C.

(8) supernatant is carefully removed, is precipitated with 75% ethanol washing.The ethyl alcohol of 1mL75% is added into EP pipe, piping and druming is mixed It is even, 12000rpm, 4 DEG C of centrifugation 5min.

(9) supernatant is carefully removed, is repeated 8. twice.It is careful to remove supernatant, precipitating is inverted in draught cupboard, 2min is done.

(10) RNA of 60 μ L is added into precipitating without enzyme water.Minus 80 DEG C of preservations.

2) according to the step labeled RNA of embodiment 8.

3) by the RNA marked in conjunction with Streptavidin MagneSphere, wherein

In conjunction with RNA amount be 50pmol, in conjunction with steps are as follows:

(1) it takes the EP of a 1.5mL to manage, the Streptavidin MagneSphere of 50 μ L is added.

(2) EP pipe is placed in magnetic frame, collects magnetic bead in one end of EP pipe, removes supernatant.

(3) Tris (pH7.5) that the 20mM of isometric (50 μ L) is added is washed, and magnetic bead is resuspended using pipette tips.

(4) step 2 and three is repeated.

(5) EP pipe is placed in magnetic frame, collects magnetic bead in the other end of EP pipe, removes supernatant.

(6) 1 × RNA Capture Buffer of isometric (50 μ L) is added, magnetic bead is resuspended using pipette tips.

(7) the labeled RNA of 50pmol is added in magnetic bead, mixes gently.

(8) room temperature rotation mixes, and is incubated for 30min.

Then:

(1) magnetic bead for combining RNA and the above-mentioned RNA prepared are incubated for, the RNA enzyme suppression of 10 μ L is added in system Preparation, 4 DEG C of incubation 60min.

(2) eluted rna-RNA combines compound, specific elution step are as follows:

One) EP pipe is placed in magnetic frame, collects magnetic bead in the other end of EP pipe, removes supernatant.

Two) compound is carried out with 1 × wash buffer of isometric (100 μ L) to wash.

Three) repeat step 1), two) twice.

Four) EP pipe is placed in magnetic frame, collects magnetic bead in the other end of EP pipe, removes supernatant.

Five) plus the Elution Buffer of 50 μ L is in magnetic bead, mixes, 37 DEG C of incubation 30min.

Six) EP pipe is placed in magnetic frame, collects magnetic bead in the other end of EP pipe, collects supernatant.For subsequent analysis.

Seven) plus 1 × sample-loading buffer is in compound.10min, minus 20 DEG C of preservations are boiled in boiling water.

Finally, the compound eluted is placed in minus 80 DEG C of preservations.

10 RNA-RNA combination compound of embodiment carries out tiny RNA sequencing

1) the step of tiny RNA sample treatment is as shown in Figure 8.

2) tiny RNA data analysis step is as shown in Figure 9.

11 lncRNA H19 target miRNA of embodiment verifying

Using the miRNA Reverse Transcriptase kit and real-time quantitative PCR kit of Takakra-clontech, to pull- MiR-19a-3p and miR-106b-5p in down product are detected.

Experimental result:

1. the template of the in-vitro transcription using H19 slow virus carrier as template amplification with T7 promoter, as a result such as Figure 10 institute Show.

2. the identification of product lncRNA H19 is transcribed in vitro:

Using the DNA with T7 promoter as template, it is carried out according to kit specification in-vitro transcription, to the RNA after transcription Agarose gel electrophoresis is carried out, is compared according to the size of relative molecular mass and marker, as a result as shown in figure 11.

The target miRNA of 3.RNA-seq detection long-chain non-coding RNA H19

Inventor carries out RNA-seq sequencing to the RNA compound that long-chain non-coding RNA H19 is pulled down, and uses simultaneously The microRNA that starbae V2.0 software prediction can interact with long-chain non-coding RNA H19, has found to predict Have in microRNA 5 with the microRNA that RNA-seq is detected be it is common, they are respectively: hsa-miR-19a-3p, Hsa-miR-106b-5p, hsa-miR-148a-3p, hsa-miR-148b-3p, hsa-miR-454-3p, as a result such as Figure 12 institute Show.

The target miRNA of 4.Real-time PCR verifying lncRNA H19

Inventor carries out miRNA reverse transcription to the product of RNA pull-down and real-time PCR is verified, and discovery produces There are miR-19a-3p and miR-106b-5p in object, as a result as shown in figure 13.

Interpretation of result:

LncRNA and miRNA interaction type include: that first, miRNA mediates lncRNA degradation.IncRNA and cell Proliferation, differentiation, aging, programmed death etc. have very close connection.Just because of lncRNA molecular chain length is special, just make There are the recognition site of many miRNA on its chain, these sites can mediate lncRNA's specifically in conjunction with miRNA Degradation, the biological function (Figure 14 A) of regulating cell.H19, miR-9 for example, let-7 degradation lincRNA-p21, let-7 degrade Degrade MALATI etc..Second: the lncRNA bait molecule controlling gene expression as miRNA.LncRNA may act as luring for miRNA Bait molecule adsorbs miRNA in such a way that target imitates (target mimicry), inhibits miRNA to the effect (figure of target 14B).Third: lncRNA is as miRNA competitiveness endogenous molecule and mRNA interaction.IncRNA can be competitive in conjunction with miRNA MRNA is directly regulated and controled (Figure 14 C) to target gene.4th: many lncRNA be the precursor of miRNA.For example, miR-206and MiR-133b is generated by Linc-MD1, and MiR-675 is generated by H19.

In this section in experiment, inventor obtains lncRNA H19 (Figure 11) in vitro, so using the method being transcribed in vitro Biotin labeling RNA is utilized afterwards, and the compound of lncRNA H19 albumen in connection is obtained using RNA pull-down technology, Compound of the lncRNA H19 in conjunction with RNA is obtained simultaneously.Starbase V2.0 software can predict miRNA-lncRNA interactions、miRNA-circRNA interactions、miRNA-sncRNA interactions、miRNA-mRNA interactions、Protein-lncRNA interactins、Protein-mRNA interactions、Protein- pseudogene interactions,Protein-sncRNA interactions.RNA-seq has detected 1134 small RNA molecule, Starbase V2.0 software prediction to the miRNA with lncRNA H19 interaction have 40, the common miRNA of the two Molecule has 5 (Figure 12), they are respectively: hsa-miR-19a-3p, hsa-miR-106b-5p, hsa-miR-148a-3p, hsa-miR-148b-3p,hsa-miR-454-3p.Obtained by verifying, the target miRNA of lncRNA H19 have miR-19a-3p and MiR-106b-5p (Figure 13).

The functional study of Part III lncRNA H19 target miRNA miR-106b-5p

1, the sequence of miR-19a-3p antisense nucleic acid is synthesized by Sangon Biotech (Shanghai) Co., Ltd.

2. cell strain

K562 cell strain, human chronic myelogenous leukemia system, is purchased from Shanghai Cell Bank of the Chinese Academy of Sciences.

3.MTT solution

In the PBS for the pH7.2 that the MTT powder of 100mg is dissolved in 20mL, it is configured to the solution that concentration is 5mg/mL, 0.22 μ M membrane filtration degerming, 4 DEG C are kept in dark place.

Influence of the 12 targeted inhibition miR-106b-5p of embodiment to K562 cell viability

Experimental method:

(1) experiment is divided into blank group (blank), random controls group (SCR), miR19a-3p antisense nucleic acid group, every group of setting 5 multiple holes.

(2) cell for taking logarithmic growth, with serum-free, antibiotic-free RPMI-1640 culture medium by concentration of cell suspension It is adjusted to 10 × 10⁴/ mL, for the cell suspension inoculation of every 50 μ L of hole in 96 orifice plates, each experimental group repeats 5 multiple holes.

(3) Lipofectamine is diluted^TM2000: according to Lipofectamine^TM2000 specifications are prepared, and are taken required Lipofectamine^TM2000 amount is diluted using Opti-MEM Optimal Medium.It is stored at room temperature 5min.

(4) antisense nucleic acid is diluted: according to Lipofectamine^TMRequirement of 2000 specifications to transfected antisense nucleic acid is prepared Antisense nucleic acid.Required antisense nucleic acid is taken, is diluted using Opti-MEM Optimal Medium

(5) Lipofectamine that above-mentioned dilution is good^TM2000 mix with antisense nucleic acid dilution according to the ratio of 1:1, It is stored at room temperature 20min.

(6) 50 μ L are taken to be slowly added dropwise in cell suspension for every group, blank group adds the Opti-MEM Optimal Medium of 50 μ L, Every group of 5 multiple holes, every hole final volume are 100 μ L.By the cell incubator that 96 orifice plates are placed in 37 DEG C, CO2 volume fraction is 5% In, after cultivating 6h under saturated humidity, every hole adds the RPMI-1640 culture medium containing 20% serum of 100 μ L, so that cell culture medium Middle serum content is 10%.

(7) after cultivating 48h, 20 μ L of MTT solution is added in every hole, is placed in incubator culture 4h, 1000rpm, is centrifuged 20min, Carefully supernatant is sucked out with liquid-transfering gun after centrifugation, every hole adds the DMSO of 150 μ L, vibrates 10min, dissolving crystallized.

(8) absorbance A 570 is measured on multi-function microplate reader.Experiment is repeated 3 times, and calculates proliferation inhibition rate.Proliferation Ability Rate (%)=(1-A experimental group/A control group) × 100%.

The influence that 13 targeted inhibition miR-106b-5p of embodiment forms K562 cell colony

Specific method is referring to embodiment 12.

The data processing of this part uses SPSS statistical software, and data are indicated with mean ± standard deviation, using single factor test side Poor analytic approach, (one-way ANOVA) analyze the conspicuousness of difference between each group of data, are to have significant difference with P ﹤ 0.05.

Experimental result:

1, the influence after targeted inhibition miR-106b-5p to K562 ability of cell proliferation

As shown in figure 15, SCR the and miR-106b-5p antisense nucleic acid that transfection concentrations are 100nM in K562 cell, 48h After detect cell viability, the cell viability of discovery miR-106b-5p antisense nucleic acid transfection group obviously weakens, and has statistical significance (P<0.05)。

2, the influence that K562 cell colony is formed after targeted inhibition miR-106b-5p

As shown in figure 16, transfection concentrations are that SCR the and miR-106b-5p antisense nucleic acid of 100nm is laggard in K562 cell Row colony forms experiment, compared with blank group and SCR group, discovery miR-106b-5p antisense nucleic acid transfection group cell Colony Forming ability weakens, and has statistical significance (P < 0.05).

This part experiment mainly has studied the function of the target miRNA miR-106b-5p of lncRNA H19, inventor's synthesis The antisense nucleic acid of miR-106b-5p, after so that it is inhibited miR-106b-5p, shadow of the research miR-106b-5p to K562 cell It rings, finds targeted inhibition miR-106b-5p, it is suppressed that the proliferation (Figure 15) of K562 cell reduces the colony of K562 cell Forming ability (Figure 16).

Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention And range.

Claims (9)

1. Application of the target miRNA miR-106b-5p of 1.lncRNA H19 in the drug for preparing and/or screening treatment tumour.
2. 2. application according to claim 1, which is characterized in that the tumour is leukaemia.
3. The application of 3.lncRNA H19 and miR-106b-5p being used in combination in the drug for preparing and/or screening treatment tumour.
4. 4. application according to claim 3, which is characterized in that the tumour is malignant tumour.
5. 5. a kind of drug for treating tumour, which is characterized in that contain the inhibitor of miR-106b-5p in the drug.
6. 6. drug according to claim 5, which is characterized in that the inhibitor is the antisense nucleic acid of miR-106b-5p.
7. 7. drug according to claim 5, which is characterized in that also containing in the drug can promote lncRNA H19 to express Reagent or carrier.
8. 8. drug according to claim 6, which is characterized in that the base sequence of the antisense nucleic acid such as t-anti-miR- Shown in 106b-5p.
9. 9. drug according to claim 5, which is characterized in that the tumour is leukemia.