CN109750040B - Application of HEATR1 gene or protein inhibitor in preparation of antitumor drugs - Google Patents

Abstract

The invention relates to application of an inhibitor of HEATR1 gene or protein in preparation of an antitumor drug. The invention designs a small interfering RNA sequence, an RNA interfering carrier and an RNA interfering slow virus aiming at a human HEATR1 gene, and further detects the influence of HEATR1 gene silencing efficiency, HEATR1-siRNA slow virus on tumor cell proliferation capacity and apoptosis level, and the result shows that the HEATR1 gene can be effectively inhibited from proliferation and growth and promoting apoptosis of tumor cells after being regulated by adopting an RNAi method, so that the HEATR1 gene is a protooncogene and can be used as a target point of tumor treatment, and the HEATR1 gene expression can be silenced by adopting an RNAi mode and can be used as an effective means for inhibiting tumor development.

Description

Application of HEATR1 gene or protein inhibitor in preparation of antitumor drugs

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of an HEATR1 gene or protein inhibitor in preparation of antitumor drugs.

Background

Along with the changes of human life habits and behavior patterns, and environmental pollution, population aging and other reasons brought by the development process of agriculture industry, malignant tumors become a main disease which is fatal to human beings. Recent data from the world health organization International cancer research center (IARC/WHO) shows that malignant tumors are on an increasing trend in global incidence, with 760 thousands of deaths from malignant tumors in 2008, 64% occurring in developing countries. In China, the situation faced by tumor prevention is also very severe, and the latest report shows that the incidence and death rate of malignant tumors in China are obviously increased in the past 30 years; it is predicted that the trend of increasing incidence and mortality of malignant tumors in China will continue to be maintained in the future 20-30 years. Thus, research on tumors is one of the continuing research hotspots in the life sciences and medical fields.

The limitations of traditional therapies for malignant tumors such as radiotherapy, chemotherapy and surgical treatment have prompted the search for new anti-tumor methods. With the deep understanding of the molecular mechanism of tumorigenesis, it has been realized that tumors are a genetic disease, and correcting defective genes may provide new hopes for tumor treatment. In recent years, many researches on gene therapy methods for tumors have been developed, but several gene drugs which have been clinically applied so far have limited clinical effects and have potential, long-term and delayed side effects, so that the search for new targets for therapy and the development of new gene therapy methods have become research directions for tumor gene therapy at home and abroad.

RNA interference (RNAi) is a gene blocking technique, which is a process by which double-stranded RNA (dsRNA) molecules block or silence the expression of specific genes at the mRNA level, i.e., sequence-specific Post-transcriptional gene silencing (PTGS). In recent years, RNAi has become an effective strategy for tumor gene therapy. The RNAi technology can inhibit the expression of protooncogenes, mutated oncogenes, cell cycle related genes, anti-apoptosis related genes and the like to inhibit the occurrence and development of tumors.

HEATR-1 (HEAT repeat containing-1) is a multi-spliced gene originally found on a different protein family including huntingtin, elongation factor 3 (EF 3), PR 65/subunit protein phosphatase 2, HEATR1 on human chromosome 1q43 with mRNA length of 8497bp encoding 2144 amino acids protein, gene being a 7kb multi-spliced gene involved in the processing of pre-18S ribosomal RNA and ribosomal biosynthesis of nucleolus, HEATR consisting of eight subunits, while folded HEATR1 has topological function, HEATR-1 contains two antiparallel helices (helix-1 and 2) in its structure and is linked by IL-2. Studies have shown that HEATR-1 may be used as a lymphocyte that regulates rRNA synthesis and cytotoxicity in glioma patients, HEATR-1 is highly expressed in glioma cell lines, and T cell antigens based on HEATR-1 are capable of inducing CTL to produce cytotoxicity. There is also literature that HEATR1 negatively regulates the activation and activation of Akt, and upregulation of Akt activity by HEATR 1-deficient pancreatic cancer patients may be associated with drug resistance to chemotherapeutic drugs. However, HEATR-1 remains largely unknown for most cellular functions, and few reports are currently made about HEATR1 in the tumor-associated field.

Disclosure of Invention

The invention aims at overcoming the defects in the prior art and providing a new application of an inhibitor of HEATR1 gene or protein.

The invention provides application of an inhibitor of HEATR1 gene or protein in preparation of an anti-lung cancer drug.

As a specific embodiment, the inhibitor is selected from siRNA, dsRNA, shRNA, miRNA, antisense nucleic acid which targets the HEATR1 protein or its transcript and is capable of inhibiting HEATR1 protein expression or gene transcription; or a construct capable of expressing or forming said siRNA, dsRNA, miRNA, antisense nucleic acid.

As a preferred example, the inhibitor is selected from one of the following:

a) siRNA, the target sequence of the siRNA is shown as SEQ ID NO. 1;

b) The sense strand of the coding sequence of the shRNA is shown as SEQ ID NO.5, and the antisense strand is shown as SEQ ID NO. 6;

c) A vector capable of expressing a) the siRNA or b) the shRNA;

d) A virus comprising the vector of c).

The invention also provides an siRNA molecule, and the target sequence of the siRNA is shown as SEQ ID NO. 1.

The invention also provides an shRNA molecule, the sense strand of the coding sequence of the shRNA is shown as SEQ ID NO.5, and the antisense strand is shown as SEQ ID NO. 6.

The invention also provides an expression vector capable of expressing the siRNA molecule or the shRNA molecule.

The invention also provides a virus comprising the expression vector.

As a specific embodiment of the present invention, the virus is a lentivirus.

The invention also provides an anti-tumor drug, which comprises the siRNA molecule, the shRNA molecule, the expression vector and/or the virus, and a pharmaceutically acceptable carrier.

By "pharmaceutically acceptable" is meant that the substance is not biologically or otherwise substantially undesirable, i.e., it can be administered to an individual without causing any substantial undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

The "carrier" material, also referred to as "excipient," includes any excipient commonly used in pharmacy and should be selected based on compatibility and the desired release profile properties of the dosage form. Exemplary carrier materials include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. "pharmaceutically acceptable carriers" may include, for example, acacia, gelatin, colloidal silica, calcium glycerophosphate, calcium lactate, dextrin-maltose complexing agent, glycerol, magnesium silicate, sodium caseinate, soybean lecithin, sodium chloride, tricalcium phosphate, dipotassium hydrogen phosphate, sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like. See, e.g., hoover, john e., remington's Pharmaceutical Sciences, mack Publishing co., easton, pa.1975.

The invention also provides application of the reagent for detecting the expression level of the human HEATR1 gene or protein in preparing lung cancer diagnosis reagent or kit.

The invention has the advantages that:

1. the invention designs a small interfering RNA (siRNA) sequence, an RNA interfering carrier and an RNA interfering slow virus aiming at a human HEATR1 gene, and further detects the silencing efficiency of the HEATR1 gene and the influence of the HEATR1-siRNA slow virus on the proliferation capacity and apoptosis level of tumor cells, and the result shows that:

(1) The RNAi method is adopted to down regulate the expression of the human HEATR1 gene, so that the proliferation and the growth of tumor cells can be effectively inhibited and the apoptosis of the tumor cells can be promoted, and the research result shows that the HEATR1 gene can promote the growth of the tumor, is a protooncogene, can be used as a target point of tumor treatment, and can be used as an effective means for inhibiting the development of the tumor by silencing the expression of the HEATR1 gene in an RNAi mode;

(2) The siRNA sequence of the invention can obviously and specifically silence the expression of endogenous HEATR1 genes in human tumor cells (the silencing efficiency is up to 85.1 percent), is obviously superior to other siRNA sequences obtained by a conventional method, and lentivirus expressing the siRNA sequence can effectively inhibit the proliferation of human tumor cells and promote the apoptosis of human tumor cells, so that the siRNA sequence, shRNA sequence, constructs and viruses thereof can be used for preparing high-efficiency antitumor drugs.

2. The invention proves that the human HEATR1 gene has obvious expression difference in lung cancer tumor tissues and paraneoplastic tissues, so the HEATR1 gene can be used as a diagnosis marker of lung cancer.

Drawings

FIG. 1 GV112vector map.

FIG. 2 agarose gel electrophoresis pictures of fragments of interest after linearization of the vector. Lane 1: GV112Vector; lane 2: GV112Vector digested with Age I and EcoR I; lane 3:1kb DNA Ladder, in order from top to bottom: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp.

FIG. 3 schematic representation of RNA interference vector construction and positive clone identification.

FIG. 4 agarose gel electrophoresis pictures of RNA interference vector positive clone identification. Lane 1: blank control (ddH) ₂ O); lane 2: negative control (empty vector self-connecting control group) to eliminate foreign nucleic acid pollution in the system to cause false positive result; lane 3:250bp DNA Ladder (bands of 5kb, 3kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp, 100bp from top to bottom in order); lanes 4-8: transformants No. 1-5.

FIG. 5 shows that HEATR1mRNA expression level is significantly reduced after 3 days of infection of lung cancer A549 cells by HEATR1-RNAi lentivirus.

FIG. 6 HEATR1-RNAi lentiviruses induced inhibition of cell proliferation 5 days after infection of human lung cancer A549 cells. Cells were plated in 96-well plates after shRNA lentivirus infection. Celigo was continuously examined for 5 days, and proliferation rate of the experimental group A549 cells was found to be significantly inhibited. Suggesting that the HEATR1 gene is significantly related to the proliferation capacity of A549 cells.

FIGS. 7 and 8.FACS detect the effect of HEATR1 gene depletion on lung cancer cell cycle. The shRNA lentivirus was passaged 3 days after infection and tested 5 days, and the number of apoptosis in the shratr 1 (PSC 37251) group was increased (P < 0.05) compared to the control group, suggesting that the hereatr 1 gene was significantly associated with apoptosis in a549 cells.

FIG. 9 differential expression of human HEATR1 gene in tumor tissue and paraneoplastic tissue of lung cancer patients.

Detailed Description

The invention is further described below in conjunction with the detailed description. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention; further, it is understood that various changes and modifications of the present invention may be made by those skilled in the art after reading the description of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto. The experimental methods without specific conditions noted in the examples and the reagents without the formulation noted were all in accordance with conventional conditions.

Example 1 preparation of RNAi lentiviruses against human HEATR1 Gene

The technical route is as follows: the human HEATR1 gene sequence is called from Genbank; predicting siRNA sites; synthesizing an effective siRNA sequence aiming at the HEATR1 gene, and double-stranded DNA Oligo with enzyme cutting sites at two ends and sticky ends; the lentiviral vector is connected with double-stranded DNA Oligo after double enzyme digestion, and RNAi plasmid expressing siRNA sequence of HEATR1 gene is constructed; the RNAi plasmid and the auxiliary vector required by lentivirus package are co-transfected into human embryo kidney cell 293T cells to generate race lentivirus particles, thus obtaining the lentivirus with high-efficiency silencing HEATR1 gene.

1RNA interference target design and double-stranded DNA oligo preparation

1.1 design synthesis of efficient siRNA targets against the human HEATR1 Gene

The HEATR1 (NM_ 018072) gene information is called from Genbank; an effective siRNA target was designed against the hetr 1 gene. Table 1 shows effective siRNA target sequences against HEATR1 gene.

TABLE 1 effective siRNA target sequences targeting the human HEATR1 Gene

1.2DNA oligo sequence Synthesis

And designing a shRNA interference sequence according to the selected target sequence, and adding proper restriction enzyme cutting sites at two ends to complete the construction of the vector. In addition, TTTTT termination signals are added at the 3 'end of the forward strand, while termination signal complements are added at the 5' end of the reverse strand. A single-stranded DNA oligo was synthesized.

TABLE 2 Single DNA Oligo containing the sticky ends of the Age I and EcoRI cleavage sites at both ends

* CCGG: an Age I cleavage site; AATTC: ecoR I cleavage site; g: ecoR I cleavage site complement.

1.3 preparation of double-stranded DNA oligo

The synthesized single-stranded DNA oligo dry powder was dissolved in an annealing buffer (final concentration 20. Mu.M) and water-bath was conducted at 90℃for 15min. After naturally cooling to room temperature, a double strand with sticky ends is formed, the sequence of which is:

positive chain: 5'-CCGGGCAGAGTTGATGGAAGATGAACTCGAGTTCATCTTCCATCAACTCTGCTTTTTG-3' (SEQ ID NO. 5);

reverse chain: 5'-AATTCAAAAATCTTTGTTTCAGGTTAATCGCCTCGAGGCAGAGTTGATGGAAGATGAA AATTCAAAAA-3' (SEQ ID NO. 6).

1.4 preparation of linearization vectors

A50. Mu.l reaction system was prepared according to NEB instructions and linearized using Age I and EcoR I double cut GV112vector (available from Shanghai Ji Kai Gene chemical technologies Co., ltd., vector map see FIG. 1). The reaction system is as follows:

the reaction is carried out for 1h at 37 ℃ (optimal temperature), and then the target fragment is recovered by cutting the gel. The agarose gel electrophoresis picture of the fragment of interest is shown in FIG. 2.

2RNA interference lentiviral vector construction

2.1 connection

A20. Mu.l reaction system was prepared according to Fermentas T4DNA library instructions and the double stranded DNA oligo was ligated to the linearized vector. The reaction system is as follows:

the ligation was reacted at 16℃for 1-3h, the ligation product was designated psc45562, after which the transformation experiment was performed.

2.2 conversion

The ligation products were transformed into E.coli competent cells, and the detailed procedure was as follows:

1) Mu.l of ligation product psc45562 was added to 100. Mu.l of E.coli competent cells and ice-bathed for 30min.

2) Heat shock at 42 ℃ for 90sec and ice bath for 2min.

3) 500. Mu.l of LB liquid medium without antibiotics was added thereto, and shaking culture was performed at 200rpm at 37℃for 1hr.

4) 150 μl of the bacterial liquid was uniformly smeared on LB solid medium containing Amp, and cultured overnight in an incubator at 37 ℃.

2.3 PCR identification of Positive clones

(1) RNA interference vector construction and positive clone identification schematic diagram

Schematic representation of RNA interference vector construction and positive clone identification is shown in FIG. 3.

(2) Primer(s)

The sequence of the identified primer is as follows:

(3) PCR amplification

A single colony is selected as a template by a sterile gun head to carry out PCR amplification, and the reaction conditions are as follows: 94 ℃ for 3min;94℃for 30s,55℃for 30s,72℃for 30s,22 cycles; and at 72℃for 5min. The PCR reaction system is as follows:

(4) Agarose gel electrophoresis detection

After completion of PCR, 5. Mu.l of the product was taken and the band was detected by 1% agarose gel electrophoresis. Agarose gel electrophoresis pictures are shown in FIG. 4. The positive clone PCR fragment size ligated into shRNA fragment was: 342bp; the empty vector clone PCR fragment size without the joining shRNA fragment was: 307bp. Thus, psc24135-1,2,4,5 was judged to be positive, and clones with correct identification were stored.

2.4 analysis of sequencing results of Positive clones

Positive clone sequencing is carried out by identifying the primer-F, and clones with the sequencing result completely consistent with the target sequence are selected for the next experiment. The sequencing result is shown as SEQ ID NO.9, wherein 198-256bp is an insert of the shRNA interference sequence, and the Age I enzyme cutting site is destroyed.

3 extraction of plasmid

The bacterial liquid with correct sequencing is transferred into 150ml LB liquid medium containing Amp antibiotics, and shake culture is carried out at 37 ℃ for overnight. Plasmids were extracted according to EndoFree Maxi Plasmid Kit instructions and plasmids that were qualified for quality testing were entered into downstream flow.

The detailed procedure for plasmid extraction was as follows:

1) Centrifuging at 8000rpm for 4min to collect thalli;

2) Adding 7ml of P1, and shaking and uniformly mixing;

3) 7ml of P3 is added, the mixture is reversed and mixed for 6 to 8 times, and the mixture is stood for 5 minutes;

4) 7ml of P4 is added, and the mixture is reversed and mixed for 6 to 8 times, and the ice bath is carried out for 10 minutes;

5) Centrifuging at 9000rpm for 10min, transferring the supernatant to a filter CS, filtering, adding 10ml isopropanol, and mixing;

6) Adding 2.5ml of balancing solution BL into the adsorption column, centrifuging at 8000rpm for 2min, pouring out the waste liquid in the collecting pipe, and putting the column back for standby;

7) Pouring the supernatant into an adsorption column twice, centrifuging at 8000rpm for 2min, and discarding the waste liquid;

8) Adding 10ml of rinsing liquid PW (absolute ethyl alcohol is added) into the adsorption column, centrifuging for 2min at the same rotating speed, discarding the waste liquid, and repeating the steps once;

9) Adding 3ml of absolute ethyl alcohol into an adsorption column, centrifuging at 8000rpm for 2min, and discarding the waste liquid;

10 9500rpm for 5min, and removing residual rinse liquid; transferring the adsorption column to a new white tube, dripping 800 μl of elution buffer TB (preheated first) into the center of the column, standing at room temperature for 5min, and centrifuging at 9500rpm for 2min;

11 Transferring the eluate in the tube to a clean 1.5ml EP tube and preserving at-20 ℃;

12 Sample electrophoresis, plasmid concentration using a spectrophotometer (thermo_nanodrop 2000), quality control;

13 Packaging the plasmids with qualified quality inspection.

4 lentivirus package

Human embryo kidney cells 293T and lentivirus packaging cells are anchorage dependent epithelioid cells, a growth medium is DMEM (containing 10% FBS), and the anchorage dependent cells are cultured and grown to form monolayer cells. Coli strain DH 5. Alpha. Is used for amplifying lentiviral vectors and helper packaging vector plasmids.

RNAi plasmids were extracted with Qiagen's plasmid extraction kit, and three plasmid DNAs (GV vector plasmid, pHelper 1.0 vector plasmid, pHelper2.0 vector plasmid) in a lentiviral packaging system were prepared as 100 ng/. Mu.l stock solution.

24h prior to transfection, 293T cells in the logarithmic growth phase were digested with trypsin and the cell density was adjusted to about 5X 10 in a medium containing 10% serum ⁶ Cell/15 ml, re-inoculated in 10cm cell culture dish, 37℃at 5% CO ₂ Culturing in an incubator. For 24 hoursThe cell density reaches 70-80% and can be used for transfection. 2h before transfection, changing to serum-free culture medium; the prepared DNA solutions (GV vector plasmid 20. Mu.g, pHelper 1.0 vector plasmid 15. Mu.g, pHelper2.0 vector plasmid 10. Mu.g) were added to a sterile centrifuge tube, the corresponding volumes of transfection reagent were mixed well, the total volume was adjusted to 1ml, and incubated at room temperature for 15min; slowly dripping the mixed solution into 293T cell culture solution, mixing, and adding 5% CO at 37deg.C ₂ Culturing in a cell incubator; after 6h incubation, the medium containing the transfection mixture was discarded, and 10ml of PBS was added for washing once, and the dish was gently shaken to wash the residual transfection mixture and discarded; slowly adding 20ml cell culture medium containing 10% serum at 37deg.C and 5% CO ₂ Culturing in incubator for 48-72 hr.

Lentivirus concentration and purification: according to the cell state, collecting 293T cell supernatant 48h after transfection (the transfection can be counted as 0 h); centrifuging at 4deg.C for 10min at 4000g to remove cell debris; the supernatant was filtered through a 0.45 μm filter in a 40ml ultracentrifuge tube; respectively balancing samples, putting the ultracentrifuge tubes with virus supernatant into a Beckman ultracentrifuge one by one, setting the centrifugation parameters to 25000rpm, and controlling the centrifugation temperature to 4 ℃ for 2 hours; after centrifugation, removing the supernatant, removing the liquid remained on the tube wall as much as possible, adding virus preservation liquid (PBS or cell culture medium can be used for replacing), and lightly and repeatedly blowing and resuspension; after full dissolution, the supernatant is taken and split-packed after high-speed centrifugation at 10000rpm for 5min.

EXAMPLE 2RT-PCR detection of silencing efficiency of HEATR1 Gene

Subjecting lung cancer A549 cells in logarithmic growth phase to pancreatin digestion to obtain cell suspension (cell number about 5×10) ⁴ Per ml), inoculated in 6-well plates, and cultured until the cell fusion degree reached about 30%. An appropriate amount of the virus prepared in example 1 was added, the medium was changed after culturing for 6 hours, and after the infection time reached 3 days, the cells were collected.

RNA extraction and cDNA synthesis:

1) Samples were collected and Trizol lysed: collecting cells, centrifuging at 2000rpm for 5min, removing supernatant, adding 1ml Trizol into cell precipitate, mixing thoroughly, standing at room temperature for 5min, and transferring to new 1.5ml EP tube;

2) 200 μl chloroform was added to each tube, the EP tube was turned upside down by hand for 15s, and left standing at room temperature for 10min;

3) Centrifuging at 4 ℃ and 12800rpm for 15min;

4) Sucking the upper liquid, transferring to a new 1.5ml EP tube, adding equal volume of precooled isopropanol, mixing uniformly, and standing at 4 ℃ for 10min;

5) Centrifuging at 12800rpm at 4deg.C for 12min, and discarding supernatant;

6) Adding 1ml of 75% ethanol (freshly prepared with DEPC water), washing the precipitate;

7) Centrifuging at 11800rpm at 4deg.C for 5min, and discarding most supernatant;

8) Centrifuging again at 4deg.C and 11800rpm for 5min, discarding supernatant, and drying at room temperature;

9) When the RNA precipitation is basically transparent, adding RNase-free water (the adding volume is determined by the RNA precipitation amount) until the RNA precipitation is completely dissolved, and analyzing and measuring the concentration and the quality of the extracted RNA by a Nanodrop 2000/2000C spectrophotometer;

10 Reverse transcription of RNA to obtain cDNA, reacting the reverse transcription system in a water bath at 42℃for 1h, and then inactivating RT enzyme in a water bath at 70℃for 10min, wherein the reverse transcription system is as follows:

RT-PCR: real-time quantitative detection was performed using the Real time PCR of MX3000p type from Agilent company. The primers for the HEATR1 gene were as follows: upstream primer 5'-TTCACTTGTCGCCTTACTTCC-3' (SEQ ID NO. 10) and downstream primer 5'-CCAGAACCATCTGTGCTTTGA-3' (SEQ ID NO. 11). The housekeeping gene GAPDH is taken as an internal reference, and the primer sequences are as follows: upstream primer 5'-TGACTTCAACAGCGACACCCA-3' (SEQ ID NO. 12) and downstream primer 5'-CACCCTGTTGCTGTAGCCAAA-3' (SEQ ID NO. 13). The reaction system is as follows:

the procedure was set as two-step Real-time PCR: pre-denaturation at 95℃for 30s,then each step is denatured at 95 ℃ for 5s; annealing and extending at 60 ℃ for 30s; a total of 45 cycles were performed. The absorbance was read each time during the extension phase. After the PCR was completed, the DNA was denatured at 95℃for 1min, and then cooled to 55℃to allow the DNA double strand to bind sufficiently. Starting from 55 ℃ to 95 ℃, increasing the temperature by 0.5 ℃ in each step, keeping for 4s, and simultaneously reading the absorbance value to prepare a melting curve. By 2 ^-ΔΔCt The assay calculates the abundance of expression of the infectious HEATR1 mRNA. The experimental results showed that the expression level of HEATR1mRNA in lung cancer A549 cells was significantly reduced, specifically 80.6% was down-regulated (FIG. 5).

Example 3 detection of proliferation potency of HEATR1-siRNA lentivirus-infected tumor cells

Subjecting lung cancer A549 cells in logarithmic growth phase to pancreatin digestion to obtain cell suspension (cell number about 5×10) ⁴ Per ml), inoculated in 6-well plates, and cultured until the cell fusion degree reached about 30%. An appropriate amount of the virus prepared in example 1 was added, the medium was changed after culturing for 6 hours, and after the infection time reached 5 days, the cells were collected. Complete medium resuspension of the adult cell suspension (2X 10) ⁴ Per ml) was seeded at a cell density of about 2000 cells per well in 96-well plates. Each group had 3 duplicate wells, 100 μl per well. After plating, the cells are placed in a cell incubator for culture. Starting from the next day after the plate is paved, the plate is read once by Celigo detection every day, and the plate is continuously read for 3-5 days; accurately calculating the number of cells with green fluorescence in each scanning hole plate by adjusting the input parameters of analysis settings; statistical plots were made on the data to plot 5 day cell proliferation curves.

Cell growth curves based on cell count values were plotted from cell count values and time points. Experimental results show that after the tumor cells of the lentivirus infected group are cultured for 5 days in vitro, the proliferation speed is obviously slowed down, which is far lower than that of the tumor cells of the control group, and the number of viable cells of the lung cancer A549 cells is reduced by 77.5% relative to that of the control group (figure 6).

Example 4 detection of tumor apoptosis level of HEATR1-siRNA lentivirus infection

When the cells of the 6-pore plates of each experimental group grow to reach the coverage rate of about 70%, the HEATR1-siRNA is used for inducing apoptosis by the interference group; pancreatin digestion, complete media resuspensionThe cell suspension and the supernatant cells are collected in the same 5ml centrifuge tube, and each group is provided with three compound holes (in order to ensure that the number of the cells on the machine is enough, the number of the cells is more than or equal to 5 multiplied by 10) ⁵ Treatment); 1300rmp for 5min, discarding supernatant, washing cell pellet with pre-cooled D-Hanks (pH=7.2-7.4) at 4deg.C; washing the cell pellet once by 1×binding buffer, centrifuging at 1300rmp for 3min, and collecting the cells; 200 μl of 1×binding buffer resuspended cell pellet; adding 10 μl Annexin V-APC for dyeing, and keeping away from light at room temperature for 10-15min; according to the cell quantity, 400-800 μl of 1×binding buffer is added, and the test is carried out on the machine.

The results show that the tumor cell apoptosis rate of the HEATR1-siRNA interference group is significantly higher than that of the control group, and as shown in the figures, in lung cancer A549 cells (figures 7, 8 and 9), figures 7 and 8 indicate that the HEATR1 expression is silenced, the cells in the G1 phase of the experimental group are significantly increased, the cells in the S phase of the experimental group are significantly reduced, the cells in the G2/M phase of the experimental group are reduced, and the HEATR1 gene is significantly related to the apoptosis of the A549 cells. Fig. 9 suggests that the experimental group apoptosis rate is significantly higher than the control group. It was shown that down-regulating HEATR1 expression levels can lead to tumor cell apoptosis.

Example 5: screening of effective siRNA sequences of targeted human HEATR1 genes

The siRNA sequence was designed against the human hetr 1 gene, and other siRNA target sequence selections designed in addition to the sequence shown in SEQ ID No.1 in example 1 were exemplified as follows:

TABLE 3 siRNA target sequences targeting the human HEATR1 Gene

Lentiviral vectors were constructed as described in examples 1-4, and the effects on HEATR1 gene silencing efficiency and on lung cancer A549 cell proliferation and apoptosis were examined and the results are shown in Table 4.

TABLE 4 silencing efficacy of siRNA sequences, effect on cell proliferation and apoptosis

Experimental results show that the silencing efficiency of the siRNA sequence in the embodiment 1 on the human HEATR1 gene, the proliferation down-regulating capability and the apoptosis promoting capability on lung cancer A549 cells are obviously superior to those of other siRNA sequences, and the siRNA sequence can be used for preparing high-efficiency antitumor drugs.

Example 6: detection of differential expression of human HEATR1 Gene in tumor tissue and paraneoplastic tissue

Tumor tissue and paraneoplastic tissue specimens of 57 lung cancer patients were obtained, of which 24 men and 33 women had an age median of 66 years (42-86 years). The relative expression levels of HEATR1mRNA of tumor tissues and tumor tissues are detected respectively, and the relative expression levels of HEATR1mRNA of tumor tissues and tumor tissues are normalized by taking beta-actin as an internal reference gene, and the result shows that the expression level of HEATR1mRNA of tumor tissues is obviously higher than that of tumor tissues (P < 0.05), as shown in figure 9. The relative expression amount median of HEATR1mRNA of tumor tissue is 2426.14 (P25-P75 is 841.18-1416.60), and the relative expression amount median of HEATR1mRNA of tumor tissue is 794.15 (P25-P75 is 479.43-620.65).

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and additions may be made to those skilled in the art without departing from the method of the present invention, which modifications and additions are also to be considered as within the scope of the present invention.

SEQUENCE LISTING

<110> Shanghai city seventh people's hospital

Application of <120> HEATR1 gene or protein inhibitor in preparation of antitumor drugs

<130> /

<160> 15

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gcctcattgc ttgtgttctg ccataccacg agacaagaat atttgtgcga gtcatacagc 600

ttctaaaaat taataattca aagcacagat ggttctggtt gttgccagtt aagcaatctg 660

gagtgccgtt agctaaagga actttgatta cccactgcta caaagatctt ggattcatgg 720

atttcatttg cagtttggtg acaaaatctg tgaaggtttt tgctgagtac ccgggcagct 780

cagctcagtt gagggtgctc ttggctttct atgcttctac catagtgtcg gcgctggtag 840

ctgcagagga cgtatcagac aatatcatcg ccaaactatt tccctatatc caaaagggat 900

tgaaatcatc tttaccagat tacagagctg caacatacat gataatatgt cagatttctg 960

tgaaagtgac catggaaaat acctttgtga attcattggc atcacagatc atcaaaacat 1020

tgaccaagat tccctctttg atcaaggatg ggttaagttg cttgatagtg ctcctgcaga 1080

gacagaagcc agagagcctt gggaaaaagc cattccctca cttatgtaat gttcctgatc 1140

ttattacaat acttcatggg atttctgaaa cttacgatgt cagtcctctt ctgcattaca 1200

tgcttcccca tctggtcgtc tccatcattc atcatgttac aggagaagaa actgaaggaa 1260

tggatggtca aatctacaag agacacttag aagctatact tacaaaaata tcactgaaga 1320

acaacttaga ccatttgttg gctagccttc tatttgaaga gtatatttca tatagttcac 1380

aggaagaaat ggattctaat aaagtgtctt tgcttaatga acaatttctt ccactcatta 1440

ggcttttaga aagcaaatac cccagaacat tagatgttgt attagaggaa cacttaaagg 1500

aaattgcaga tctgaaaaaa caagagcttt tccatcagtt tgtttctctt tctacaagtg 1560

gaggaaagta tcagttttta gcagattctg atacttcttt gatgctcagc ctgaatcatc 1620

cacttgctcc tgtgagaatt ctggccatga atcatttgaa aaagatcatg aaaacatcaa 1680

aggagggtgt tgatgaatct ttcataaaag aagctgtttt agcccgatta ggtgatgata 1740

atatagatgt tgttttgtcg gctataagtg cttttgagat tttcaaagaa cacttcagtt 1800

cagaagtgac gatttcaaat cttctgaatc tctttcaaag agcagaactt tcaaagaatg 1860

gagaatggta cgaggtactt aagatagccg ctgacatatt aattaaagaa gagatactga 1920

gtgaaaatga tcagttgtca aatcaggtgg ttgtatgttt gctgccattt atggttatca 1980

ataatgatga tacggaatct gctgagatga aaattgctat atatttatca aaatcaggaa 2040

tctgctccct gcaccctcta ttaagaggct gggaagaagc tcttgaaaat gtaattaaaa 2100

gcacaaagcc aggaaaacta atcggtgtag caaatcagaa gatgattgag ttgttggctg 2160

ataatataaa tttaggagat ccttcttcaa tgttaaagat ggtggaggat ttaataagcg 2220

tgggtgagga ggagtccttt aacctgaagc agaaagtaac gtttcatgtg atcctgtctg 2280

tgctcgtctc ttgttgttca tctttaaaag aaacccactt tccatttgcg ataagagtct 2340

tcagtttgtt gcagaaaaaa ataaagaagc ttgaaagtgt cattactgca gtggaaatcc 2400

cctcagaatg gcacattgaa ctgatgttag acagagggat cccagtggag ctgtgggcac 2460

attatgtaga agagctcaac agcactcaga gggtggccgt ggaggactcg gtttttcttg 2520

tattttcctt gaaaaaattt atttatgcac tgaaagctcc taaatctttt cctaaaggtg 2580

atatatggtg gaatcctgaa caactgaaag aagacagcag ggactatctg cacttgctca 2640

ttgggctgtt tgagatgatg ctcaatggtg ccgatgctgt tcatttcaga gttctgatga 2700

aacttttcat aaaggtgcat ctagaagatg tttttcagtt attcaagttc tgttctgttt 2760

tatggaccta tggttctagc ctttcaaatc cactaaactg cagtgtgaaa acagtgctgc 2820

agactcaagc tctttatgtg ggctgtgcaa tgctttcttc tcagaagaca cagtgtaaac 2880

accaactggc atccatatct tctccagtgg tgacatcttt actcattaac ctgggaagcc 2940

ccgtaaaaga agttcgtagg gctgccattc agtgtctcca ggccctcagt ggagtggcat 3000

ccccgtttta tctgataata gatcatttga tttctaaagc agaggagatc acttcagatg 3060

ctgcctatgt tattcaggat ttggctactt tatttgagga actacagaga gaaaagaaac 3120

tgaaatctca tcagaagttg tctgaaactt tgaaaaactt acttagttgt gtgtatagtt 3180

gcccatctta tatagcaaaa gatttgatga aagtacttca gggagtcaac ggtgagatgg 3240

tgctttctca gctattgcct atggctgaac aactgctaga aaagatccag aaggagccca 3300

cagctgtgct gaaagatgag gccatggttc tgcatctcac tctgggaaag tataatgaat 3360

tttcagtttc ccttttaaat gaggatccga agagtctaga tatatttata aaagctgtgc 3420

acacaacaaa ggaactttac gcgggaatgc caaccattca gatcacagcc cttgaaaaga 3480

ttacaaaacc attttttgca gccatatcag atgaaaaagt tcagcagaag cttttaagaa 3540

tgttgtttga tttattggtg aactgtaaaa actcacattg tgctcagact gtcagcagtg 3600

tttttaaagg gatttccgtt aatgctgaac aagtccgaat agaactggag ccaccagata 3660

aagctaaacc cttgggcaca gttcagcaaa aaagaaggca aaaaatgcag cagaaaaaat 3720

cacaagatct agaatctgtt caggaagttg gaggttctta ctggcaaaga gtaactctca 3780

tcctggaatt actgcagcac aaaaagaagc tcagaagtcc tcagatattg gtgccaactc 3840

tttttaactt gctatcaaga tgtttagaac ccttgccaca agagcaggga aatatggaat 3900

acaccaaaca attaattctt agttgtctgc tcaacatctg ccaaaaacta tctccagatg 3960

gtggcaaaat acccaaagat attttagatg aggagaagtt caacgtggag ttgatagttc 4020

agtgcatccg cctttcggag atgccgcaga cccatcacca tgccctttta cttttgggca 4080

ctgttgctgg aatatttccg gataaagttt tacacaatat catgtctatt tttacattta 4140

tgggagccaa tgtcatgcgc ctagatgata cttacagttt tcaagttatt aacaagacag 4200

tgaaaatggt tattcccgca cttattcagt ctgatagtgg agattctata gaagtttcaa 4260

gaaacgttga agagattgtg gtaaaaatca ttagtgtatt tgtggatgcg ctgccacacg 4320

tcccggagca caggcgcctg cccatccttg ttcaacttgt tgatacactg ggtgcagaga 4380

aattcctctg gattctcctc atcttgcttt ttgaacagta tgtcacaaaa acagtgctgg 4440

cggctgccta tggcgaaaag gatgctattt tagaagcaga cactgaattt tggttttcag 4500

tctgttgtga gtttagtgtc cagcatcaga tacaaagctt gatgaatatc ctccagtact 4560

tactaaagct gccagaggaa aaagaagaaa ccattcccaa agcagtgtca tttaataaga 4620

gtgaatcaca agaagaaatg ctacaggttt ttaatgtaga gactcacact agcaagcaac 4680

tgcggcattt taaatttttg tcagtgtcct tcatgtctca gctcctgtct tccaataatt 4740

ttctgaaaaa ggtagttgag agtggtggtc ctgagatttt aaaaggcctt gaagagaggt 4800

tgctggagac cgttctcggc tatatcagtg cagttgcaca gtccatggaa aggaacgcag 4860

acaaactcac cgtgaagttc tggcgcgcgc tccttagtaa agcttacgac ctgttagata 4920

aggtcaatgc cttgctgccc acagagacat tcattcctgt gatcagaggg ctggtgggca 4980

atcccctgcc atctgttcgc cgcaaagcgc tggacctttt gaataacaag ctgcagcaaa 5040

atatatcctg gaagaagaca atagttaccc gtttcctaaa actggttcca gaccttttgg 5100

ccattgtgca gcgtaagaaa aaggaagggg aagaagaaca agcaatcaac agacagacag 5160

cgttgtatac cttaaagctt ttatgcaaga attttggtgc agaaaatcca gatccttttg 5220

tcccagtgct gaacactgct gtgaaactga ttgctccaga gagaaaggag gagaagaatg 5280

tcctgggaag cgcgctgctg tgcatagcag aggtgacctc caccctggag gcgctggcca 5340

tcccccagct tcccagcctg atgccatcgt tgctgacaac aatgaagaac accagcgagc 5400

tggtctccag cgaggtctac ctgctcagtg ccttggctgc tctgcagaag gttgtggaga 5460

ctctcccgca cttcatcagc ccctatctgg aaggcattct ctcccaggtg attcatctgg 5520

agaaaatcac tagtgaaatg ggttctgcgt cacaggctaa tatccgtctc acatctctta 5580

aaaagacact ggctaccaca cttgcacccc gagtcctgtt gcccgccatc aaaaaaactt 5640

acaagcagat tgagaagaac tggaagaatc acatgggtcc gtttatgagc atcttgcaag 5700

agcatattgg ggtgatgaag aaggaagagc tcacctccca tcagtctcag ctaaccgcct 5760

ttttcctgga agccctggac ttccgagccc agcactctga gaacgatctg gaggaagttg 5820

gaaaaacgga aaattgtatc attgactgtc tagtagccat ggttgtcaaa ctttccgagg 5880

tcacattcag gcccctgttc ttcaagctgt ttgattgggc taaaacagaa gatgccccaa 5940

aggacaggtt gttgacattt tacaacttgg cagattgcat tgctgaaaag ctgaaagggc 6000

tttttactct gtttgccggc cacttagtga agccttttgc tgacaccttg aaccaggtga 6060

acatctccaa aacagatgaa gcattttttg actctgaaaa tgaccctgaa aagtgctgct 6120

tgctgttgca gtttattttg aactgtttat acaaaatctt cctttttgat acccagcatt 6180

ttataagtaa agagagagca gaagccttga tgatgcctct ggtggatcag ctggaaaaca 6240

ggcttggggg agaagagaaa ttccaggaac gggtgacaaa gcacctgata ccatgcatcg 6300

cacagttttc ggtggccatg gcggatgact ctctttggaa accactgaac taccagattc 6360

tgctaaagac gagagactcc tcgcctaagg ttcgatttgc tgctttgatt actgtgttag 6420

cactggctga aaaactaaag gagaattata ttgtcttgct accagaatcc attcctttct 6480

tagcagagtt gatggaagat gaatgtgaag aagtagaaca tcagtgccaa aagactattc 6540

agcaactgga aactgtcctg ggagagccac tccagagcta tttctaagac tttctgtggt 6600

gtttcatact ctactcagag ttcacactca tatttcatat ttttattttt gggtgttggg 6660

tgccatgtta cttttggtgc cttaatacac ctacttggat tacttacaaa tgttttatca 6720

cttctttaca aaatccccac ctggcttgtg ctgccacata agcctctcct gcctatcgta 6780

tagagctgca gaaagagtaa atgatacacg gtatttttat acagactgct gtgtttgttt 6840

aaacatttat tattctcttc ctgattgatg gtaataatat tagacttgtt aattttagca 6900

cccaaagctg acgcctcatt tgcactgtaa gccttaactc ttctgtacag cagtatctta 6960

catacatggt atccatgttg cagatttcac tcaaagttgc tctatttcaa gaaaatgaag 7020

ttatttagca atcaacagaa gtacttttga ctgtaaagcc tacttttcat tttgggtagg 7080

cgaacttcag ccttcgtttc tttgttgtgc ccataaagag aagtggttct ggaatgcttt 7140

ttttaaccca ggagtgtgac tgtcaccttt atcctttgtt cttttgggaa acgggagaga 7200

tgaaggcaac acgctgcttc taaaacagct cacacctggc tgctcacaca gagggcccag 7260

aaacactggg tggcacgagg aagctcctcc aggattcaga atgaacccag ttccattggt 7320

ggttaactaa gaactacttg tctaagaaag taagtatcag tagatttttt tcaatgcttt 7380

gaagtgccct aatctctagt actgggtcat ggtgaagttg gaaagtgagg gttcaaataa 7440

aattagatct gccccctttt taaaggcatc taagaacatc ccgtagaatg ttcgcattga 7500

gtttaaaagc ctttgaagct aatatagaag tttaatgcag aaatgtggca agcaaaaggc 7560

caacttgcat tttagagaca gagcttgcta gtatagaagt gcatatttct aagaaatgtc 7620

tcagtaaaat tcattaataa atactaatag ctttttaaat ttttttcctc cacgtaaaat 7680

gaaaatgaac ttaagactta caaggaatga tgtgagtggg ctattttttt cctaaatgcc 7740

tcacagtctc caacagctca gttaagcact ctgatggtct ttatggagaa aaataactct 7800

gggggattct cgagtcttct tgccttctac agctttccct gtggcaccct ccacccccaa 7860

ccagtgtctt tgagcttggt gagactctgc atacattaat atgaaaagct ggaaaagaat 7920

taagggctag cgatttctac actgagtact tttaaaaaaa taagattgac aatggtattc 7980

ttttcaatat atggatagaa ataatatcta aaatctgttc tgaaaatatt cctaaatcct 8040

ggcagagttc tgccccagtc taatgtgata aaatgatcct cactgactga gcttttttcc 8100

ctccccactc ccaaaccttt gaaatttcct gtaacaagat ctgacctgta aagatcccat 8160

atacatggaa cttctgccaa ctcccctcaa ataatagaaa aatcacagaa atggaaccat 8220

ttaacttgct gatacaggtt tgattttaat cacgtaaagt ggaagagtgc tttgatctca 8280

gttgctaatg atgatggact aatctttaga agaacggagg ccttgattgg tgatttcatg 8340

ggaacaaaac agatttctgc attacctctg cctgctttgt gggtccctta gttggcgtac 8400

actttctgat agtgcctgcc ttatgaatgc ctgaagtcta atcctgacct tactttgttg 8460

cccataaata tctttcaaat tgaaaaaaaa aaaaaaagtc gtaacaactc 8510

<210> 10

<211> 20

<212> DNA

<213> artificial sequence

<400> 10

cccgaaggca gacgaaatcc 20

<210> 11

<211> 20

<212> DNA

<213> artificial sequence

<400> 11

tgaggaaagt cccgctggtg 20

<210> 12

<211> 21

<212> DNA

<213> artificial sequence

<400> 12

tgacttcaac agcgacaccc a 21

<210> 13

<211> 21

<212> DNA

<213> artificial sequence

<400> 13

caccctgttg ctgtagccaa a 21

<210> 14

<211> 19

<212> DNA

<213> artificial sequence

<400> 14

caccgctggc gattaacct 19

<210> 15

<211> 18

<212> DNA

<213> artificial sequence

<400> 15

caccggatca tgaacgaa 18

Claims (7)

1. Use of an inhibitor of the HEATR1 gene or protein for the preparation of an anti-lung cancer medicament, said inhibitor being selected from one of the following:

   a) siRNA, the target sequence of the siRNA is shown as SEQ ID NO. 1;

   b) The sense strand of the coding sequence of the shRNA is shown as SEQ ID NO.5, and the antisense strand is shown as SEQ ID NO. 6;

   c) A vector capable of expressing a) the siRNA or b) the shRNA;

   d) A virus comprising the vector of c).
2. 2. An siRNA molecule, which is characterized in that the target sequence of the siRNA is shown as SEQ ID NO. 1.
3. 3. The shRNA molecule is characterized in that the sense strand of the coding sequence of the shRNA is shown as SEQ ID NO.5, and the antisense strand is shown as SEQ ID NO. 6.
4. 4. An expression vector capable of expressing the siRNA molecule of claim 2 or the shRNA molecule of claim 3.
5. 5. A virus comprising the expression vector of claim 4.
6. 6. The virus of claim 5, wherein the virus is a lentivirus.
7. 7. An anti-neoplastic agent comprising the siRNA molecule of claim 2, the shRNA molecule of claim 3, the expression vector of claim 4 and/or the virus of claim 5, and a pharmaceutically acceptable carrier.

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