CN101920020B - New application of human MED19 gene in tumor therapy - Google Patents

Abstract

The invention belongs to the field of biotechnology, and in particular relates to the new application of human MED19 gene in tumor treatment. Current studies have shown that MED19 has an important biological function in the development of tumors. Compared with normal tissues, MED19 gene is widely and highly expressed in tumor tissues. The RNA interference lentivirus designed for the human MED19 gene specifically interferes and down-regulates the expression of the MED19 gene, which can inhibit the growth and invasion of tumor cells, promote the apoptosis of tumor cells, and inhibit the tumorigenesis of tumor cells. MED19 gene can be used as a molecular target for tumor therapy. siRNA silencing against MED19 gene can be used as a novel tool for tumor therapy.

Description

New application of human MED19 gene in tumor therapy

technical field

The invention belongs to the field of biotechnology, and in particular relates to the new application of human MED19 gene in tumor treatment.

Background technique

The phenomenon of RNA interference is a conserved defense mechanism in biological evolution that arises autonomously within cells. Considered to be the main effector of RNA interference, siRNA has become a powerful tool to study the function of mammalian gene products by inhibiting the expression of target genes. Direct transfection of synthetic siRNA can specifically inhibit the expression of homologous genes in mammalian cells, but siRNA in cells is easily degraded, so this method cannot achieve stable RNA interference. Lentiviral vectors have high infection efficiency in mammals, low immunogenicity, and can infect both dividing and non-dividing cells. Due to these characteristics of viral vectors, lentivirus-mediated RNA interference can stably express siRNA in various mammalian cells for a long time and inhibit gene expression; it has the characteristics of high efficiency, stability, strong specificity, and wide application range, and has become the preferred choice of RNA interference technology. Primary research tool. The application of RNA interference technology in biomedical research has added a powerful means to the applied basic research of clinical tumors.

The MED19 gene is located at the 11q12.1 position of the chromosome, and encodes a protein homologous to the transcriptional mediator MED19 that promotes transcription in yeast. There is no clear functional report in mammals.

Contents of the invention

The inventors of the present invention have found that: compared with normal tissues, MED19 gene is significantly highly expressed in tumor tissues, suggesting that the expression of MED19 genes may be closely related to the occurrence and development of tumors. The invention provides a series of effective targets for interfering with the MED19 gene, and constructs a lentivirus specifically interfering with the human MED19 gene.

The inventors found through research that the RNA interference lentivirus designed for the human MED19 gene specifically interferes with and down-regulates the expression of the MED19 gene, which can inhibit the growth and invasion of tumor cells, promote the apoptosis of tumor cells, and inhibit the tumorigenesis of tumor cells. It shows that the MED19 gene can be the target of tumor treatment, down-regulating the expression of MED19 gene can effectively control the process of tumor (Take the tumor cell AGS as an example).

Design idea of the present invention is:

The expression level of MED19 gene in tumor tissue, normal tissue and normal tissue around tumor was detected by immunohistochemical method. Studies have found that the expression of MED19 in tumor tissues is significantly higher than that in normal tissues and normal tissues around tumors. It is suggested that MED19 may be an oncogene that plays an important role in the occurrence and development of tumors; reducing the expression of MED19 may inhibit the progress of tumors; MED19 may become a target for tumor therapy; the expression level of MED19 gene may be a marker for tumor diagnosis.

The present invention screens and obtains a human MED19 gene RNA interference lentivirus through the following methods: the human MED19 gene sequence is obtained from the genebank; the siRNA site is predicted; the siRNA effective target site of the MED19 gene is synthesized, and both ends contain enzyme cleavage sites Double-stranded DNA oligo with cohesive ends; lentiviral vector double-digested and ligated with double-stranded DNA oligo; construct interfering lentiviral plasmid and MED19 overexpression plasmid with interfering sequence; co-transfect 293T cells with interfering plasmid and MED19 overexpression plasmid; Western -Blot detection of the inhibitory effect of these interfering plasmids on the protein expression level of the MED19 gene, and screening of interfering viruses with a protein level inhibition effect of more than 90%; the two auxiliary vectors required for packaging the interfering plasmids and lentivirus - pHelper1.0 vector and After the pHelper 2.0 vector is co-transfected into 293T cells to produce recombinant lentiviral particles, a lentivirus that efficiently interferes with the MED19 gene can be produced.

Based on the above method, the present invention provides a series of effective targets for interfering with the MED19 gene, and constructs a lentivirus that specifically interferes with the human MED19 gene.

At the same time, the invention also discloses a human MED19 gene RNA interference lentivirus (GCG-973-siRNA) and its preparation and application.

This study found that the use of lentivirus-mediated RNA interference method to reduce the expression of MED19 gene in tumor cells can inhibit the growth and invasion of tumor cells, promote the apoptosis of tumor cells, and inhibit the tumorigenesis of tumor cells. It shows that MED19 has an important biological function in the occurrence and development of tumors, MED19 gene can be the target of tumor therapy, and the specific silencing of MED19 gene can be used as a new tool for tumor therapy.

The first aspect of the present invention provides a use of the human MED19 gene in tumor treatment, that is, the use of the human MED19 gene in the preparation of drugs or preparations for treating or diagnosing tumors.

Preferably, the human MED19 gene is used as a target for tumor cells in the preparation of drugs or preparations for treating or diagnosing tumors.

Further preferably, the MED19 gene of tumor cells is used as the target of RNA interference.

The second aspect of the present invention provides an RNA interference lentivirus of human MED19 gene, which is obtained by cloning the sequence expressing shRNA into a lentiviral vector.

Preferably, the sequence for expressing shRNA includes two short inverted repeat sequences separated by a stem-loop sequence; wherein, the two short inverted repeat sequences are respectively the siRNA target sequence of human MED19 gene and its complementary sequence.

More preferably, the siRNA target sequence of the human MED19 gene is shown in SEQ ID NO:1.

More preferably, the stem-loop sequence in the sequence expressing shRNA is preferably as shown in SEQ ID NO:2.

Further preferably, the sense strand sequence of the above shRNA expression sequence is shown in SEQ ID NO: 3, and the antisense strand sequence is shown in SEQ ID NO: 4.

The two ends of the expressed shRNA sequence contain restriction endonuclease enzyme cutting site sticky ends.

Preferably, the sticky ends of the restriction endonucleases are those of Hpa I and Xho I.

Preferably, the lentiviral vector is pGCL-GFP (provided by Jisheng Company).

The human MED19 gene RNA interference lentivirus described in the present invention can be used to prepare drugs or preparations for treating or diagnosing tumors. At the same time, the lentivirus can also be used in the form of a composition to prepare drugs or preparations for treating or diagnosing tumors, and the composition contains 0.001 to 99.999% by weight of the aforementioned human MED19 gene RNA interference lentivirus and an acceptable carrier , diluent or excipient.

Preferably, the composition is a pharmaceutical composition, and the carrier, diluent or excipient is a pharmaceutically acceptable carrier, diluent or excipient.

Preferably, the tumors include: colorectal cancer, breast cancer, prostate cancer, liver cancer and lung cancer.

The present invention is based on the finding that the MED19 gene is abnormally expressed in various malignant tumors. Down-regulating the expression of MED19 by RNA interference can effectively control the tumor process, indicating that the MED19 gene can be a target for tumor treatment. The RNA interference lentivirus designed for the human MED19 gene can reduce the expression of the MED19 gene in cells, which can inhibit the growth and invasion of tumor cells, promote the apoptosis of tumor cells, and inhibit the tumorigenesis of tumor cells. Therefore, aiming at reducing the expression of MED19 RNA can be used as one of the methods of tumor gene therapy. The inhibition efficiency of the human MED19 gene RNA interference lentivirus of the present invention is greater than 90% at the mRNA level, and greater than 90% at the protein level. Tests show that it has obvious inhibitory effect on the growth and metastasis of tumor cells.

Description of drawings

Figure 1: Schematic diagram of the design of siRNA against human MED19 gene and the preparation of high-efficiency interference lentivirus.

Figure 2: Schematic diagram of the results of detecting the difference in expression of MED19 in normal hyperplastic tissue and cancerous prostate tissue by immunohistochemistry;

A is benign prostatic hyperplasia tissue;

B is prostate cancer tissue.

Figure 3: Schematic diagram of the results of detecting the proliferation ability of tumor cells infected with GCG-973-siRNA by MTT assay.

Figure 4: Schematic diagram of the results of detecting the proliferation ability of tumor cells infected with GCG-973-siRNA by BrdU method.

Figure 5: Schematic diagram of the results of flow cytometry (FACS) detection of cell cycle changes in tumor cells infected with GCG-973-siRNA.

Figure 6: Schematic diagram of the results of colony formation assay of tumor cells infected with GCG-973-siRNA for colony formation ability.

Figure 7: Gray histogram of apoptosis in Control group detected by TTP after tumor cells infected with GCG-973-siRNA.

Fig. 8: Gray histogram of apoptosis of tumor cells infected with GCG-973-siRNA by TTP detection and control interference (NC group).

FIG. 9 : Gray histogram of apoptosis of tumor cells infected with GCG-973-siRNA (KD group) detected by TTP.

Figure 10: Schematic diagram of TTP detection of apoptosis of tumor cells infected with GCG-973-siRNA.

Fig. 11: Schematic diagram of the results of detecting the invasion ability of tumor cells infected with GCG-973-siRNA by the invasion chamber.

Fig. 12: Tumor formation test in nude mice to detect tumor formation in adult prostate cancer cells infected with GCG-973-siRNA.

Figure 13: Schematic diagram of the results of tumorigenesis in nude mice to detect changes in the tumorigenic ability of prostate cancer cells infected with GCG-973-siRNA.

Detailed ways

After extensive and in-depth research, the inventors of the present invention found that the MED19 gene was significantly highly expressed in tumor tissues; RNA interference down-regulated the expression of the MED19 gene, which could effectively inhibit the proliferation of tumor cells, promote cell apoptosis, and reduce tumor cell proliferation. Cell invasion and metastasis, etc., can effectively control the growth process of tumors, indicating that MED19 gene can be a target for tumor therapy. The present inventors further synthesized and tested various siRNAs of MED19, screened out siRNAs that could strongly inhibit the expression of MED19 and further inhibit tumor progression, and completed the present invention on this basis.

The present invention is further set forth below in conjunction with embodiment. It should be understood that the examples are only used to illustrate the present invention, not to limit the scope of the present invention. The experimental method of unspecified specific condition in the embodiment and the reagent of unspecified formula all are according to conventional conditions such as people such as Sambrook, molecular cloning: the condition described in test handbook (New York: Cold Spring Harbor Laboratory Press, 1989) or conditions recommended by the manufacturer or configured.

Example 1: Preparation of human MED19 gene efficient interference lentivirus

The gene information of MED19 (NM_153450) was retrieved from genbank; an effective siRNA target for MED19 was designed by using the design software genechem accumulated and summarized by Jikai Gene Chemical Technology Co., Ltd. for many years. The siRNA target sequence is: GGTGAAGGAGAAGCTAAGT.

Synthesize double-stranded DNA oligo sequences with sticky ends containing Hpa I and Xho I restriction sites at both ends for the siRNA target, as shown in the table below;

The double-stranded DNA oligo with sticky ends containing Hpa I and Xho I restriction sites at both ends is:

NO. 5' STEM Loop STEM 3' 1-F T GGTGAAGGAGAAGCTAAGT TTCAAGAGA ACTTAGCTTTCTCTTCACC TTTTTTC 1-R TCGAGAAAAAAA GGTGAAGGAGAAGCTAAGT TCTCTTGAA ACTTAGCTTTCTCTTCACC A

Construction of lentivirus-GCG-973-siRNA containing MED19 interfering double-stranded DNA oligo (see Figure 1);

293T cells were co-transfected with GCG-973-siRNA and MED19 overexpression plasmid:

Digest 293T cells with trypsin and inoculate them in 24-well culture plates at a cell density of 60% to 70%. Replace with OMEM the next day, add GCG-973-siRNA and MED19 overexpression plasmids to the culture plate, and transfect Change fresh medium after 6 hours (set up two plasmid transfection dosage groups: low group——GCG-973-siRNA:MED19 overexpression plasmid amount=0.25:1; high group——GCG-973-siRNA:MED19 overexpression Plasmid amount = 0.5:1). After 24 hours of transfection, the fluorescence was observed under a fluorescent microscope, and the transfection rate was greater than 70%. After 24 hours of continuous culture, the samples were collected and Western Blot was used to screen targets to obtain highly efficient GCG-973-siRNA.

Western Blot detection: Compared with the control interference (negative control, NC), after GCG-973-siRNA infected (KD) tumor cells, the reduction efficiency of endogenous MED19 mRNA level reached 90%.

Example 2: The method of immunohistochemistry detects the expression difference of MED19 in normal hyperplastic tissue and cancerous tissue (taking prostate tissue as an example)

There were 30 patients with prostate tumors and 10 patients with benign prostatic hyperplasia. Paraffin sections of surgically resected prostate tissue were dewaxed and rehydrated with xylene and graded ethanol, boiled in citrate buffer for antigen retrieval, blocked with 10% BSA at 37°C for 1 hr, and MED19 primary antibody was added at a working concentration of 1 μg/ml , and incubated overnight at 4°C with the primary antibody. The corresponding secondary antibody was added for staining, the secondary antibody was HRP-labeled antibody against the species of the primary antibody, and the working concentration was diluted 1:100. 37 degrees Celsius, one hour. After washing with PBS, develop color with DAB solution, room temperature for 3-5 minutes, rinse with tap water and perform H&E staining, dehydrate with graded ethanol xylene and seal with neutral gum.

As shown in the immunohistochemical results in Figure 2, the expression of MED19 was hardly detected in the benign prostatic hyperplasia tissue; while in the prostate cancer tissue, there was a very strong high expression of MED19.

We also did a variety of other tumor tissues and got similar results.

Example 3: MTT method to detect the proliferation ability of tumor cells infected with GCG-973-siRNA

pko (colorectal cancer cell line) cells were trypsinized and seeded in a 12-well plate at a cell density of 10-15%. The next day, replace with fresh medium containing 5ug/ml polybrene. Add GCG-973-siRNA lentivirus to the culture plate according to MOI 10, and change the fresh medium after 12-24 hours of infection. After 72 hours of infection, the fluorescence was observed under a fluorescence microscope, and the infection efficiency reached 90%.

Trypsinize the virus-infected cells in the logarithmic growth phase, resuspend the complete medium into a cell suspension; inoculate in a 96-well plate, 100 μl per well; culture in a 5% CO2 incubator at 37°C; before the termination of the culture Add 10μl 5mg/ml MTT at 4h; add 100μl DMSO to terminate the reaction after discarding the culture medium; detect the OD value with a microplate reader at 570nm; use the software SPSS12.0 for statistical drawing of the data;

As shown in the results of the MTT colorimetric method in Figure 3, taking pko (colorectal cancer cell line) as an example, compared with no interference (control) and control interference (NC group), RNA interference reduces the expression of MED19 gene (KD group ), the increase rate of tumor cell number was significantly reduced, indicating that the proliferation rate of tumor cells was significantly reduced after down-regulating the expression of MED19 gene.

We also did a variety of other tumor cell lines and got similar results.

Example 4: BrdU method to detect the proliferation ability of tumor cells infected with GCG-973-siRNA tumor cells

MCF-7 (breast cancer cell line) cells were trypsinized and seeded in a 12-well plate at a cell density of 10-15%. The next day, replace with fresh medium containing 5ug/ml polybrene. Add GCG-973-siRNA lentivirus to the culture plate according to MOI 10, and change the fresh medium after 12-24 hours of infection. After 72 hours of infection, the fluorescence was observed under a fluorescence microscope, and the infection efficiency reached 90%.

Trypsinize the virus-infected cells in the logarithmic growth phase, resuspend the complete medium into a cell suspension; inoculate in a 96-well plate, 100 μl per well; culture in a 5% CO ₂ incubator at 37°C; terminate the culture Add Brdu in the first 4h. After the action of Brdu, the culture medium was discarded, and the cells were fixed with fixative solution for 30 min. Then wash the plate, block with 5% FBS (diluted in plate washing buffer) for 30min, discard the blocking solution, add Brdu primary antibody and incubate for 1h, wash the plate, then incubate with peroxidase-labeled secondary antibody for 1h, and finally add TMB to block light After reacting for 30 minutes, the reaction was terminated, and the OD value was detected by a microplate reader at 450nm; the data was statistically drawn using software SPSS12.0.

As shown in Figure 4, the BrdU method is used to detect the proliferation ability of tumor cells infected with GCG-973-siRNA tumor cells. Taking MCF-7 (breast cancer cell line) as an example, compared with uninterrupted (control) and control interference (NC group), After RNA interference reduced the expression of MED19 gene (KD group), the proliferation ability of MCF-7 (breast cancer cell line) tumor cells was significantly reduced. It shows that the proliferative ability of tumor cells is significantly reduced after down-regulating the expression of MED19 gene.

We also did a variety of other tumor cell lines and got similar results.

Example 5: Flow cytometry (FACS) detection of cell cycle after tumor cells were infected with GCG-973-siRNA

SaoS-2 (osteosarcoma cell line) cells were trypsinized and seeded in a 12-well plate at a cell density of 10-15%. The next day, replace with fresh medium containing 5ug/ml polybrene. Add GCG-973-siRNA lentivirus to the culture plate according to MOI 10, and change the fresh medium after 12-24 hours of infection. After 72 hours of infection, the fluorescence was observed under a fluorescence microscope, and the infection efficiency reached 90%.

After the cells in the logarithmic growth phase were digested with trypsin, the complete medium was resuspended into a cell suspension; inoculated in a 6-well plate, 2ml per well; cultured in a 5% CO2 incubator at 37°C; the cells were collected at a specific time after infection, Centrifuge at 1200rmp for 5min; wash the cell pellet twice with 4°C pre-cooled PBS (pH=7.2~7.4); fix the cells with 4°C pre-cooled 70% ethanol; centrifuge at 1500rmp for 5min to remove the fixative, resuspend in PBS; filter with 400-mesh sieve 1 time, centrifuge at 1200rmp for 5min, discard PBS; 1ml PI staining, 4°C in the dark for 30min, for flow detection.

As shown in Fig. 5, the results obtained by detecting the cell cycle changes of tumor cells infected with GCG-973-siRNA by flow cytometry (FACS), taking SaoS-2 (osteosarcoma cell line) as an example. Compared with no interference (control) and control interference (NC group), after RNA interference reduced the expression of MED19 gene (KD group), the proportion of tumor cells in the prophase of mitosis (G1 phase) increased, while in the middle/late phase of mitosis (G2 phase). /M phase) decreased, indicating that the cell cycle changes after inhibiting the expression of MED19. , found that the proportion of tumor cells in G1 phase increased after downregulation of MED19 gene expression.

We also did a variety of other tumor cell lines and got similar results.

Example 6: Detection of cell clone formation ability after GCG-973-siRNA infection of tumor cells

DU145 (prostate cancer cell line) cells were trypsinized and seeded in a 12-well plate at a cell density of 10-15%. The next day, replace with fresh medium containing 5ug/ml polybrene. Add GCG-973-siRNA lentivirus to the culture plate according to MOI 20, and change the fresh medium after 12-24 hours of infection. After 72 hours of infection, the fluorescence was observed under a fluorescence microscope, and the infection efficiency reached 90%.

After the virus-infected cells in the logarithmic growth phase were trypsinized, the complete medium was resuspended into a cell suspension; the cells were counted and seeded in a 6-well plate (200 cells/well), and the seeded cells were placed in Continue culturing in the incubator until 14 days or until the number of cells in most of the individual clones is greater than 50, the medium is changed every 3 days and the state of the cells is observed; the cell clones are photographed under a fluorescent microscope before the end of the experiment; Cells were fixed with formaldehyde, washed with PBS, stained with Giemsa, and photographed.

As shown in Figure 6, the results of the colony formation experiment to detect the colony formation ability of tumor cells after infection with GCG-973-siRNA are schematic diagrams, taking DU145 (prostate cancer cell line) as an example, compared with the non-interference (control) and control interference (NC group) Compared with that, after RNA interference reduced the expression of MED19 gene (KD group), the number of clonal plaques formed by tumor cells was significantly reduced, and the volume of clonal plaques was significantly reduced; indicating that MED19 silencing leads to a decline in the ability of tumor cells to form clones.

Plate colony formation assay to detect the colony formation ability of DU145 (prostate cancer cell line) tumor cells after reducing the expression of MED19

We also did a variety of other tumor cell lines and got similar results.

Example 7: Detection of cell apoptosis level after GCG-973-siRNA infection of tumor cells

SMMC7721 (liver cancer cell line) cells were trypsinized and seeded in a 12-well plate at a cell density of 10-15%. The next day, replace with fresh medium containing 5ug/ml polybrene. Add GCG-973-siRNA lentivirus to the culture plate according to MOI 20, and change the fresh medium after 12-24 hours of infection. After 72 hours of infection, the fluorescence was observed under a fluorescence microscope, and the infection efficiency reached 90%.

After the cells in the logarithmic growth phase were digested with trypsin, the complete medium was resuspended into a cell suspension; inoculated in a 96-well plate, 100ul per well; cultured in a 5% CO2 incubator at 37°C; after culturing for 36 hours, discard the culture medium , fix the cells with 85% ethanol pre-cooled at 4°C; wash the plate twice with PBS; treat the cells with RNase, stain with PI, and protect from light for 15 minutes. The 96-well plate is scanned and analyzed on the TTP instrument using the preset template for analyzing the subG1 phase, and the results are obtained.

As shown in the schematic diagram results of TTP detection of apoptosis of tumor cells infected with GCG-973-siRNA in FIGS. 7-10 , SMMC7721 (liver cancer cell line) was taken as an example. PI staining-TTP method was used to detect the changes in the proportion of apoptotic bodies (subG1 phase) of 7721 (hepatoma cell line) tumor cells after reducing the expression of MED19. It was found that the apoptotic ratio of tumor cells increased after down-regulating the expression of MED19 gene. Compared with non-interference (control) and control interference (NC group), RNA interference reduced the expression of MED19 gene (KD group), and the number of apoptotic bodies produced by apoptotic tumor cells increased significantly; indicating that MED19 silencing leads to tumor cell apoptosis . PI staining-TTP method was used to detect the changes in the proportion of apoptotic bodies (subG1 phase) of 7721 (hepatoma cell line) tumor cells after reducing the expression of MED19.

We also did a variety of other tumor cell lines and got similar results.

Example 8: Detection of cell invasion level after GCG-973-siRNA infection of tumor cells

Put the invasion chamber in the incubator to reach room temperature; sterilize the tweezers with 70% ethanol, and use the tweezers to treat the transwell chamber; add 300 μl of warm serum-free medium to the chamber, and place it at room temperature for 1-2 hours to allow the ECM layer (Extracel lular Matrix) to regenerate. Hydration; prepare 1.0×10 ⁶ /ml (serum-free medium) cell suspension; after rehydration in step 3, carefully remove the medium from the small chamber; add 500 μl of culture base containing 10% FBS to the lower chamber; Add 300 μl of the cell suspension prepared in step 4 to each small chamber; incubate in the tissue culture incubator for 48 hours; gently remove the non-invasive cells with a cotton swab; add 500 μl of staining solution to the empty well of the plate; place the small chamber Soak in the staining solution for 20 minutes, and stain the invading cells on the lower surface of the membrane; soak the chamber in a large water cup and rinse several times. Dry the chamber in the air; photograph the membrane under a microscope; dissolve the membrane in acetic acid to detect the OD value.

As shown in Figure 11, the invasion ability of tumor cells infected with GCG-973-siRNA was detected by the invasion chamber. Taking A549 (lung cancer cell line) as an example, compared with no interference (control) and control interference (NC group), RNA interference decreased After the expression of MED19 gene (KD group), it was found that the invasion ability of tumor cells decreased after down-regulating the expression of MED19 gene.

We also did a variety of other tumor cell lines and got similar results.

Example 9: After GCG-973-siRNA infects tumor cells, the tumorigenic ability of nude mice

Prostate cancer cells were trypsinized and seeded in 12-well plates at a cell density of 10-15%. The next day, replace with fresh medium containing 5ug/ml polybrene. Add GCG-973-siRNA lentivirus to the culture plate according to MOI 20, and change the fresh medium after 12-24 hours of infection. After 72 hours of infection, the fluorescence was observed under a fluorescence microscope, and the infection efficiency reached 90%.

The cells are in exponential growth phase. The cells were digested and pipetted to make a single cell suspension. The single cell suspension was adjusted to a cell concentration of 2.5×10 ⁷ /ml, and 0.2ml of the single cell suspension was inoculated subcutaneously on one side of the back of each nude mouse. The subcutaneous nodule with a diameter of more than 0.5 cm was used as the standard for tumor formation, and the tumor formation rate was 100% in about 2 weeks. After 25 days, the nude mice were killed by neck dislocation, and the tumors were taken out and weighed.

As shown in the results in Figure 12, the nude mouse tumor formation experiment detected the tumor formation photos of prostate cancer cells infected with GCG-973-siRNA in adults. As shown in FIG. 13 , it is a schematic diagram of the results of nude mouse tumorigenesis assay to detect changes in the tumorigenic ability of prostate cancer cells infected with GCG-973-siRNA in adults. Compared with non-interference (control) and control interference (NC group), after RNA interference reduces the expression of MED19 gene (KD group), the volume and weight of tumor cells formed by tumor cells in nude mice are significantly reduced.

We also did a variety of other tumor cell lines and got similar results.

Sequence Listing

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Claims (5)

1. The application of human MED19 gene in the preparation of medicines for treating or diagnosing rectal cancer, breast cancer, prostate cancer and liver cancer, characterized in that the human MED19 gene is used as a target for tumor cells in the preparation of treatment or In the medicine for diagnosing tumors, the target for tumor cells is an RNA interference target. 2. The application of human MED19 gene according to claim 1, characterized in that the RNA interference lentivirus containing human MED19 gene is used in the medicine for treating or diagnosing tumors. 3. The application of an RNA interference lentivirus of the human MED19 gene in the preparation of a drug for treating or diagnosing rectal cancer, breast cancer, prostate cancer, or liver cancer, wherein the RNA interference lentivirus clones the sequence expressing shRNA into Obtained after the lentiviral vector, the expression shRNA sequence includes two short inverted repeat sequences separated by a stem-loop sequence in the middle; wherein, the two short inverted repeat sequences are the siRNA target sequence of the human MED19 gene and its complementary Sequence; The siRNA target site sequence of the human MED19 gene is shown in SEQ ID NO:1, and the stem-loop sequence is shown in SEQ ID NO:2. 4. the application of the RNA interference lentivirus of human MED19 gene as claimed in claim 3, is characterized in that, the sense strand sequence of the sequence of described expression shRNA is as shown in SEQ ID NO:3, and antisense strand sequence is as SEQ ID NO :4 shown. 5. The application of the RNA interference lentivirus of the human MED19 gene as claimed in claim 4, wherein the lentiviral vector is pGCL-GFP.

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