CN110872599A

CN110872599A - AURKA/UHRF1 double-gene co-interference lentivirus and application thereof in drugs for treating colorectal cancer

Info

Publication number: CN110872599A
Application number: CN201810999454.2A
Authority: CN
Inventors: 杨晶; 郑葵阳; 汤仁仙; 韩静
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2018-08-29
Filing date: 2018-08-29
Publication date: 2020-03-10

Abstract

The invention relates to an AURKA/UHRF1 double-gene co-interference lentivirus and a preparation method thereof, wherein a U6 promoter, an interference target sequence of a targeted human AURKA gene, a U6 promoter and an interference target sequence of a targeted human UHRF1 gene are respectively inserted behind an enzyme cutting site of a vector, an obtained plasmid is transferred into an expression strain to be cultured and screened to obtain an ampicillin-tolerant cloned strain, and the ampicillin-tolerant cloned strain is obtained by shaking bacteria and then packaging to obtain the AURKA/UHRF1 double-gene co-interference lentivirus; the AURKA/UHRF1 double-gene co-interference lentivirus disclosed by the invention is used for infecting a colon cancer SW480 cell, Aurora-A and UHRF1 are used as synergistic drug delivery targets, the expression levels of Aurora-A and UHRF1 in a tumor cell can be reduced, the proliferation of the colon cancer cell is remarkably inhibited, and the apoptosis of the tumor cell is promoted.

Description

AURKA/UHRF1 double-gene co-interference lentivirus and application thereof in drugs for treating colorectal cancer

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to an AURKA/UHRF1 double-gene co-interference lentivirus and application thereof in treating colorectal cancer.

Background

Colon cancer is a common malignancy of the digestive tract that occurs in the colon, accounting for the second place of gastrointestinal tumors. At present, the treatment effect of the colon cancer is not ideal. Aurora-A is an important serine/threonine protein kinase participating in regulation of cell mitosis, and a large number of researches show that Aurora-A is abnormally and highly expressed in tumors such as colorectal cancer, breast cancer, pancreatic cancer and the like. In addition, this region expansion is also considered to be associated with poor patient prognosis.

However, the Aurora-a inhibitors are not ideal in preclinical testing for therapeutic efficacy. Ubiquitin-like PHD-containing and Ring finger domain 1(UHRF1) is a newly discovered oncogene. It is involved in maintaining DNA methylation, cell proliferation, cycle regulation, and the like. Although inhibition of UHRF1 can inhibit cell proliferation and promote apoptosis, in vitro experimental results show that: blocking UHRF1 alone is not effective in inhibiting tumor cell growth.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides an AURKA/UHRF1 double-gene co-interference lentivirus and application thereof in treating colorectal cancer. The AURKA/UHRF1 double-gene co-interference lentivirus takes Aurora-A and UHRF1 as cooperative drug delivery targets, can reduce the expression level of Aurora-A and UHRF1 in tumor cells, remarkably inhibits the proliferation of colon cancer cells and promotes the apoptosis of the tumor cells.

The technical scheme adopted by the invention is as follows:

a method for preparing AURKA/UHRF1 double-gene co-interference lentivirus comprises the following steps:

(1) respectively inserting a U6 promoter, an interference target sequence of a targeted human AURKA gene, a U6 promoter and an interference target sequence of a targeted human UHRF1 gene behind the enzyme cutting site of the vector to obtain plasmids;

(2) transferring the plasmid into an expression strain, and culturing and screening to obtain an ampicillin-resistant clone strain;

(3) shaking the ampicillin resistant cloned strain in the step (2) to obtain AURKA/UHRF1 double-gene co-interference plasmid;

(4) and (4) packaging the AURKA/UHRF1 double-gene co-interference plasmid in the step (3) to obtain the AURKA/UHRF1 double-gene co-interference lentivirus.

In the step (1), the vector is CV147, and the enzyme cutting sites of the vector are Pac I and Nhe I.

In the step (2), the expression strain is an escherichia coli competent cell.

In the step (2), the specific operations for culturing and screening ampicillin-resistant clonal strains are as follows:

dispersing the plasmid in double distilled water to prepare the plasmid with the concentration of 100 ng/mul in the double distilled water; the number of cells was 5X 10⁶The competent expression strains were suspended in 100. mu.l of 0.1mol/L CaCl containing 15% glycerol₂To the solution, 1. mu.l of double distilled water with dispersed plasmid was added to 100. mu.l of CaCl with suspended competent expression strain₂Putting the solution on ice for 20 min; then hot compressing for 90s at 42 ℃, rapidly placing in ice for 5min, and adding 600 mu lLB culture solution; shaking at 37 deg.C and 220r/min for 1 hr, centrifuging, spreading on LB plate containing 50-100 μ g/ml ampicillin, and performing inverted culture at 37 deg.C overnight to obtain ampicillin-resistant clone strain.

In the step (3), the specific operation of shaking the ampicillin resistant clone strain is as follows:

taking ampicillin-resistant clonal strains on an LB plate to inoculate in a test tube containing 3ml of LB culture solution containing 50 mu g/ml of ampicillin, shaking at 37 ℃ and 220r/min overnight; the next day, inoculating into 30ml LB culture solution containing 50. mu.g/ml ampicillin at a volume ratio of 1:100, shaking at 37 deg.C and 220r/min until thallus OD600 is 0.6-0.8, and extracting to obtain plasmid, i.e. AURKA/UHRF1 double-gene co-interference plasmid.

In the step (4), the packaging of the AURKA/UHRF1 double-gene co-interference plasmid is specifically performed as follows:

fully and uniformly mixing the packaged mixed plasmid with the AURKA/UHRF1 double-gene co-interference plasmid in the step (3), adding 60 mu l of PEI and 400 mu l of OPTI-MEM, and keeping the temperature at room temperature for 10 min; adding 5ml of 10% FBS, fully and uniformly mixing, pouring into a 293T cell culture dish, changing the liquid after 4h, collecting the supernatant after 48h, and performing high-speed centrifugation and concentration to obtain the AURKA/UHRF1 double-gene co-interference lentivirus.

The packaging mixed plasmid is pHelper1.0 and pHelper2.0, and the mass ratio of pHelper1.0, pHelper2.0 to the AURKA/UHRF1 double-gene co-interference plasmid is 20:15: 10.

The packaging mixed plasmid is PMD2G and PSPAX2, and the mass ratio of the PMD2G, the PSPAX2 and the AURKA/UHRF1 double-gene co-interference plasmid is 1:1: 1.

The AURKA/UHRF1 double-gene co-interference lentivirus prepared by the method.

The AURKA/UHRF1 double-gene co-interference lentivirus can be used for preparing or screening medicaments for treating colorectal cancer or preparing tumor diagnosis medicaments.

The invention has the beneficial effects that:

the invention relates to a preparation method of AURKA/UHRF1 double-gene co-interference lentivirus, which is characterized in that a U6 promoter, an interference target sequence of a targeted human AURKA gene, a U6 promoter and an interference target sequence of a targeted human UHRF1 gene are respectively inserted behind the enzyme cutting site of a vector, an obtained plasmid is transferred into an expression strain to be cultured and screened to obtain an ampicillin resistant clone strain, and the AURKA/UHRF1 double-gene co-interference lentivirus is obtained by packing after shaking bacteria; the AURKA/UHRF1 double-gene co-interference lentivirus disclosed by the invention is used for infecting a colon cancer SW480 cell, Aurora-A and UHRF1 are used as synergistic drug delivery targets, the expression levels of Aurora-A and UHRF1 in a tumor cell can be reduced, the proliferation of the colon cancer cell is remarkably inhibited, and the apoptosis of the tumor cell is promoted.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is an immunoblot of SW480 cells infected with different viruses;

FIG. 2 is a diagram showing the formation of cell clones with a cell number of more than 50.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.

Example 1

The embodiment provides a preparation method of AURKA/UHRF1 double-gene co-interference lentivirus, which comprises the following steps:

(1) respectively inserting a U6 promoter, an interference target sequence of a targeted human AURKA gene, a U6 promoter and an interference target sequence of a targeted human UHRF1 gene into enzyme cutting sites Pac I and NheI of a vector CV147 (provided by Shanghai JikKa Gene chemistry technology Co., Ltd.) to obtain a CV147-sh AURKA/UHRF1 plasmid; the oligo information of the CV147-sh AURKA/UHRF1 plasmid is shown in Table 1;

TABLE 1 oligo information on the plasmids

(2) Transferring the CV147-sh AURKA/UHRF1 plasmid into a TOP10 clone strain, and culturing and screening to obtain an ampicillin-resistant clone strain, wherein the concrete operation is as follows:

the CV147-sh AURKA/UHRF1 plasmid was dispersed in double distilled water (concentration 100 ng/. mu.l) and the competent expression strain (cell number 5X 10)⁶TOP10 clone) was suspended in 100. mu.l of 0.1mol/L CaCl containing 15% glycerol₂To the solution, 1. mu.l of double distilled water in which CV147-sh AURKA/UHRF1 plasmid was dispersed was added to 100. mu.l of CaCl in which competent expression strain was suspended₂Putting the solution on ice for 20 min; then hot compressing for 90s at 42 ℃, rapidly placing in ice for 5min, and adding 600 mu lLB culture solution; shaking at 37 deg.C and 220r/min for 1 hr, centrifuging, coating on LB plate containing 50 μ g/ml ampicillin, and performing inverted culture at 37 deg.C overnight to obtain ampicillin-resistant clone strain;

(3) shaking the ampicillin resistant cloned strain in the step (2) to obtain AURKA/UHRF1 double-gene co-interference plasmid; the specific operation is as follows:

taking ampicillin-resistant clonal strains on an LB plate to inoculate in a test tube containing 3ml of LB culture solution containing 50 mu g/ml of ampicillin, shaking at 37 ℃ and 220r/min overnight; inoculating the mixture into 30ml LB culture solution containing 50 mug/ml ampicillin according to the volume ratio of 1:100 the next day, shaking at 37 ℃ for 220r/min until the OD600 of the thallus is 0.6-0.8, and extracting the plasmid, namely AURKA/UHRF1 double-gene co-interference plasmid;

(4) packaging the AURKA/UHRF1 double-gene co-interference plasmid in the step (3) to obtain AURKA/UHRF1 double-gene co-interference lentivirus; the specific operations for packaging are as follows:

fully and uniformly mixing the packaged mixed plasmids pHelper1.0 and pHelper2.0 with the AURKA/UHRF1 double-gene co-interference plasmid in the step (3) according to the mass ratio of 20:15:10, adding 60 mu l of PEI and 400 mu l of OPTI-MEM, and keeping the temperature for 10 min; adding 5ml of 10% FBS, fully and uniformly mixing, pouring into a 293T cell culture dish, changing the liquid after 4h, collecting the supernatant after 48h, and performing high-speed centrifugation and concentration to obtain the AURKA/UHRF1 double-gene co-interference lentivirus.

After PCR identification, sequencing and alignment analysis are carried out, and the successful construction of the co-interfering lentivirus containing the AURKA/UHRF1 double genes is verified.

Example 2

the CV147-sh AURKA/UHRF1 plasmid was dispersed in double distilled water (concentration 100 ng/. mu.l) and the number of cells was 5X 10⁶Personal feelingThe expression strain (TOP10 clone) was suspended in 100. mu.l of 0.1mol/L CaCl containing 15% glycerol₂To the solution, 1. mu.l of double distilled water in which CV147-sh AURKA/UHRF1 plasmid was dispersed was added to 100. mu.l of CaCl in which competent expression strain was suspended₂Putting the solution on ice for 20 min; then hot compressing for 90s at 42 ℃, rapidly placing in ice for 5min, and adding 600 mu lLB culture solution; shaking at 37 deg.C and 220r/min for 1 hr, centrifuging, coating on LB plate containing 50 μ g/ml ampicillin, and performing inverted culture at 37 deg.C overnight to obtain ampicillin-resistant clone strain;

fully and uniformly mixing the packaged mixed plasmids PMD2G and PSPAX2 with the AURKA/UHRF1 double-gene co-interference plasmid in the step (3) according to the mass ratio of 1:1:1, adding 60 mu l PEI and 400 mu l OPTI-MEM into the mixture, wherein the total amount of the plasmids is 15 mu g, and keeping the temperature at 10 min; adding 5ml of 10% FBS, fully and uniformly mixing, pouring into a 293T cell culture dish, changing the liquid after 4h, collecting the supernatant after 48h, and performing high-speed centrifugation and concentration to obtain the AURKA/UHRF1 double-gene co-interference lentivirus.

Example 3

the CV147-sh AURKA/UHRF1 plasmid was dispersed in double distilled water (concentration 100 ng/. mu.l) and the number of cells was 5X 10⁶Each competent expression strain (TOP10 clone) was suspended in 100. mu.l of 0.1mol/L CaCl containing 15% glycerol₂To the solution, 1. mu.l of double distilled water in which CV147-sh AURKA/UHRF1 plasmid was dispersed was added to 100. mu.l of CaCl in which competent expression strain was suspended₂Putting the solution on ice for 20 min; then hot compressing for 90s at 42 ℃, rapidly placing in ice for 5min, and adding 600 mu lLB culture solution; shaking at 37 deg.C and 220r/min for 1 hr, centrifuging, spreading on LB plate containing 100 μ g/ml ampicillin, and performing inverted culture at 37 deg.C overnight to obtain ampicillin-resistant clone strain;

Examples of the experiments

The AURKA/UHRF1 double-gene co-interfering lentivirus obtained in example 1 was used to infect colon cancer SW480 cells and the effect of the virus on SW480 cell proliferation was observed. The specific operation is as follows:

1x10⁵SW480 cells were seeded in 6-well plates in a/ml format. The next day, the medium was discarded, and 5. mu.l of shcontrol (purchased from control group, Shanghai Jika), shAURKA (knocked-down AURKA group, purchased from Shanghai Jika), shUHRF1 (knocked-down UHRF1 group, purchased from Shanghai Jika), and shAURKA/UHRF1 (AURKA/UHRF 1 double-gene co-interfering lentivirus obtained in example 1) were added to a centrifuge tube containing 500. mu.l of Opti-MEM medium and 495. mu.l of 10% FBS-containing DMEM, respectively, mixed well, and polybrene was added thereto to give a final concentration of 5. mu.l/ml. After 24 hours of culture, puromycin2mg/ml is screened for 48 hours, and protein is extracted to detect the expression condition of the target protein.

1. The protein extraction comprises the following steps:

(1) adherent cells in 6-well plates were washed 3 times with 1ml of 4 ℃ pre-cooled PBS and the 6-well plates were placed on ice. Preparing a cell lysate, mixing the IP cell lysate, PMSF and phosphatase inhibitor according to the volume of 100:1:1, and preparing the required volume.

(2) Adding 100 mul of cell lysate into each culture well, then quickly scraping off adherent cells by using a cell scraper, sucking the scraped cell suspension into a 1.5ml EP tube, placing the EP tube on ice, and uniformly mixing for 15min by using a shaking table to ensure that the cell lysate fully cracks the cells. The cell suspension was centrifuged at 4 ℃ and 10000g for 10 min. After centrifugation, the supernatant was aspirated with a 200. mu.l pipette tip into a new EP tube and the pellet discarded.

(3) The soluble protein concentration in the supernatant was determined by BCA method. The method comprises the following steps:

adding 1.2ml of the protein standard preparation solution into a tube of protein standard (30mgBSA), and fully dissolving to prepare a 25mg/ml protein standard solution. The preparation can be used immediately after preparation, and can be stored at-20 deg.C for a long time after use.

Taking a proper amount of 25mg/ml protein standard, and diluting to a final concentration of 0.5 mg/ml.

According to the number of samples, a proper amount of BCA working solution is prepared by adding 50 volumes of BCA reagent A and 1 volume of BCA reagent B (50:1), and the mixture is fully mixed.

The standard was added to the standard well of a 96-well plate in an amount of 0. mu.l, 1. mu.l, 2. mu.l, 4. mu.l, 8. mu.l, 12. mu.l, 16. mu.l, 20. mu.l, and the standard dilution was added to make up to 20. mu.l.

Add the appropriate volume of sample to the sample wells of a 96 well plate and standard dilutions to 20. mu.l.

200. mu.l of BCA working solution was added to each well, and the mixture was left at 37 ℃ for 30 min.

The OD value (A570) is detected by a microplate reader, and the protein concentration of the sample is calculated according to the standard curve.

(4) After the protein concentration in each EP tube was determined, the tubes were made up into soluble protein solutions of the same concentration using cell lysate, and the protein solution in each well was mixed with 5 Xloading buffer at a volume of 4:1 and stored at-20 ℃ until use.

(5) Protein electrophoresis

Preparing 7.5% and 10% separation gel according to the raw material ratio in table 2, mixing uniformly, adding into gel plate, and adding ddH₂Taking O as a line pressing liquid, pouring out the upper layer liquid for about 40min, sucking the residual liquid by using filter paper, preparing 4% concentrated glue according to the mixture ratio of the raw materials in the table 3, pouring the upper layer, adding a forming comb, and polymerizing for 30min at room temperature.

TABLE 2 proportions of the raw materials of the separation gel

Reagent	7.5％	10％
			ddH₂O	7.840m1	6.300ml
30％Arc-Bis	4.000m1	5.340ml
			1.5MTris-HCl	4.000ml	4.000ml
10％SDS	0.160m1	0.160ml
			10% ammonium persulfate	0.080m1	0.080ml
TEMED	0.008m1	0.008ml

TABLE 3 proportions of the respective raw materials of the concentrated gums

Removing the forming comb, adding electrophoresis buffer solution, adding about 60-100 μ g of prepared sample into each gel hole, and adding standard protein product into one hole. And (3) performing electrophoresis at 60V for 30min, changing the voltage to 120V when the sample is electrophoresed to the boundary of the concentrated gel, and stopping electrophoresis when bromophenol blue in the separation gel runs to the bottom edge of the separation gel.

(6) Film transfer: and (3) shearing the PVDF membrane with the same size as the glue, firstly putting the PVDF membrane into methanol for soaking for 10s to fully activate the membrane, and then putting the PVDF membrane into a transfer buffer solution for soaking for 15-30 min.

A pad of the same size as the gel was cut and soaked in the transfer buffer.

The film transfer was carried out in a wet-to-electric transfer apparatus. The plate arrangement sequence is: whiteboard-foam-pad-filter paper-membrane-glue-filter paper-foam-pad-blackboard.

Switching on a power supply, stabilizing current by 80mA, and performing electrophoresis for 1 h.

And after the transfer is finished, the power supply is turned off.

(7) And (3) sealing: placing the membrane in plastic packaging membrane, adding blocking solution (5% skimmed milk powder), and incubating for 4-6 hr at room temperature by shaking table.

(8) Primary antibody incubation:

the primary antibody was diluted with 1% BSA, IL-34 primary antibody at 1:500 dilution, and GAPDH at 1:1000 dilution. After the sealing is finished, the membrane is simply cleaned, then the membrane is placed into a plastic packaging membrane, primary antibody is added, and after 30min of shaking table incubation at room temperature, the membrane is placed at 4 ℃ overnight. The next day, the membrane was removed, re-warmed 30min in a shaker at room temperature, and washed 5min x 3 times with 1 x wash buffer.

(9) Incubation with secondary antibodies (HRP-labeled goat anti-rabbit, goat anti-mouse antibodies)

The film was placed in a plastic packaging film and secondary antibodies were added. The secondary antibody was still diluted with 1% BSA at a dilution of 1: 2000. Incubated for 2h at room temperature with shaking. The membrane was removed and washed 3min × 5 times with 1 × wash buffer.

(10) ECL (chemiluminescence) color development and observation

The results are shown in the immunoblot alignment chart of FIG. 1, from which it can be seen that: the shAURKA or/and shUHRF1 of SW480 cells are knocked down, so that the expression of Aurora-A, UHRF1 of SW480 cells is reduced.

2. Cell clone formation assay

1×10⁵SW480 cells were seeded in 6-well plates in a/ml format. The next day, the medium was discarded, and 5. mu.l of shcotrl (control group, purchased from Kshikah, Shanghai), shAURKA (knock-down AURKA group, purchased from Kshikah, Shanghai), shUHRF1 (knock-down UHRF1 group, purchased from Kshikah), and shAURKA/UHRF1 (AURKA/UHRF 1 double-gene co-interfering lentivirus obtained in example 1) were added to centrifuge tubes containing 500. mu.l of Opti-MEM medium and 495. mu.l of 10% FBS, respectively, mixed, and added theretopolybrene was added to a final concentration of 5. mu.g/ml. After 24 hours of culture, puromycin2mg/ml was selected for 48 hours, the cells were collected, and the collected cells were resuspended in a DMEM medium containing 2mg/ml puromycin, seeded in 6-well plates at 600 cells per well, and cultured for another 10-14 days. The plate was placed under a microscope at low magnification to count clones greater than 50 cells, and the results are shown in FIG. 2. As can be seen from fig. 2: after the shaaurka and the shUHRF1 of the SW480 cells are knocked down independently, the clone forming capability of the SW480 cells is weakened; when shaaurka and UHRF1 were knocked down simultaneously, the clonogenic capacity of SW480 cells showed a significant decrease.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims

1. A preparation method of AURKA/UHRF1 double-gene co-interference lentivirus is characterized by comprising the following steps:

2. The method for preparing the AURKA/UHRF1 double-gene co-interfering lentivirus of claim 1, wherein in step (1), the vector is CV147, and the restriction enzyme cutting sites of the vector are Pac I and Nhe I.

3. The method for preparing the AURKA/UHRF1 double-gene co-interfering lentivirus of claim 1, wherein in the step (2), the expression strain is an Escherichia coli competent cell.

4. The method for preparing the AURKA/UHRF1 double-gene co-interfering lentivirus according to claim 1, wherein the specific operation of culturing and screening ampicillin resistant cloned strains in the step (2) is as follows:

dispersing the plasmid in double distilled water to prepare the plasmid with the concentration of 100 ng/mul in the double distilled water; the number of cells was 5X 10⁶The competent expression strain was suspended in 100. mu.l of 0.1mol/L CaCl containing 15% glycerol₂To the solution, 1. mu.l of double distilled water with dispersed plasmid was added to 100. mu.l of CaCl with suspended competent expression strain₂Putting the solution on ice for 20 min; then hot compressing for 90s at 42 ℃, rapidly placing in ice for 5min, and adding 600 mu lLB culture solution; shaking at 37 deg.C and 220r/min for 1 hr, centrifuging, spreading on LB plate containing 50-100 μ g/ml ampicillin, and performing inverted culture at 37 deg.C overnight to obtain ampicillin-resistant clone strain.

5. The method for preparing the AURKA/UHRF1 double-gene co-interfering lentivirus as claimed in claim 1, wherein the ampicillin resistant cloned strain in the step (3) is specifically shaken as follows:

6. The method for preparing AURKA/UHRF1 double-gene co-interfering lentivirus according to claim 1, wherein the packaging of the AURKA/UHRF1 double-gene co-interfering plasmid in step (4) is performed as follows:

7. The method of claim 6, wherein the packaged plasmid mixture is pHelper1.0 and pHelper2.0, and the mass ratio of pHelper1.0, pHelper2.0 to AURKA/UHRF1 double-gene co-interfering plasmid is 20:15: 10.

8. The method for preparing AURKA/UHRF1 bi-gene co-interfering lentivirus according to claim 6, wherein the packaged hybrid plasmid is PMD2G and PSPAX2, and the mass ratio of the PMD2G, PSPAX2 and the AURKA/UHRF1 bi-gene co-interfering plasmid is 1:1: 1.

9. An AURKA/UHRF1 bi-gene co-interfering lentivirus produced by the method of any one of claims 1 to 8.

10. Use of the AURKA/UHRF1 double-gene co-interfering lentivirus of claim 9 for the preparation or screening of drugs for the treatment of colorectal cancer, or for the preparation of drugs for the diagnosis of tumors.