A shRNA targeting and inhibiting MZF1 gene expression and its application

Abstract

The present invention relates to the field of genetic engineering technology, specifically to a shRNA for targeted inhibition of MZF1 gene expression and its use. The shRNA's target sequence is selected from the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. The shRNA is capable of sustained, stable, efficient, and specific inhibition of MZF1 gene expression, providing a highly specific, stable, and reliable new strategy for gene therapy of colon cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of gene engineering, and more particularly to shRNA for targeting and inhibiting MZF1 gene expression and application thereof. Background Art

[0002] Colon cancer is one of the most common and lethal malignant tumors worldwide. According to the latest Global Cancer Report, the incidence and mortality rates of colon cancer are both among the highest among malignant tumors, posing a serious threat to human health. The occurrence and development of colon cancer involves a variety of complex factors, including genetic background, environmental factors, and lifestyle habits. Among them, gene mutation, as one of the key influencing factors, not only determines the biological characteristics of the tumor, but is also directly related to the patient's treatment plan selection and prognosis assessment. Although some genes and markers related to colon cancer have been discovered, there are still few colon cancer markers with high specificity, and the early diagnosis and treatment of colon cancer still face challenges. Therefore, in-depth exploration of the molecular mechanisms of colon cancer occurrence and the search for new therapeutic targets are of great scientific significance and clinical value.

[0003] In biological processes, gene expression is primarily regulated at the transcriptional level. Tumor development and progression is a complex regulatory process, accompanied by significant disturbances in gene expression, particularly abnormalities in transcriptional regulation. Transcription factors, as a key class of regulatory molecules, play a vital role in the transcription of genes from DNA to RNA by recognizing specific DNA sequences. These transcription factors exhibit selectivity, specificity, and multiple functions, regulating the expression of downstream genes, thereby influencing pathophysiological processes such as cell proliferation, differentiation, and apoptosis, as well as tumor development and progression.

[0004] Among numerous transcription factors, MZF1 (myeloid zinc finger 1), a zinc finger transcription factor, has garnered increasing attention in recent years in the field of oncology. Early research on MZF1 focused primarily on myeloid differentiation and leukemia, but recent studies have shown that MZF1 also participates in the development and progression of various tumors. However, due to its tissue-specific nature and the complexity of MZF1's functions, the role of MZF1 in tumors remains unclear. Whether it functions as a tumor promoter or suppressor remains controversial, and the specific mechanisms remain unclear.

[0005] shRNA (short hairpin RNA) is an RNA molecule with a specific structure. By forming a hairpin structure and being processed by the RNAi (RNA interference) pathway, it can specifically inhibit the expression of target genes. An shRNA expression vector is a tool for cloning shRNA sequences into a plasmid vector. It can stably express shRNA in cells, thereby achieving sustained inhibition of the target gene. In recent years, with the development of gene editing and RNAi technologies, shRNA expression vectors have become increasingly widely used in cancer research.

[0006] In light of the potential role of the MZF1 gene in colon cancer, this patent proposes to construct an MZF1 shRNA expression vector and explore its application in colon cancer cells. By inhibiting MZF1 expression, changes in colon cancer cell biological behaviors such as proliferation, apoptosis, migration, and invasion can be observed, thereby revealing the mechanism of action of the MZF1 gene in the occurrence and progression of colon cancer. Furthermore, the MZF1 shRNA expression vector could serve as a potential therapeutic tool, providing new strategies and methods for gene therapy of colon cancer. Summary of the Invention

[0007] Based on this, the purpose of the present invention is to provide a shRNA expression vector and an expression method thereof that can effectively and stably inhibit the expression of the MZF1 gene, which has important application value.

[0008] A shRNA that targets and inhibits MZF1 gene expression, wherein the target sequence of the shRNA is selected from the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0009] Preferably, the single-stranded nucleotide sequence of the shRNA is SEQ ID NO: 4 or SEQ ID NO: 5.

[0010] A lentiviral expression vector comprising the aforementioned shRNA target sequence.

[0011] Preferably, it further comprises a puromycin resistance gene.

[0012] A use of the aforementioned shRNA and lentiviral expression vector in the preparation of a drug for inhibiting the proliferation of colon cancer cells.

[0013] Preferably, the colon cancer cells are selected from CACO2 or HCT15 cell lines.

[0014] A drug comprising the aforementioned shRNA or lentiviral expression vector that targets and inhibits MZF1 gene expression.

[0015] Preferably, the drug reduces the proliferation activity of colon cancer cells by inhibiting the transcription or translation of the MZF1 gene.

[0016] The present invention has the following beneficial effects: By designing shRNA sequences (SEQ ID NO: 1 and SEQ ID NO: 2) that specifically target the MZF1 gene and constructing a lentiviral expression vector, the present invention achieves efficient and stable silencing of the MZF1 gene in colon cancer cells. Experimental data demonstrate that the shRNA provided by the present invention significantly reduces the expression levels of MZF1 mRNA and protein, and its biological effects are verified by inhibiting cancer cell proliferation and weakening colony formation. This provides a novel, highly targeted and effective tool for gene therapy of colon cancer, with potential for clinical translation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the backbone map of the MZF1-shRNA plasmid vector;

[0018] Figure 2 This is a diagram showing the knockdown effect of the MZF1 gene at the protein and mRNA levels after CACO2 and HCT15 cells were transfected with the lentiviral expression vector in Example 4;

[0019] Figure 3 This is a statistical diagram of cell proliferation in CACO2 and HCT15 cells after knockdown of the MZF1 gene in Example 5;

[0020] Figure 4 This is a statistical graph of plate colony formation in CACO2 and HCT15 cells after knocking down the MZF1 gene in Example 6;

[0021] Figure 5 Statistical graph of soft agar colony formation after knockdown of MZF1 gene in CACO2 and HCT15 cells in Example 7. DETAILED DESCRIPTION

[0022] To make the objectives, features, and advantages of the present invention more readily apparent, the following detailed description of the present invention is provided with reference to the accompanying drawings. The accompanying drawings illustrate several embodiments of the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein.

[0023] The materials and reagents used in the following examples were obtained from the following sources: MZF1-shRNA1, MZF1-shRNA2, and MZF1-shRNA3 sequences and plasmid vectors were designed, constructed, and synthesized by Guangzhou Paizhen Biotechnology Co., Ltd., and the transfection reagent Lipofectamine 2000 was purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0024] In the following examples, all experiments were conducted under conventional conditions or those recommended by the manufacturer unless otherwise specified. Reagents and instruments used without manufacturer identification are commercially available. Unless otherwise noted, all techniques employed in this invention are based on existing technologies in the field.

[0025] Example 1: Design of shRNA oligonucleotide sequences for the human MZF1 gene

[0026] The full mRNA sequence of MZF1 (NM_003422.2) was found in the GenBank database (http: / / www.ncbi.nlm.nih.gov / genbank). Specificity was confirmed by BLAST homology comparison. MZF1 mRNA target nucleotide sequences (target sequences) were designed using the ThermoFisher website (https: / / rnaidesigner.thermofisher.com / rnaiexpress / insert.do). The target mRNA sequences were evaluated and three target sequences were obtained. See Table 1.

[0027] Table 1. Nucleotide sequences of specific shRNA target sites of the MZF1 gene

[0028]

[0029] Design the shRNA interference sequence based on the selected target sequence as shown in Table 2. Add AgeI and EcoRI restriction endonuclease sites at both ends. Send the resulting shRNA to Guangzhou Paizhen Biotechnology Co., Ltd. for synthesis of single-stranded DNA oligos and preparation of double-stranded DNA oligos. The shRNA single-stranded nucleotide sequence consists of the Sense (underlined) sequence, the Loop sequence, the Antisense (underlined) sequence, and the Terminator sequence.

[0030] Table 2. Single-stranded nucleotide sequences of shRNA of MZF1 gene

[0031]

[0032] Example 2: Lentiviral packaging of shRNA of human MZF1 gene

[0033] The double-stranded oligonucleotide short fragments were ligated to the lentiviral vector linearized by adding AgeI and EcoRI restriction enzymes. Figure 1 As shown, the vector carries ampicillin resistance and can be used for plasmid screening, and the plasmid expresses puromycin resistance and can be used for gene screening. The above plasmids were designed, constructed and synthesized by Guangzhou Paizhen Biotechnology Co., Ltd.

[0034] The MZF1 lentiviral interference plasmid was used to transfect the tool cell 293T cells for lentiviral packaging and lentiviral supernatant extraction. Among them, shcontrol represents the negative control virus, and shMZF1-1, shMZF1-2, and shMZF1-3 are MZF1 interference lentiviruses.

[0035] The specific steps are as follows:

[0036] (1) Human embryonic kidney cells (293T) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Culture conditions: DMEM medium + 10% fetal bovine serum + 100 U / ml penicillin + 100 μg / ml streptomycin, cultured at 37°C in a 5% CO2 cell culture incubator.

[0037] (2) One day before transfection, subculture 293T cells into a 6 cm culture dish at an appropriate ratio. When the cells grow to 70% to 80% of their size, prepare for transfection. 1 to 2 hours before transfection, replace the cells to be transfected with 5 ml of fresh culture medium.

[0038] (3) Take a sterile 1.5ml centrifuge tube, add 250ul serum-free DMEM, and according to the previously measured plasmid concentration, add 8ug target plasmid, 1.7pg Gag, 2.5ug Rev, and 2.5ug VSVG respectively, and mix well; take another 1.5ml sterile centrifuge tube, add 240ml serum-free DMEM, and then add 10ul transfection reagent Lipofectamine 2000, mix well, add the solution containing Lipofectamine 2000 to the solution containing the plasmid, invert it upside down 3-4 times to mix well, and let it stand at room temperature for 15-20 minutes.

[0039] (4) Add the transfection reagent-plasmid mixture to a 6 cm 293T culture dish and culture in a CO2 incubator: observe the cell status after 4-6 hours. If the cell status deteriorates, change the medium in time. If the cell status is good, wait until 12 hours;

[0040] (5) 12 hours after transfection, carefully aspirate the cell culture medium, then add 6 ml of fresh complete medium and continue culturing. After 48 hours of changing the medium, use a 20 ml syringe to aspirate the 293T cell culture medium containing the lentivirus, pass the filtrate through a 0.45 μm filter, transfer the filtrate into the virus concentration column, centrifuge at 2500 rpm for 20 minutes, and the virus concentrate will remain in the upper layer of the concentration column; the virus can be aliquoted and stored at -80 ° C, or the virus concentrate can be added to the target cells to be infected.

[0041] Example 3: CACO2, HCT15 cell culture and lentiviral infection

[0042] Colon cancer cell lines CACO2 and HCT15 were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. CACO2 / HCT15 culture medium consisted of DMEM / RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 100 μg / ml of penicillin / streptomycin. Cultures were maintained at 37°C in a cell culture incubator containing 5% CO2. When the cell density reached approximately 30%, either the shMZF1 interfering lentivirus or the shControl negative control virus was added at the corresponding MOI. After 24 hours of infection, the cells were replaced with a new medium.

[0043] Colorectal cancer cells CACO2 and HCT15 were infected with lentiviral supernatant, and stably infected cells were selected with puromycin. Stably infected cells were named CACO2-shMZF1-1, CACO2-shMZF1-2, CACO2-shMZF1-3, HCT15-shMZF1-1, HCT15-shMZF1-2, and HCT15-shMZF1-3, while controls were named CACO2-shcontrol and HCT15-shcontrol.

[0044] The specific steps are as follows:

[0045] (1) On the first day, cells were plated (e.g., 24-well plates) according to experimental needs. The cell density should be approximately 50% of that on the second day and cultured at 37°C overnight.

[0046] (2) Before infection the next day, remove the lentiviral supernatant from the -80°C freezer and thaw on ice. Dilute the virus to the desired concentration using fresh complete culture medium according to the viral titer and MOI value. Note: Do not add antibiotics to the complete culture medium to avoid affecting the infection efficiency of the lentiviral supernatant.

[0047] (3) Aspirate the original culture medium of the cells and add the diluted virus solution to the cells; if the MOI value is large, such as above 20, 5 μg / ml Polybrene can be added during infection to improve the efficiency of virus infection;

[0048] (4) 24 hours after infection, remove the culture medium containing the lentivirus and replace it with fresh culture medium; continue to culture for 24 to 48 hours, add puromycin to screen for successfully infected cells; about 7 days later, collect cells as needed to detect the expression of target gene mRNA or protein.

[0049] Example 4: Detection of MZF1 protein and mRNA levels after shMZF1 virus infection

[0050] (1) The virus-infected CACO2 and HCT15 cells obtained in Example 3 were collected, and total protein was extracted and the protein concentration was determined. The protein was denatured and then transferred to a gel. The membrane was blocked with 5% skim milk for 2 hours, and then incubated with MZF1 antibody and GAPDH antibody overnight. The membrane was washed with TBST solution at room temperature for 3 times, each time for 15 minutes, and then incubated with a secondary antibody of the corresponding property for 2 hours at room temperature. After the antibody incubation, the membrane was washed with TBST solution at room temperature for 3 times, each time for 5 minutes, and then developed using an ultrasensitive ECL chemiluminescence kit on a chemiluminescence imaging system. Western Blot was used to detect the expression of the target protein MZF1 in different groups of cells. The detailed information of the target protein antibodies involved is shown in Table 3.

[0051] Table 3. Detailed information of target protein antibodies

[0052]

[0053] (2) Cells were collected and RNA was extracted. After conversion to cDNA, RT-qPCR experiments were performed. The mRNA expression level was detected using the SYBR Green Pro TaqHS premixed qPCR kit in the QuantStudio™ 1 real-time fluorescence quantitative PCR system (Thermo Fisher). The primer sequences of MZF1 and GAPDH are shown in Table 4.

[0054] Table 4. Detailed information of target protein antibodies

[0055]

[0056] The test results are as follows:

[0057] like Figure 2 As shown in A and C, Western blot was used to detect the protein levels of MZF1 in five cell lines. The results showed that the protein levels of MZF1 in the HCT15 and CACO2 interference MZF1 groups (shMZF1-1, shMZF1-2) were significantly downregulated.

[0058] like Figure 2As shown in Figures B and D, qPCR was used to measure MZF1 mRNA levels in HCT15 and CACO2 blank control groups (Mock), HCT15 and CACO2 transfected with control vectors (sh-control), and HCT15 and CACO2 MZF1 knockout groups (shMZF1-1, shMZF1-2, and shMZF1-3). β-actin was used as an internal reference (i.e., MZF1 mRNA levels were normalized to 1.0), and relative expression levels were calculated using 2^-ΔΔCT. The results showed that the normalized relative mRNA levels of MZF1 in the CACO2 MZF1 knockout groups (shMZF1-1 and shMZF1-2) were approximately 0.11 and 0.08, respectively, while the normalized relative mRNA levels of MZF1 in the HCT15 MZF1 knockout groups (shMZF1-1 and shMZF1-2) were approximately 0.22 and 0.28, respectively. That is to say, compared with the control group, the mRNA level of MZF1 in the HCT15 and CACO2 interference MZF1 groups (shMZF1-1, shMZF1-2) was significantly downregulated, and the difference was statistically significant.

[0059] In summary, the MZF1 gene was successfully knocked down in CACO2 and HCT15 cells, which also demonstrated that the shRNA of Example 1 and the lentiviral expression vector constructed in Example 2 can stably knock down the MZF1 gene in colon cancer cells.

[0060] Example 5: Effect of MZF1 gene knockdown on the proliferation activity of colon cancer cells

[0061] The CCK8 assay uses the CCK8 kit from Nanjing KeyGen Biotech Co., Ltd., which is a highly sensitive, non-radioactive colorimetric assay for determining the number of viable cells during cell proliferation.

[0062] The specific steps are as follows:

[0063] (1) Cell processing: Digest the target cells with routine trypsin and gently pipette to make them into single cells, centrifuge, resuspend and count;

[0064] (2) Seeding: Use a 96-well plate, generally seeding 2000-3000 cells per well, 200 μl of culture medium per well, and each group of cells contains three replicate wells at each time point. Culture in a 37°C, 5% CO2 cell culture incubator;

[0065] (3) After the cells have attached to the wall, measure the absorbance at 0 h: aspirate the culture medium in the wells, add 100 μl of the detection solution mixed with empty culture medium and CCK8 at a ratio of 9:1 to each well, and place in a 37°C, 5% CO2 cell culture incubator for 1 h before taking out;

[0066] (4) Measurement: Use a WD-2102A automatic microplate reader to detect the absorbance of each well at a wavelength of 450 nm and record the value; then return the well to the incubator for continued cultivation, and then measure the absorbance of each well at a wavelength of 450 nm at 24 h, 48 h, 72 h, and 96 h.

[0067] (5) Data analysis: Calculate the average absorbance of the three replicate wells at 0 h in each group, and divide the absorbance at each time point in each group by the average absorbance at 0 h in the group. Based on this, the absorbance at 0 h is 1, as well as the relative absorbance at each time point. Calculate the standard deviation of each time point in each group and draw a line graph.

[0068] The test results are as follows:

[0069] CCK8 proliferation assay was used to detect the cell proliferation ability of HCT15, CACO2 blank control group (Mock), HCT15, CACO2 transfected control vector group (sh-control), and HCT15, CACO2 interference MZF1 group (shMZF1-1, shMZF1-2). Figure 3 As shown in the results, compared with the control group, the cell proliferation ability of the HCT15 and CACO2 interference MZF1 groups decreased, and the difference was statistically significant.

[0070] Example 6: Effect of MZF1 gene knockdown on colony formation ability of colon cancer cells

[0071] This experiment can detect the proliferation ability of cells in vitro. After a single cell proliferates continuously for more than 6 generations in vitro, it forms a clone or colony. At this time, the proliferation ability of a single cell can be quantitatively analyzed by counting the number of clones formed.

[0072] The specific steps are as follows:

[0073] (1) Cell processing: routine digestion and gentle pipetting to make single cells, centrifuge, resuspend and count, and inoculate 400 cells / 2ml / well in a 6-well plate and culture at 37°C, 5% CO2 incubator;

[0074] (2) Observe daily. When visible clones are observed after about 10-14 days, terminate the culture. Discard the culture medium and wash twice with PBS buffer. Be careful to be gentle.

[0075] (3) After aspirating the PBS buffer, add 1 ml of 4% paraformaldehyde to each well and fix for 15-30 minutes;

[0076] (4) Aspirate 4% paraformaldehyde, wash twice with PBS buffer, add 0.1% crystal violet staining solution, and let it stand for 15 minutes;

[0077] (5) Aspirate the crystal violet stain (reusable), wash twice with PBS buffer, and rinse gently with running water. Stop rinsing when the running water does not turn purple. Purple dot-like colonies can be seen at the bottom of the well.

[0078] (6) Place the plate in an oven to dry. After drying, take photos and count the colonies. Calculate the number of colonies formed in each group and perform statistical analysis.

[0079] (7) Crystal violet formula (0.5% stock solution): 0.5g crystal violet + 100ml methanol. Crystal violet formula (0.1% working solution): Dilute with normal saline.

[0080] The test results are as follows:

[0081] HCT15 and CACO2 blank control group (Mock), HCT15 and CACO2 transfected control vector group (sh-control), and HCT15 and CACO2 MZF1 interference group (shMZF1-1 and shMZF1-2) were plated in 6-well plates at a density of 300 cells / well and incubated in an incubator for 14 days before being fixed with 4% paraformaldehyde and stained with crystal violet. Figure 4 The left panel shows photos of clone formation in each group, and the right panel shows statistical analysis of the number of clones formed in each group. The results suggest that compared with the control group, the proliferation capacity of cells in the HCT15 and CACO2-interfered MZF1 groups decreased, and the difference was statistically significant.

[0082] Example 7: Effect of MZF1 gene knockdown on soft agar colony formation ability of colon cancer cells

[0083] Certain non-adherent malignant tumor cells can not only proliferate in an adherent state, but also in a suspended state. In soft agar, the cells are kept in a non-adherent and single-cell state, simulating the growth of cells in vivo. Therefore, this experiment is often used to detect the malignant proliferation ability of tumor cells in vitro.

[0084] The specific steps are as follows:

[0085] (1) Prepare 20 ml of 2.5% Argrose and 1.5% Argrose. For Argrose, use BIOWEST REGULAR Argrose G10 (4°C) and ddH2O as the solvent. After autoclaving for 30 minutes, maintain at 42°C to prevent solidification.

[0086] (2) Before the experiment, place the water bath in a clean bench, irradiate with UV light, set the temperature to 42°C, and preheat the complete culture medium;

[0087] (3) Laying gel (final concentration is 0.6% Argrose): Prepare a 50ml centrifuge tube in a 42℃ water bath, mix 2.5% Argrose gel and preheated complete culture medium in a ratio of 1:3, quickly add 1.5ml of the mixture to each well of a 6-well plate, mix gently, and let it stand at room temperature until the gel solidifies (be gentle when adding the mixture to avoid creating bubbles);

[0088] (4) Cell counting: Take cells in the logarithmic growth phase, digest them with 0.25% trypsin and gently pipette them to make them single cells. Count the viable cells and adjust the cell density to above 40×10^4 / ml with complete culture medium. The volume added is less than 100μl and will not affect the dilution of other components. Plate 1×10^4 cells per well, and set up three replicate wells for each group of cells.

[0089] (5) Top coat (final concentration 0.3% Argrose): Prepare a 10ml centrifuge tube in a 42℃ water bath, mix 0.7% Argrose top coat and preheated complete culture medium in a ratio of 1:3, then add the counted cell suspension to the above mixture, mix well and quickly add 1ml to each well of a 6-well plate. After the top layer of agar solidifies, place in 5% CO2, 37℃, and culture for 2-3 weeks;

[0090] (6) Add 200 μl of complete culture medium every 2 days to prevent excessive drying;

[0091] (7) For staining, prepare 1 ml of 1% (w / v) nitrosoiodine violet per well and store at 4°C overnight.

[0092] (8) Calculate the number of clones formed in each group and perform statistical analysis.

[0093] The test results are as follows:

[0094] HCT15 and CACO2 blank control group (Mock), HCT15 and CACO2 transfected control vector group (sh-control), and HCT15 and CACO2 MZF1 interference group (shMZF1-1 and shMZF1-2) were plated in the upper soft agar gel of 6-well plates at a density of 10^4 cells / well and incubated in an incubator for 14 days before staining. Figure 5 As shown, the left part is a photo of the clone formation in each group, and the right part is a statistical analysis of the number of clones formed in each group. The results showed that compared with the control group, the cell proliferation ability of the HCT15 and CACO2 interference MZF1 group decreased, and the difference was statistically significant.

[0095] In summary, the results of Examples 1 to 7 demonstrate that the present invention successfully achieved efficient silencing of the MZF1 gene in colon cancer cells by designing specific shRNA sequences (shMZF1-1 and shMZF1-2) and constructing lentiviral expression vectors. Western blot and RT-qPCR (Example 4) showed that shMZF1-1 and shMZF1-2 significantly reduced MZF1 protein and mRNA levels (knockdown efficiency exceeding 70%), while shMZF1-3 had a lesser effect. Functional experiments (Examples 5-7) further confirmed that knockdown of MZF1 significantly reduced the proliferation activity (CCK8 assay), adherent colony formation ability (plate colony formation assay), and non-adherent malignant proliferation ability (soft agar assay) of colon cancer cells (CACO2 and HCT15). Based on the above results, the present invention draws the following conclusions: The MZF1 gene plays a key role in promoting proliferation and malignant phenotypes in colon cancer cells, and the shRNA lentiviral vector targeting silencing MZF1 can effectively inhibit tumor cell growth, providing a highly specific, stable and reliable new strategy for gene therapy of colon cancer.

[0096] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A shRNA targeting and inhibiting the expression of MZF1 gene, characterized in that: The target sequence of the shRNA is selected from the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; the single-stranded nucleotide sequence of the shRNA is SEQ ID NO: 4 or SEQ ID NO:

5.

2. A lentiviral expression vector, characterized in that: Comprising the shRNA according to claim 1.

3. The lentiviral expression vector according to claim 2, wherein It also contains the puromycin resistance gene.

4. Use of the shRNA according to claim 1 or the lentiviral expression vector according to claim 2 or 3 in the preparation of a drug for inhibiting the proliferation of colon cancer cells.

5. The use according to claim 4, characterized in that The colon cancer cells are selected from CACO2 or HCT15 cell lines.

6. A drug, characterized in that It includes the shRNA targeting and inhibiting MZF1 gene expression as claimed in claim 1, or the lentiviral expression vector as claimed in claim 2 or 3.

7. The drug according to claim 6, characterized in that The drug reduces the proliferation activity of colon cancer cells by inhibiting the transcription or translation of the MZF1 gene.

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