Application of MDFI-knocked-down short hairpin RNA in preparation of medicine for treating colorectal cancer

Abstract

The invention relates to the field of tumor treatment, in particular to application of short hairpin RNA with MDFI knocked down in preparation of medicine for treating colorectal cancer. The short hairpin RNA with the MDFI knocked down can inhibit colorectal cancer proliferation, induce cell cycle arrest, induce apoptosis and enhance the sensitivity of colorectal cancer cells to oxaliplatin and 5-fluorouracil. According to the invention, the MDFI-knocked-down short hairpin RNA is used for inhibiting the proliferation of colorectal cancer cells, inducing the apoptosis of the colorectal cancer cells and improving the sensitivity of the colorectal cancer cells to chemotherapeutic drugs oxaliplatin and 5-fluorouracil, and can be used for preparing drugs for treating colorectal cancer.

Description

Technical Field

[0001] The present invention relates to the field of tumor treatment, and specifically refers to the application of short hairpin RNA for knocking down MDFI in the preparation of drugs for treating colorectal cancer. Background Art

[0002] Colon cancer is the third most common digestive tract malignancy in the world, and ranks third and second in the incidence of colon cancer in male and female cancer patients worldwide, respectively. 20% of patients have already developed metastasis and lost the opportunity for surgery when they are first diagnosed with colon cancer, while other patients often have local or distant metastasis of the tumor even after surgical treatment. In addition, colon cancer is highly malignant and the tumor progresses rapidly, so its 5-year survival rate is only 64-67%. So far, there is a strong demand for new targeted drugs that can solve the problem of colorectal cancer in clinical practice, and traditional chemotherapy drugs oxaliplatin and 5-fluorouracil often encounter the problem of insufficient response in clinical treatment. Therefore, finding potential drugs for the treatment of colorectal cancer is of great clinical significance. Similarly, finding drugs that can enhance the sensitivity of colorectal cancer to chemotherapy drugs oxaliplatin and 5-fluorouracil is also of great clinical value.

[0003] MyoD Family Inhibitor (MDFI) is an important myogenic inhibitory protein in early embryonic development and is generally believed to regulate the development of biological segments by inhibiting myogenesis. However, no relevant studies have yet conducted in-depth research on the role of MDFI in colon cancer.

[0004] So far, it is still unclear whether the short hairpin RNA knocking down MDFI can regulate the proliferation of colorectal cancer cells, affect the cell cycle of colorectal cancer cells, induce apoptosis of colorectal cancer cells, and affect the sensitivity of colorectal cancer cells to chemotherapeutic drugs. The present invention will evaluate the effects of the short hairpin RNA knocking down MDFI on the proliferation of colorectal cancer cells, the cell cycle of colorectal cancer cells, the apoptosis of colorectal cancer cells, and the sensitivity of colorectal cancer cells to chemotherapeutic drugs through in vivo and in vitro models, confirm its effects, and propose technical solutions related to the invention. Summary of the invention

[0005] The purpose of the present invention is to reveal the effect of short hairpin RNA knocking down MDFI on the proliferation of colorectal cancer cells, the cell cycle of colorectal cancer cells, the apoptosis of colorectal cancer cells, and the sensitivity of colorectal cancer cells to chemotherapeutic drugs through in vitro studies. Therefore, the main contents of the present invention are summarized as follows: the short hairpin RNA knocking down MDFI can inhibit the proliferation of colorectal cancer cells, induce the cell cycle arrest of colorectal cancer cells, induce the apoptosis of colorectal cancer cells, and improve the sensitivity of colorectal cancer cells to chemotherapeutic drugs, and the short hairpin RNA knocking down MDFI can be used to prepare drugs for treating colorectal cancer.

[0006] The details of the present invention are as follows:

[0007] 1. We revealed for the first time that the gene expression level of MDFI was significantly upregulated in colon cancer tissues compared with normal tissues, and that the expression level of MDFI in tumor tissues was positively correlated with the overall survival and disease-free survival of colon cancer patients.

[0008] 2. We first revealed that MDFI has the effects of improving cell viability, promoting proliferation and anti-apoptosis in colorectal cancer cells, and MDFI affects tumor progression in vivo;

[0009] 3. We first revealed that knocking down MDFI can increase the sensitivity of colorectal cancer cells to chemotherapeutic drugs, improve the effect of chemotherapy and delay tumor progression;

[0010] 4. We revealed for the first time that MDFI and ITGB4, as well as MDFI and LAMB3 proteins, directly interact in colorectal cancer cells, jointly regulating the downstream AKT / GSK3β / β-catenin pathway to affect cell phenotype.

[0011] 5. We first used short hairpin RNA to knock down MDFI to reduce the expression level of MDFI protein, and the sensitivity of colorectal cancer cells to chemotherapy drugs increased. In the in vivo model, the combined use of short hairpin RNA chemotherapy drugs to knock down MDFI had a significant effect on delaying tumor progression. It can be seen that short hairpin RNA to knock down MDFI can be used to prepare drugs for the treatment of colorectal cancer.

[0012] Based on the above results, we propose the following solution:

[0013] 1. Application of short hairpin RNA for knocking down MDFI in the preparation of drugs for the treatment of colorectal cancer.

[0014] 2. The sequences of the short hairpin RNA are shown in the sequence listing SEQ ID NO: 1 and SEQ ID NO: 2.

[0015] 3. Application of short hairpin RNA that knocks down MDFI in the preparation of drugs that enhance the sensitivity of colorectal cancer to chemotherapeutic drugs.

[0016] 4. The sequences of the short hairpin RNA are shown in the sequence listing SEQ ID NO: 1 and SEQ ID NO: 2.

[0017] 5. The chemotherapy drugs are oxaliplatin and 5-fluorouracil.

[0018] 6. Use of a combination of short hairpin RNA for knocking down MDFI and oxaliplatin in the preparation of drugs for treating colorectal cancer.

[0019] 7. The sequences of the short hairpin RNA are shown in the sequence listing SEQ ID NO: 1 and SEQ ID NO: 2.

[0020] 8. Use of a combination of short hairpin RNA for knocking down MDFI and 5-fluorouracil in the preparation of drugs for treating colorectal cancer.

[0021] 9. The sequences of the short hairpin RNA are shown in the sequence listing SEQ ID NO: 1 and SEQ ID NO: 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 .Expression of MDFI in colon cancer tissue samples: (A) Based on the colon cancer sample data (GSE144735) provided by the TCGA database, the gene expression level of MDFI in colon tumor tissues (275 cases) and normal tissues (41 cases) of colon cancer patients was analyzed. (B) Based on the Kaplan-Meier survival curve of colon cancer patients obtained from the GEPIA dataset information (n=275), the correlation between the MDFI expression level and the overall survival and disease-free survival of colon cancer patients was analyzed. *P<0.05.

[0023] Figure 2 .Relative expression level of MDFI in colon cancer cells after shRNA transfection for 48h: (A) qPCR assay to detect the mRNA level of MDFI in HCT116 cells transfected with shRNA for 48h. (B) Western blot assay to detect the protein expression level of MDFI in HCT116 cells transfected with shRNA for 48h. (C) qPCR assay to detect the mRNA level of MDFI in SW620 cells transfected with shRNA for 48h. (D) Western blot assay to detect the protein expression level of MDFI in SW620 cells transfected with shRNA for 48h. The bar graph on the right side of the protein representative band presents a quantitative bar graph of the relative expression of the target protein. *P<0.05; **P<0.01; ***P<0.001.

[0024] Figure 3.Relative expression level of MDFI in colon cancer cells 72 hours after lentiviral transfection: (A) qRT-PCR assay to detect the mRNA level of MDFI in HCT116 cells 72 hours after lentiviral transfection. (B) Western blot assay to detect the protein expression level of MDFI in HCT116 cells 72 hours after lentiviral transfection. (C) qRT-PCR assay to detect the mRNA level of MDFI in SW620 cells 72 hours after lentiviral transfection. (D) Western blot assay to detect the protein expression level of MDFI in SW620 cells 72 hours after lentiviral transfection. The bar graph on the right side of the protein representative band presents the quantitative results of the relative expression of the target protein. *P<0.05; **P<0.01; ***P<0.001.

[0025] Figure 4 .Changes in cell viability of colon cancer cells after knockdown or overexpression of MDFI: (A) HCT116 cells were treated with CCK8 reagent after knockdown of MDFI for 24h, 48h, and 72h, and the absorbance at a wavelength of 450nm was measured by an ELISA instrument. The OD value of the control cells after 24h of transfection was used as a reference to obtain the relative survival level of cells at different times after transfection. (B) SW620 cells were treated with CCK8 reagent after knockdown of MDFI for 24h, 48h, and 72h, and the absorbance at a wavelength of 450nm was measured by an ELISA instrument. The OD value of the control cells after 24h of transfection was used as a reference to obtain the relative survival level of cells at different times after transfection. (C) HCT116 cells were treated with CCK8 reagent after overexpression of MDFI for 24h, 48h, and 72h, and the absorbance at a wavelength of 450nm was measured by an ELISA instrument. The OD value of the control cells after 24h of transfection was used as a reference to obtain the relative survival level of cells at different times after transfection. (D) SW620 cells overexpressing MDFI were treated with CCK8 reagent for 24h, 48h, and 72h, and the absorbance at a wavelength of 450nm was measured by an ELISA instrument. The relative survival level of cells at different times after transfection was obtained by taking the OD value of the control cells at 24h of transfection as a reference. **P<0.01; ***P<0.001.

[0026] Figure 5 .Changes in the proliferation ability of colon cancer cells after knocking down MDFI: (A) HCT116 cells with low MDFI expression were obtained after shRNA transfection for 48 hours, and the cell proliferation level was detected by clone formation experiment. (B) SW620 cells with low MDFI expression were obtained after shRNA transfection for 48 hours, and the cell proliferation level was detected by clone formation experiment. HCT116 and SW620 were uniformly plated at a density of 600 cells / well and 1000 cells / well, respectively, and fixed, stained, photographed and counted after 10-14 days. **P<0.01; ***P<0.001.

[0027] Figure 6 .Changes in the proliferation ability of colon cancer cells after overexpression of MDFI: (A) HCT116 cells overexpressing MDFI were obtained after lentiviral transfection for 72 hours, and the cell proliferation level was detected by clone formation experiment. (B) SW620 cells overexpressing MDFI were obtained after lentiviral transfection for 72 hours, and the cell proliferation level was detected by clone formation experiment. HCT116 and SW620 were uniformly plated at a density of 600 cells / well and 1000 cells / well, respectively, and fixed, stained, photographed and counted after 10-14 days. **P<0.01; ***P<0.001.

[0028] Figure 7 .Effect of knockdown of MDFI on apoptosis in colon cancer cells: (A) HCT116 cells with low MDFI expression were obtained after shRNA transfection for 48 hours, and the apoptosis levels of the knockdown group (shMDFI#1, shMDFI#2) and the control group (shcontrol) were detected by flow cytometry. (B) SW620 cells with low MDFI expression were obtained after shRNA transfection for 48 hours, and the apoptosis levels of the knockdown group (shMDFI#1, shMDFI#2) and the control group (shcontrol) were detected by flow cytometry. (C) Histogram of apoptosis of HCT116 and SW620 cells after MDFI knockdown for 48 hours. ***P<0.001.

[0029] Figure 8 .Effect of MDFI overexpression on apoptosis in colon cancer cells: (A) HCT116 cells overexpressing MDFI were obtained after lentiviral transfection for 72 hours, and the apoptosis levels of the overexpression group (shMDFI#1, shMDFI#2) and the control group (shcontrol) were detected by flow cytometry. (B) SW620 cells overexpressing MDFI were obtained after lentiviral transfection for 72 hours, and the apoptosis levels of the overexpression group (shMDFI#1, shMDFI#2) and the control group (shcontrol) were detected by flow cytometry. (C) Histogram of apoptosis of HCT116 and SW620 cells after MDFI overexpression for 72 hours. ***P<0.001.

[0030] Fig. 9.Effect of knockdown of MDFI in colon cancer cells on their cell cycle: (A) HCT116 cells with low MDFI expression were obtained after shRNA transfection for 48 hours, and the cell cycle distribution of the knockdown group (shMDFI#1, shMDFI#2) and the control group (shcontrol) was detected by flow cytometry. (B) SW620 cells with low MDFI expression were obtained after shRNA transfection for 48 hours, and the cell cycle distribution of the knockdown group (shMDFI#1, shMDFI#2) and the control group (shcontrol) was detected by flow cytometry. (C) Cell cycle bar graph of HCT116 and SW620 cells after MDFI knockdown for 48 hours. ***P<0.001.

[0031] Fig.10 .The effect of overexpression of MDFI on the cell cycle in colon cancer cells: (A) HCT116 cells overexpressing MDFI were obtained after lentiviral transfection for 72 hours, and the cell cycle distribution of the overexpression group (lvMDFI) and the control group (lvcontrol) was detected by flow cytometry. (B) SW620 cells overexpressing MDFI were obtained after lentiviral transfection for 72 hours, and the cell cycle distribution of the overexpression group (lvMDFI) and the control group (lvcontrol) was detected by flow cytometry. (C) Cell cycle bar graph of HCT116 and SW620 cells after MDFI overexpression for 72 hours. ***P<0.001.

[0032] Fig.11 Effect of MDFI on the progression of colon cancer in vivo: (A) Tumor growth curves obtained by subcutaneously bearing tumors in nude mice (8 nude mice / group) with HCT116 cells transfected with overexpressed MDFI and empty vector. Tumor volume was measured every three days, and the volume was calculated as length*width*width / 2(mm3). (B) When the tumor volume grew to about 2000mm3, the nude mice were euthanized in real time and the tumors were removed to measure the final tumor volume. (C) Representative images of Ki67 expression in tissues detected by immunohistochemistry after processing of xenograft tumor tissues. Magnification was ×400. Data are expressed as mean ± SD, **p < 0.01.

[0033] Fig.12Effects of chemotherapeutic drugs on the viability of colon cancer cells: (A) HCT116 and SW620 cells with silencing MDFI were treated with different concentrations of oxaliplatin for 24 hours, and then CCK8 detection was performed. The OD value of the shcontrol group under 0μM oxaliplatin treatment was used as a reference to obtain the relative cell survival level at each concentration. (B) HCT116 and SW620 cells with silencing MDFI were treated with different concentrations of 5-Fu for 24 hours, and then CCK8 detection was performed. The OD value of the shcontrol group under 0μM 5-Fu treatment was used as a reference to obtain the relative cell survival level at each concentration. HCT116 and SW620 cells were transfected with shRNA for 48 hours and then seeded in 96-well plates at a density of 10,000 cells / well. Six replicate wells were set up for each group of cell samples, and the experiment was repeated. *P<0.05; **P<0.01; ***P<0.001.

[0034] Fig.13 . Effect of oxaliplatin on proliferation of HCT116 cells after knocking down MDFI: HCT116 cells with low MDFI expression, shMDFI#1 (A) and shMDFI#2 (B), were obtained after shRNA transfection for 48 hours, and then exposed to 0.1μM oxaliplatin and subjected to clone formation experiment. Cells were plated evenly at a density of 600 cells / well, and after 24 hours of treatment with 0.1μM oxaliplatin, they were cultured in normal complete medium for 7-14 days, fixed, stained, photographed and counted. *P<0.05; **P<0.01; ***P<0.001.

[0035] Fig.14 . Effect of oxaliplatin on the proliferation of SW620 cells after knocking down MDFI: SW620 cells with low MDFI expression, shMDFI#1 (A) and shMDFI#2 (B), were obtained after shRNA transfection for 48 hours, and then exposed to 0.1μM oxaliplatin and subjected to clone formation experiments. The cells were plated evenly at a density of 1000 cells / well, and after 24 hours of treatment with 0.1μM oxaliplatin, they were switched to normal complete medium and cultured for 7-14 days, fixed, stained, photographed and counted. *P<0.05; **P<0.01; ***P<0.001.

[0036] Fig.15.The effect of 5-FU on the proliferation of HCT116 cells after knocking down MDFI: MDFI-silenced HCT116 cells shMDFI#1 (A) and shMDFI#2 (B) were obtained by shRNA transfection for 48 hours, and then exposed to 1μM 5-FU and subjected to clone formation experiment. The cells were plated evenly at a density of 600 cells / well, and after 24 hours of treatment with 1μM 5-FU, they were cultured in normal complete medium for 7-14 days, fixed, stained, photographed and counted. **P<0.01; ***P<0.001.

[0037] Fig.16 .The effect of 5-FU on the proliferation of SW620 cells after knocking down MDFI: SW620 cells shMDFI#1 (A) and shMDFI#2 (B) with MDFI silenced were obtained after shRNA transfection for 48 hours, and then exposed to 1μM 5-FU treatment and clone formation experiment was performed. The cells were plated evenly at a density of 1000 cells / well, and after 24 hours of treatment with 1μM 5-FU, they were cultured in normal complete medium for 7-14 days, fixed, stained, photographed and counted. **P<0.01; ***P<0.001. DETAILED DESCRIPTION

[0038] Experimental materials and methods involved in the examples

[0039] 1.1. Experimental Materials and Reagents

[0040] 1.1.1. Cell source:

[0041] Human colon cancer cell lines HCT116 and SW620 were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0042] 1.1.2. Experimental animals:

[0043] The experimental mice were 5-6 weeks old male Balb / c-nu nude mice purchased from the Model Animal Institute of Nanjing University. The experimental mice were fed with experimental mouse maintenance feed and raised in the Animal Experiment Center of Nanjing University Medical School.

[0044] 1.1.3. Experimental consumables

[0045] Consumables such as PCR plates, PCR eight-tube strips, and protective equipment such as gloves were purchased from Biotech; consumables such as cell culture dishes, pipettes, cryopreservation tubes, centrifuge tubes, and multi-well cell culture plates were purchased from CORNING, USA; and micropipette tips were purchased from Axygen, USA.

[0046] 1.1.4. Main reagent formula:

[0047] 1) Phosphate Buffered Saline (PBS):

[0048] 10×PBS: 1L system contains 80g sodium chloride, 2g potassium chloride, 14.4g disodium hydrogen phosphate, and 2.4g potassium dihydrogen phosphate. Add appropriate amount of double distilled water to fully dissolve them and adjust the pH value to 7.2. Add double distilled water to make up to volume, sterilize with high-pressure steam, and store at room temperature.

[0049] 1×PBS: dilute 10×PBS solution with double distilled water at a ratio of 1:9.

[0050] 2) NP40 cell lysate:

[0051] 1.5mL 5M NaCl solution, 2ml 1M Tris-HCl, 0.5ml 1% NP40 solution, 1 tablet of Protease Inhibitor Cocktail, mix well, add double distilled water to 50ml, and store at 4℃. Add a small amount of PMSF and mix well before use.

[0052] 3) 10% SDS solution:

[0053] Dissolve 5g SDS in an appropriate amount of double distilled water and make up to 50ml, then store at room temperature.

[0054] 4) 10% ammonium persulfate solution (AP):

[0055] 1g AP was dissolved in an appropriate amount of double distilled water and made up to 10ml, and stored at 4℃.

[0056] 5) SDS Running Buffer:

[0057] 10×Running Buffer: 303g Tris, 1440g glycine, and 100g SDS are fully dissolved in an appropriate amount of double distilled water and the volume is adjusted to 10L. Store at room temperature.

[0058] 1× Running Buffer: dilute 10× Running Buffer with double distilled water at a ratio of 1:9.

[0059] 6) Transfer Butter:

[0060] 10×Transfer Butter: 303g Tris, 1440g glycine, 100g SDS, add appropriate amount of double distilled water to fully dissolve and then make up to 10L, and store at room temperature.

[0061] 1×Transfer Butter: dilute 10×Running Buffer solution with double distilled water at a ratio of 1:9. After dilution, add 20% volume of anhydrous ethanol and mix well before use.

[0062] 7)TBS buffer-Tween (TBS-T):

[0063] Dissolve 175.4 g sodium chloride and 31.6 g Tris-HCl in an appropriate amount of double distilled water and adjust the pH value to 7.4. Add 10 ml Tween-20 and double distilled water to make up to 20 L and store at room temperature.

[0064] 8) Protein blocking solution:

[0065] Dissolve 5g skimmed milk powder in an appropriate amount of TBS-T solution, place on a shaker to fully dissolve it, and use it immediately after preparation.

[0066] 1.2 Experimental methods

[0067] 1.2.1. Cell recovery

[0068] 1) Preheat the water bath to 37℃, quickly take out the cryotube from the liquid nitrogen tank, and place it in a 37℃ water bath for 2 minutes until it is completely thawed. The thawed cell suspension is quickly transferred to a culture dish containing culture medium and transferred to a constant temperature cell culture incubator.

[0069] 2) Centrifuge the cell suspension at 300g for 4-5 minutes in a tabletop centrifuge; discard the supernatant and resuspend the pellet evenly in 2mL complete culture medium and transfer to a cell culture dish and place in a 5% CO 2 , cultured in a 37℃ constant temperature cell culture incubator.

[0070] 3) After 6 hours of cell culture, observe the cell adhesion status, discard the upper floating cells, replace with fresh culture medium and continue culturing.

[0071] Cell culture

[0072] HCT116 cells were cultured in McCoy's 5A medium, and SW620 cells were cultured in RPMI1640 medium. 10% FBS and 1% penicillin-streptomycin solution were added to the culture medium. The cells were incubated at 5% CO 2 , cultured in a 37℃ constant temperature cell culture incubator.

[0073] Observe the cell growth status, replace fresh culture medium from time to time, and subculture when the cell density in the culture dish reaches 80-90%.

[0074] 1.2.3. Cell passaging

[0075] 1) Cells can be subcultured when the cell growth density is about 80-90%.

[0076] 2) After removing the upper culture medium, add sterile PBS to wash away a small amount of residual culture medium, repeat twice; digest the cells with 2 ml of trypsin for 2-3 minutes, tap the culture dish gently to see that the cells have a large range of cell wall detachment, and add 2 ml of complete culture medium to terminate the digestion.

[0077] 3) Use a pipette to blow the cells off the dish wall to completely remove them, and centrifuge the cell suspension at 300g for 4-5 minutes.

[0078] 4) Discard the supernatant and add an appropriate amount of culture medium to resuspend the cell pellet, add culture medium according to the subculture ratio and transfer it to the cell culture dish; place the culture dish in 5% CO 2 ,37℃ constant temperature incubator.

[0079] 1.2.4. Cell cryopreservation

[0080] 1) After removing the upper culture medium, add sterile PBS to wash away a small amount of residual culture medium, repeat twice; digest the frozen cells with 2ml trypsin for 2-3min. After a large range of cell wall detachment occurs, add 2ml complete culture medium to terminate the digestion.

[0081] 2) Use a pipette to gently blow against the dish wall to completely detach the cells, and centrifuge the cell suspension at 300g in a desktop centrifuge for 4-5 minutes.

[0082] 3) After centrifugation, discard the supernatant, aspirate 1-2 ml of complete medium to evenly resuspend the cells and transfer them to a cryopreservation tube, add 10% volume of DMSO and pipette evenly; the cryopreservation tube is placed in a 4°C and -20°C refrigerator for 20 min, then placed in a -80°C refrigerator overnight, and finally placed in liquid nitrogen for long-term storage.

[0083] 1.2.5. Construction and transfection of shRNA plasmid vector

[0084] 1.2.5.1. Construction of shRNA plasmid vector:

[0085] shRNA was synthesized by GeneCare. According to the design principles of RNA interference and the characteristics of plasmid vectors, two sequences, shMDFI#1 and shMDFI#2, were constructed as MDFI gene interference fragments, and a non-functional fragment, shcontrol, was used as a negative control.

[0086] 1.2.5.2.shRNA plasmid transfection steps:

[0087] 1) Cell culture: Before transfection, target cells were cultured in serum-containing medium in 12-well plates, with 1×10 cells per well. 4 When the cell confluence reached 30-40%, the medium was replaced with serum-free medium.

[0088] 2) Prepare transfection solution: according to the instructions of the plasmid manual, take an appropriate amount of lyophilized powder and add 50 μl Opti-MEM medium to mix thoroughly to obtain shRNA dilution solution, and let it stand at room temperature for 5 minutes; according to the instructions of Lipofectamine 3000 manual, pipette an appropriate amount of Lipofectamine RNAi-MAX into an EP tube (shake gently before taking), add an equal volume of Opti-MEM medium to the shRNA dilution solution to dilute Lipofectamine RNAi MAX, and let it stand at room temperature for 5 minutes.

[0089] 3) shRNA transfection: Use a pipette to repeatedly pipette and mix the shRNA and Lipofectamine RNAi MAX diluent, and let stand at room temperature for 15 minutes; add all the mixed solution to the cell culture plate in 2).

[0090] 4) Cell culture medium replacement: 5% CO 2 Incubate the cells in a 37°C incubator for 6 hours and then replace with fresh complete culture medium; after continuing to incubate for 24-48 hours, qPCR and Western blot experiments can be performed to identify the expression level of the target gene.

[0091] 1.2.6. Construction and transfection of lentiviral vectors

[0092] 1.2.6.1. Lentiviral vector construction:

[0093] The lentivirus was synthesized by GeneCare and included lvMDFI for overexpressing the MDFI gene and lvcontrol, a non-functional control.

[0094] 1.2.6.2. Lentiviral vector transfection steps:

[0095] 1) Cell culture: Before lentiviral infection, 1×10 4 The target cells were seeded into 12-well plates at a density of cells / well and incubated at 37°C.

[0096] 2) Virus transfection: When the cell confluency reaches 20-30%, select the appropriate MOI and virus titer to dilute the virus according to the virus manual, and the calculation formula is: virus volume = (MOI × number of cells) / virus titer; then infect the cells with the virus dilution and culture at 37°C. The culture medium is serum-free medium.

[0097] 3) Cell medium replacement: 16 hours after lentiviral infection, replace with complete medium and continue culture.

[0098] 4) Cell screening: Detect the cell fluorescence intensity and distribution under a fluorescence microscope 48-72 hours after lentiviral infection. Add 2 μg / ml puromycin to screen cells that successfully overexpress the target gene. Replace the culture medium with fresh culture medium containing the same concentration of puromycin every 2 days and continue screening for 2 weeks.

[0099] 1.2.7. Cell counting

[0100] The digested cells were diluted evenly in an appropriate ratio, and 20 μl of the cell suspension was mixed evenly with an equal volume of trypan blue solution; 20 μl of the mixture was added to a cell counting plate, and cells in three fields of view were randomly selected for counting using Count Star software to obtain the average cell concentration.

[0101] 1.2.8. Cell viability assay (CCK8 method)

[0102] 1) Each group of cells was inoculated in a 96-well plate, with 100 μl of cell suspension in each well, and the cell concentration was 1×10 5 / ml, set up 6 replicate wells and incubate at 37℃.

[0103] 2) Cell activity was detected 24h, 48h, and 72h after cell attachment.

[0104] 3) According to the instructions of the CCK8 kit, slowly add 10% (10 μl) CCK8 reagent to each well along the well wall and incubate at 37°C for 1-2 h.

[0105] 4) The culture medium is removed and the cell absorbance at a wavelength of 450 nm is measured using an enzyme reader; a survival curve is drawn.

[0106] 1.2.9. Colony formation

[0107] 1) HCT116 cells stably transfected with shRNA were seeded at 600 cells / well and SW620 cells stably transfected with shRNA were seeded at 1000 cells / well in 12-well plates, with 4 replicate wells for each group of cells; cells stably transfected with lentivirus were seeded at 5000 cells / well in 6-well plates, with 3 replicate wells for each group of cells; shake gently to mix.

[0108] 2) Incubate at 37°C for 7-14 days, replace with fresh culture medium every 4 days, and terminate the culture when clear cell colonies appear.

[0109] 3) Discard the supernatant, wash with PBS 1-2 times to remove excess culture medium, and fix the colonies with 1 ml 4% paraformaldehyde solution / well for 20 minutes; after fixation, discard the fixative, and stain the colonies with 1 ml 0.1% crystal violet solution / well for 20 minutes; finally, recover the crystal violet solution and wash with PBS.

[0110] 4) Determine the number of colonies containing more than 50 cells under a microscope, count and take photos.

[0111] Flow cytometry

[0112] 1.2.10.1. Cell cycle detection:

[0113] 1) Cell collection: Each group of cells was cultured to the logarithmic growth phase, and 2×10 cells were collected after trypsin digestion and cell counting according to 2.2.7. 5 cells; centrifuge at 400 g for 5 min, discard the supernatant and wash with PBS, centrifuge again and discard the supernatant, repeat 1-2 times to obtain a cell pellet.

[0114] 2) Cell staining: Add 1 ml DNA Staining solution and 10 μl Permeabilization solution to the cell pellet, gently vortex for 10 seconds to mix the cells, transfer the cell suspension to the flow cytometry loading tube, and incubate at room temperature in the dark for 30 minutes.

[0115] 3) Flow cytometry: FL2-W and FL2-A channels were preset for flow cytometry analysis; the lowest loading speed was selected in the flow cytometer, and 1×10 were counted per group each time. 4 Before collecting cells, gently tap the tube wall to resuspend the cells in the tube.

[0116] 4) Software analysis: The images were processed using flow cytometry analysis software Flowjo V10 to analyze the cell cycle distribution of each group.

[0117] 1.2.10.2. Apoptosis detection:

[0118] 1) Cell collection: Each group of cells was cultured to the logarithmic growth phase, and 2×10 cells were collected after trypsin digestion and cell counting according to 2.2.7. 5 cells (including cells in the culture supernatant); centrifuge at 400 g for 5 min, discard the supernatant and wash with PBS.

[0119] 2) Cell staining: 5× Binding Buffer and double distilled water were mixed in a volume ratio of 1:4 to obtain 1× Binding Buffer diluent. 500 μl of diluent was prepared for each tube to resuspend the cells. 5 μl Annexin V-FITC and 10 μl PI were added to each tube. The mixture was gently vortexed for 5-10 seconds to mix well and then transferred to the flow cytometry loading tube. The cells were incubated at room temperature in the dark for 5 min.

[0120] 3) Flow cytometry: Adjust the voltage of the fluorescence channel on the flow cytometer and the compensation under this voltage. Preset the FITC and PI detection channels for flow analysis. Select the lowest speed for sample loading in the flow cytometer, and count 1×10 per group each time.4 Before collecting cells, gently tap the tube wall to resuspend the cells in the tube.

[0121] 4) Software analysis: The images were processed using flow cytometry software Flowjo V10 to analyze the apoptotic ratio of each group.

[0122] 1.2.11. Real-time quantitative polymerase chain reaction (Realtime PCR)

[0123] 1.2.11.1. Template RNA extraction

[0124] 1) Digest the cells to be tested in the logarithmic growth phase and take 5×10 6 cells; the cell suspension was centrifuged at 350 g for 3 minutes, and the cell pellet was washed 1-2 times with PBS; the operating table was sprayed with alcohol to maintain an enzyme-free environment.

[0125] 2) Use a pipette to absorb 1 ml of Trizol and gently blow the cells, transfer the cell suspension to a 1.5 ml EP tube, and place it on ice for 5 minutes to allow the cells to fully lyse.

[0126] 3) Add 200 μl of chloroform and shake up and down for 15 seconds to mix, then let stand at room temperature for 5 minutes; precool the high-speed centrifuge to 4°C, centrifuge at 12,000 g for 15 minutes; transfer the upper aqueous phase (about 500 μl) to an enzyme-free EP tube and discard the lower phenol phase.

[0127] 4) Add an equal volume of isopropanol (about 500 μl) and mix thoroughly by inversion. Let stand at room temperature for 5-10 minutes. Centrifuge at 4°C, 12,000 g for 15 minutes, carefully remove the supernatant, and the RNA precipitate is at the bottom of the tube. Invert the tube to drain.

[0128] 5) Add about 500 μl of 75% ethanol and gently shake the EP tube. Centrifuge at 8000 g for 5 min at 4°C and carefully remove the supernatant.

[0129] 6) Repeat step 5) 1-2 times to purify RNA, turn the tube upside down to dry at room temperature or place it in a clean bench to air dry (avoid over-drying).

[0130] 7) Add 50 μl of enzyme-free DEPC water to the EP tube, place it in a 65°C drying oven and heat for 5-10 minutes to fully dissolve the RNA; store it in a -80°C refrigerator until use.

[0131] 1.2.11.2. RNA Reverse Transcription

[0132] 1) Heat the RNA in 2.2.11.1 7) at 65°C for 5 min and measure the RNA concentration using a spectrophotometer.

[0133] 2) Prepare the following reverse transcription system (20 μl):

[0134]

[0135] 3) Reverse transcription of the system in 2) using a PCR instrument (remix the system before reverse transcription), the reaction conditions are:

[0136]

[0137] 4) The reverse transcription product was used for qPCR immediately or stored at -80℃ for a long time.

[0138] 1.2.11.3.RT qPCR

[0139] 1) Prepare the following system (20 μl) in an eight-tube strip in a dark environment:

[0140]

[0141] 2) Centrifuge the eight-tube strip and then place it in the ABI QuantStudio 5 Real-time PCR instrument for qPCR reaction. The reaction conditions are as follows:

[0142]

[0143] 1.2.12. Western blot

[0144] 1.2.12.1. Target protein extraction

[0145] 1) Digest the cells to be tested after culture to the logarithmic growth phase with trypsin, centrifuge at 350g for 3 min, discard the supernatant and wash with PBS, and centrifuge again to obtain the cell pellet.

[0146] 2) Use a pipette to absorb 1 ml of Trizol and gently blow the cells. Transfer the cell suspension to a 1.5 ml EP tube and place it in an ice box for 5 minutes to allow the cells to fully lyse.

[0147] 3) Centrifuge the EP tube at 4°C, 12000g for 15 min, and take a small amount of supernatant to determine the protein concentration; add 25% of the supernatant volume of 5× protein loading buffer to the remaining supernatant, transfer them to a new EP tube and vortex evenly; heat in a metal bath at 95°C for 5 min, and use the sample immediately after it cools to room temperature or store it at -80°C for later use.

[0148] 1.2.12.2. Protein concentration determination (BCA method)

[0149] 1) Prepare a standard protein solution with a concentration of 0.5 mg / ml using sterile PBS.

[0150] 2) Add 0, 2, 4, 8, 12, 16, and 20 μl of standard protein solution to a 96-well plate in sequence, and add 20, 18, 16, 12, 8, 4, and 0 μl of NP40 solution to each well to make the total volume 20 μl; add 4 μl of the supernatant of the protein to be tested and 16 μl of NP40 solution to the 96-well plate.

[0151] 3) Mix solution A and solution B in the BCA test kit at a volume ratio of 50:1, and add 100 μl / well to the small and medium wells in 2); incubate the 96-well plate in a 65°C drying oven for 30 minutes.

[0152] 4) The protein absorbance value at a wavelength of 560 nm was measured using a multifunctional microplate reader; a protein standard curve was drawn and the target protein concentration was calculated.

[0153] 1.2.12.3. SDS-PAGE gel electrophoresis

[0154] 1) Wash the glass glue plate, dry it and assemble it on the glue rack.

[0155] 2) Prepare separation gel:

[0156] The separation gel ratios for each concentration are as follows:

[0157] After adding AP and TEMED, quickly shake the mixed solution and pour it between two gel plates. Add 300-500μl of anhydrous ethanol to seal the separation gel mixture. After the separation gel solidifies, pour out the anhydrous ethanol and turn the gel plate upside down to control drying.

[0158] 3) Prepare concentrated gel (100 ml system):

[0159]

[0160] Take 5 ml of the prepared concentrated gel solution for each gel plate, add 50 μl of 10% AP and 7.5 μl of TEMED and mix well, pour it into the upper layer of the solidified separation gel, insert the electrophoresis comb teeth and wait for the concentrated gel to solidify.

[0161] 4) Protein preparation: Heat the frozen target protein again at 95°C for 5 min, and cool to room temperature before loading.

[0162] 5) Protein loading: After the concentrated gel has solidified, assemble the gel plate and electrophoresis rack and place them in the electrophoresis tank; slowly pour in the running buffer until the inner tank is full and the liquid level in the outer tank is higher than the electrodes, and gently pull out the electrophoresis comb.

[0163] Heat the protein sample at 95°C in a metal bath heater for 5 minutes, and add 20-30 μg of protein to each well when it cools to room temperature. Add 2 μl of protein marker on both sides of the protein sample channel. Vortex mix each group of proteins before loading.

[0164] 6) Electrophoresis: Connect the electrophoresis stand to the electrophoresis instrument correctly, set the constant voltage to 80V for 30 minutes, then switch the voltage to 120V and continue electrophoresis for 60 minutes.

[0165] 7) Transfer: Prepare PVDF membrane and filter paper of appropriate size and appropriate amount of transfer buffer; soak the PVDF membrane in anhydrous ethanol to activate it, and then soak it in transfer buffer together with the filter paper. Carefully cut the separation gel containing protein from the electrophoresis plate, and place the filter paper, separation gel, PVDF membrane and filter paper in the wet transfer device in order. Fill the electrophoresis tank with transfer buffer and add an ice box to maintain low temperature conditions.

[0166] Correctly connect the positive and negative electrodes of the electrophoresis apparatus and transfer for 70 minutes at a constant current of 275mA.

[0167] 8) Blocking: TBST and skimmed milk powder were mixed evenly to prepare a 5% blocking solution. After the transfer was completed, the PVDF membrane was blocked at room temperature for 2 hours.

[0168] 9) Protein incubation: Cut the target protein band from the PVDF membrane against the marker indicator band and wash it in TBST; dilute the primary antibody with TBST at a ratio of 1:1000, pipette an appropriate amount of the primary antibody dilution into the antibody hybridization bag and place the target protein band in it, and incubate at 4°C overnight.

[0169] The target protein band was washed in TBST for 15 min, and repeated 3 times; the secondary antibody was diluted with TBST at a ratio of 1:5000, and the secondary antibody dilution solution was aspirated to fully cover the target protein band, and incubated on a horizontal shaker at room temperature for 2 h.

[0170] After incubation, the target protein band was washed in TBST for 15 min and repeated 3 times.

[0171] 10) Development: Prepare the developer according to the instructions, place the target protein band in the developing chamber with the front side facing up, drop an appropriate amount of developer on the band, and adjust the parameters to develop the target protein band.

[0172] 1.2.13. Xenotransplantation

[0173] 1) Balb / c nude mice were routinely housed in an SPF barrier environment and randomly divided into 2 groups, with 8 mice in each group.

[0174] 2) Digest the control and MDFI-overexpressing colon cancer cells grown to the logarithmic growth phase; refer to the counting method in 2.2.7, and take 5×10 cells from each group. 7 After diluting with 1 ml serum-free medium, an appropriate amount of matrix gel was added to obtain 5×10 7 The cell suspension of 1.5 cells / ml was placed on ice until use.

[0175] 3) Tumor cells (5×10 6 ) was injected subcutaneously into the right armpit of nude mice. After the tumor emerged, the length and width of the tumor were measured with a vernier caliper every 3 days and the tumor volume was calculated. The tumor volume was calculated as follows: Tumor volume (mm 3 ) = length * width * width / 2. The tumor volume is generally greater than 2000mm 3 The mice were killed, the xenograft tumors were removed, and photos were taken.

[0176] 4) The tumor tissue was immersed in 4% paraformaldehyde and stored at -80°C.

[0177] 1.2.14. In vivo administration

[0178] The mice in the treatment group were intraperitoneally injected with oxaliplatin at a concentration of 10 mg / kg per mouse, once a week for 3-4 weeks. The mice in the control group were intraperitoneally injected with the same volume of PBS solution each time. 3 Stop medication around.

[0179] Immunohistochemistry

[0180] 1) The frozen xenograft tumor tissue was embedded in paraffin, thawed in a freezing microtome (-20°C) and sliced ​​to a thickness of 10 μm.

[0181] 2) Soak the slices in xylene for 10 min, repeat twice.

[0182] 3) The slices were hydrated in alcohol of different concentrations, and were immersed in 100%, 95%, and 95% alcohol for 5 minutes, respectively; then immersed in 90% and 80% alcohol for 3 minutes, respectively; and finally immersed in 70% alcohol for 2 minutes. Then, immersed in double distilled water for 1 minute, and repeated 3 times.

[0183] 4) Soak the slices in repair solution and heat them in a microwave oven at medium heat for 15 minutes. Cool the slices to room temperature and rinse with PBS three times, 5 minutes each time.

[0184] 5) Add a small amount of 3% hydrogen peroxide solution to the slices and incubate at room temperature for 10 minutes; rinse with PBS three times, 5 minutes each time; the hydrogen peroxide solution should be prepared and used immediately.

[0185] 6) Add a small amount of BSA solution to the slices, incubate at room temperature for 30 minutes, and remove excess BSA.

[0186] 7) Dilute the primary antibody according to the instructions, add a small amount to the slices, place in a humidified box, and incubate overnight at 4°C; incubate at room temperature for 2 hours the next day; rinse with PBS 3 times, 5 minutes each time.

[0187] 8) Prepare ABC complex at 4°C, add a small amount to the slices, incubate at room temperature for 2 hours; rinse with PBS three times, 5 minutes each time.

[0188] 9) Prepare DAB colorimetric reagent and add a small amount to the slices. Incubate at room temperature in the dark for 30 minutes. Wash with PBS for 5 minutes. Repeat 3 times.

[0189] 10) The sections were stained with iron hematoxylin for 10 min and rinsed with double distilled water several times until clean.

[0190] 11) The sections were placed in 75%, 95%, and 100% ethanol in turn for 3 min to dehydrate.

[0191] 12) Soak the slices in xylene for 10 min, repeat twice.

[0192] 13) Place the slices on filter paper, add a small amount of neutral resin to the center of the slide to seal the slide, cover with a coverslip (to remove bubbles) (keep it level), and store in a cool and ventilated place.

[0193] 14) Observe under a microscope and take photos.

[0194] Statistical analysis

[0195] Each set of experimental data was obtained through three independent repetitions, and the experimental results were expressed as mean ± standard deviation. The data were analyzed using IBM SPSS Statistics 20 software.

[0196] Example 1 Expression of MDFI gene in colon cancer tissue and its relationship with the prognosis of colon cancer patients

[0197] Based on the colon cancer sample data provided by the TCGA database (GSE144735), the gene expression levels of MDFI in colon tumor tissues and normal tissues of colon cancer patients were analyzed. The results showed that the gene expression level of MDFI in colon cancer tissues was significantly upregulated compared with that in normal tissues (P < 0.05, Figure 1 A).

[0198] GEPIA (http: / / gepia.cancer-pku.cn / ) was used to analyze the correlation between the gene expression level of MDFI in colon cancer patients and their survival time. The results showed that compared with colon cancer patients with high MDFI expression, the overall survival and disease-free survival of colon cancer patients with low MDFI expression in tumor tissues were significantly increased (P=0.029, Figure 1 B; P = 0.062, Figure 1C). The above results suggest that the expression level of MDFI may be associated with the occurrence, development and prognosis of colon cancer, high expression of MDFI may promote the occurrence and development of colon cancer, and high expression of MDFI may be associated with poor prognosis of patients with colon cancer.

[0199] Example 2 Expression of MDFI in colon cancer cells after shRNA transfection

[0200] The shRNA vectors (shMDFI#1, shMDFI#2) that knocked down MDFI and the control vector (shcontrol) were transfected into HCT116 and SW620 cells, respectively. The results of qRT-PCR experiments 48 hours after transfection showed that the mRNA expression level of MDFI in the MDFI knockdown group of HCT116 cells was significantly downregulated compared with the control group (p<0.001, Figure 2 A), there was no significant difference between the shMDFI#1 and shMDFI#2 groups. Western blot results showed that the expression level of MDFI protein in HCT116 cells in the MDFI knockdown group was significantly downregulated compared with the control group (p<0.001, Figure 2 B), there was no significant difference between shMDFI#1 and shMDFI#2 groups. The trend of MDFI expression in mRNA and protein levels of SW620 after transfection was consistent with that of HCT116 (p<0.05, Figure 2 C; p<0.001, Figure 2 D). The above results show that shMDFI#1 and shMDFI#2 successfully interfered with colon cancer cells to reduce the expression of MDFI gene.

[0201] Example 3 Expression of MDFI in colon cancer cells after lentiviral transfection

[0202] The lentiviral vector (lvMDFI) that can overexpress MDFI and the control vector (lvcontrol) were transfected into HCT116 and SW620 cells, respectively. The results of qRT-PCR test 72 hours after transfection showed that the mRNA expression level of MDFI in the HCT116 overexpression group was significantly upregulated compared with the control group (p<0.01, Figure 3 A). Western blot results showed that the MDFI protein level in the HCT116 overexpression group was significantly upregulated compared with the control group (p<0.01, Figure 3 B), there was no significant difference between the experimental groups. The trend of MDFI in mRNA and protein expression levels of SW620 after transfection was consistent with that of HCT116 (p<0.001, Figure 3 C; p<0.05, Figure 3D). The above results show that the lentivirus was successfully transfected into colon cancer cells to overexpress MDFI.

[0203] Example 4 Relationship between MDFI and survival of colon cancer cells HCT116 and SW620

[0204] The CCK8 assay detected the relative cell viability of HCT116 and SW620 at 24h, 48h, and 72h after knockdown or overexpression of MDFI. The experiment found that compared with the control group, the survival rates of HCT116 in the shMDFI#1 group and shMDFI#2 group decreased by 25.65±1.7% and 41.53±1.9%, respectively (p<0.001, Figure 4 A), the survival rates of SW620 in the shMDFI#1 group and shMDFI#2 group decreased by 8.69±1.7% and 10.97±1.6%, respectively (p<0.01, Figure 4 B), the cell survival rates in the MDFI knockdown groups of HCT116 and SW620 were significantly decreased compared with the control group. The survival rate of HCT116 cells after 48h of overexpression of MDFI increased by 29.34±5.8% compared with the control group, and the survival rate of SW620 cells after 72h of overexpression of MDFI increased by 21.30±2.3% compared with the negative control group (p<0.01, Figure 4 C; p<0.001, Figure 4 D), the survival rates of the MDFI overexpression groups of HCT116 and SW620 cells were significantly higher than those of the control group. The results showed that the expression level of MDFI affects the survival of colon cancer cells, low expression of MDFI reduces the survival of colon cancer cells, and overexpression of MDFI enhances the survival of colon cancer cells.

[0205] Example 5 Relationship between MDFI and proliferation ability of colon cancer cells HCT116 and SW620

[0206] To explore the effect of MDFI on the proliferation of colon cancer cells, this study used colon cancer cells with knockdown or overexpression of MDFI to perform cloning experiments. The results of the cloning experiments showed that the number of colonies in the shMDFI#1 group and shMDFI#2 group of HCT116 was reduced by 54.60±5.6% and 59.2±5.8% respectively compared with the number of colonies in the control group, and there was no significant difference between the shMDFI#1 group and the shMDFI#2 group (p<0.001, Figure 5 A). The colony numbers of SW620 in the shMDFI#1 group and shMDFI#2 group were reduced by 51.94±3.6% and 50.80±4.4% respectively compared with the control group. There was no significant difference between the shMDFI#1 group and the shMDFI#2 group (p<0.001, Figure 5B). The results showed that the colony numbers of the MDFI knockdown groups of HCT116 and SW620 were significantly lower than those of the control groups.

[0207] The number of colonies in the MDFI overexpression group of HCT116 increased by 97.38±11.0% compared with the control group (p<0.01, Figure 6 A), the number of colonies in the SW620 MDFI overexpression group increased by 90.59±2.3% compared with the control group (p<0.001, Figure 6 B), the results showed that the proliferation ability of colon cancer cells in the MDFI overexpression group was significantly enhanced compared with the control group.

[0208] The above results indicate that the expression level of MDFI affects the proliferation of colon cancer cells. High expression of MDFI enhances the proliferation ability of colon cancer cells, while reducing the expression level of MDFI inhibits the proliferation of colon cancer cells.

[0209] Example 6 Effect of MDFI on apoptosis of colon cancer cells HCT116 and SW620

[0210] In this study, flow cytometry was used to detect the apoptosis level of colon cancer cells with knockdown of MDFI. The results of flow cytometry on apoptosis showed that the apoptosis percentages of HCT116 cells in the shMDFI#1 group and shMDFI#2 group were 5.13±0.08% and 4.18±0.02%, respectively, which were significantly higher than 0.48±0.03% in the control group (p<0.001, Figure 7 A / C). The apoptotic percentages of SW620 cells in the shMDFI#1 group and shMDFI#2 group were 4.30±0.06% and 4.43±0.33%, respectively, which were significantly higher than that in the control group (2.30±0.25%) (p<0.001, Figure 7 B / C). The above results indicate that knocking down MDFI can enhance the apoptosis of colon cancer cells.

[0211] The results of apoptosis of HCT116 and SW620 cells overexpressing MDFI showed that the percentage of apoptosis of HCT116 cells in the lvMDFI group was 0.25±0.01%, which was significantly lower than that in the control group (0.51±0.02%) (p<0.001, Figure 8 A / C). The apoptosis percentage of SW620 cells in the lvMDFI group was 0.64±0.14%, which was significantly lower than that in the control group (2.14±0.26%) (p<0.001, Figure 8 B / C). The apoptosis ratio of HCT116 and SW620 MDFI overexpression groups was significantly lower than that of the control group. Overexpression of MDFI can significantly reduce the apoptosis of colon cancer cells.

[0212] The above results indicate that the expression level of MDFI affects the apoptosis of colon cancer cells. High expression of MDFI inhibits the apoptosis of colon cancer cells, while reducing the expression level of MDFI enhances the apoptosis of colon cancer cells, suggesting that MDFI has a pro-cancer protective effect on colon cancer cells.

[0213] Example 7 Effect of MDFI on the cell cycle progression of colon cancer cells HCT116 and SW620

[0214] In order to further explore the promoting effect of MDFI on the proliferation of colon cancer cells, this study used flow cytometry to evaluate the effect of MDFI on the cell cycle of colon cancer cells. Flow cytometry was used to detect the proportion of HCT116 and SW620 cells in different stages of the cell cycle (G0 / G1 phase, S phase, and G2 / M phase) after knockdown and overexpression of MDFI.

[0215] The results of cell cycle detection of HCT116 and SW620 cells with MDFI knockdown showed that in HCT116, the proportion of cells in the G2 / M phase in the shMDFI#1 group and shMDFI#2 group were 43.8±1.4% and 46.7±0.4%, respectively, which were significantly higher than 37.3±3.2% in the control group (p<0.001, Fig. 9 A). In SW620, the percentage of cells in the G2 / M phase in the shMDFI#1 group and shMDFI#2 group were 20.5±0.5% and 24.1±0.4%, respectively, which were significantly higher than that in the control group (16.6±0.2%) (p<0.001, Fig. 9 B). In HCT116, the percentage of cells in the G0 / G1 phase in the shMDFI#1 group and shMDFI#2 group was 31.2±1.0% and 31.5±0.5%, respectively, which was lower than 34.3±0.6% in the control group; in SW620, the percentage of cells in the G0 / G1 phase in the shMDFI#1 group and shMDFI#2 group was 39.1±0.7% and 37.6±2.0%, respectively, which was lower than 44.8±0.8% in the control group (p<0.5, Fig. 9 C). The results showed that after knocking down MDFI, the number of colon cancer cells HCT116 and SW620 arrested in the G2 / M phase increased significantly, while the number of cells in the G0 / G1 phase decreased significantly.

[0216] The cell cycle results of HCT116 and SW620 cells overexpressing MDFI showed that the proportion of cells in the G2 / M phase in the lvMDFI group and the control group of HCT116 cells were 23.5±0.4% and 26.7±0.8%, respectively (p<0.05, Fig.10A); The percentage of cells in the G2 / M phase of SW620 cells in the lvMDFI group and the control group were 24.1±0.2% and 26.3±0.2%, respectively (p<0.001, Fig.10 B), the percentage of G2 / M phase cells in the MDFI overexpression groups of HCT116 and SW620 was significantly lower than that in the control group. The results showed that after overexpression of MDFI, the percentage of cells blocked in the G2 / M phase of colon cancer cells HCT116 and SW620 was significantly reduced. The percentage of cells in the G0 / G1 phase was significantly increased ( Fig.10 C).

[0217] The above experimental results show that inhibiting MDFI expression can promote G2 / M phase arrest in colon cancer cells, thereby hindering the normal cell cycle process of colon cancer cells, namely cell proliferation. On the contrary, overexpression of MDFI can promote more colon cancer cells to shift from G2 / M phase to G0 / G1 phase, thereby promoting colon cancer cell proliferation.

[0218] Example 8 Effect of MDFI on the progression of colon cancer in vivo

[0219] Existing results have confirmed the proliferation-promoting effect of MDFI on colon cancer cells. This study further confirmed the tumor-promoting ability of MDFI in vivo by constructing a tumor xenograft model. The experiment used HCT116 stably transfected cells overexpressing MDFI and empty vectors to subcutaneously load nude mice, and compared and recorded the changes in tumor growth and the volume of the xenografts finally removed. The experimental results show that the growth rate of the tumor volume formed by the HCT116 stably transfected cell line overexpressing MDFI in nude mice was significantly faster than that of the control group (p<0.01, Fig.11 A). When the tumor volume of nude mice in the experimental group grows to 2000mm 3 The experiment was ended at about 1:10 pm, all mice were euthanized and xenograft tumors were removed. The results showed that the average volume of the transplanted tumors in the MDFI overexpression group was significantly higher than that in the control group (p<0.01, Fig.11 B).

[0220] Subsequently, immunohistochemical detection was performed on the HCT116 xenografts. The results showed that the expression level of Ki67, a proliferation marker, was significantly increased in HCT116 tumor tissues overexpressing MDFI compared with that in the control group. Fig.11 C), the above results all suggest that MDFI promotes the progression of colon cancer in vivo.

[0221] Example 9 Effect of MDFI on the Chemotherapy Efficacy of Colon Cancer Cells

[0222] In order to explore whether MDFI affects the therapeutic effect of chemotherapy drugs on colon cancer cells, this experiment used different gradient concentrations of oxaliplatin and pentafluorouracil to treat HCT116 and SW620 cells with silenced MDFI, and then performed CCK8 experiments and clone formation experiments.

[0223] The results of CCK8 showed that after 24 hours of treatment with gradient concentrations of oxaliplatin, the cell survival rates of HCT116 and SW620 shMDFI#1 and shMDFI#2 groups at each concentration were significantly decreased compared with their shcontrol groups (p<0.001, Fig.12 A). After 24 hours of treatment with gradient concentrations of 5-fluorouracil, the cell survival rates of HCT116 and SW620 cells in the shMDFI#1 and shMDFI#2 groups at each concentration were significantly decreased compared with the shcontrol group (p<0.001, Fig.12 B). The above results indicate that MDFI significantly inhibits the therapeutic effects of oxaliplatin and pentafluorouracil on colon cancer cells.

[0224] To further explore the effect of MDFI on the efficacy of chemotherapy drugs, we further used clone formation experiments to explore the effects of oxaliplatin and pentafluorouracil on the proliferation of colon cancer cells after knocking down and overexpressing MDFI. The results of the clone formation experiment showed that after 24 hours of treatment with 0.1μM oxaliplatin, the number of cell colonies in the HCT116 shMDFI#1 group and shMDFI#2 group decreased by 36.35±8.8% and 74.47±8.3%, respectively, compared with the HCT116 control cells (p<0.01, Fig.13 A and B). After 24 h of treatment with 0.1 μM oxaliplatin, the number of cell colonies in the SW620 shMDFI#1 group and shMDFI#2 group decreased by 36.87±18.2% and 21.22±4.5%, respectively, compared with the SW620 control cells (p<0.01, Fig.14 A and B). Knockdown of MDFI further inhibited the proliferation of colon cancer cells under the action of oxaliplatin, indicating that MDFI can inhibit the anti-proliferative effect of oxaliplatin on colon cancer cells.

[0225] After 24 h of treatment with 1 μM 5-fluorouracil, the number of cell colonies in the HCT116 shMDFI#1 and shMDFI#2 groups was significantly reduced by 54.45±5.9% and 79.2±3.1% ( Fig.15 A and B). After 24 h of treatment with 1 μM 5-fluorouracil, the number of cell colonies in the SW620 shMDFI#1 group and shMDFI#2 group decreased significantly by 32.30±5.0% and 22.86±5.9% respectively compared with the SW620 control cells ( Fig.16A and B). Knockdown of MDFI further inhibited the proliferation of colon cancer cells under the action of oxaliplatin, indicating that MDFI can inhibit the anti-proliferative effect of oxaliplatin on colon cancer cells.

[0226] Summary of the invention

[0227] Based on the above research results, we revealed for the first time that MDFI has a regulatory effect on the malignant behavior of colorectal cancer, and it is mainly manifested in the regulation of cell proliferation. Since the role of MDFI in the progression of colorectal cancer was not clear in this field before, our findings are of great significance to this field. It is particularly important that we used short hairpin RNA interference to reduce the expression of MDFI in HCT116 and SW620 cells for the first time to explore the effect of this gene on the malignant phenotype of colon cancer. The experimental results showed that MDFI reduction can significantly inhibit the proliferation of colon cancer cells, promote apoptosis of colon cancer cells, and inhibit the transition of the cell cycle G2 / M phase to G0 / G1 phase, and knocking down MDFI can significantly promote the therapeutic effects of oxaliplatin and pentafluorouracil in colorectal cancer, confirming that MDFI plays an important role in the occurrence and development of colon cancer. In summary, the specific short hairpin RNA for knocking down MDFI provided by the present invention can be used to prepare drugs for the treatment of colorectal cancer. Sequence Listing <110> Jinan Microecology Biomedicine Provincial Laboratory <120> Application of short hairpin RNA for knocking down MDFI in the preparation of drugs for the treatment of colorectal cancer <130> 20220614 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 58 <212> RNA <213> Artificial sequence (.) <400> 1 ccggcaccgg aaguugcaga cgcaucucga gaugcgucug caacuuccgg uguuuuug 58 <210> 2 <211> 58 <212> RNA <213> Artificial sequence (.) <400> 2 ccggcugaac agcauugacc ucgaucucga gaucgagguc aaugcuguuc aguuuuug 58

Claims

1. Application of short hairpin RNA for knocking down MDFI in the preparation of drugs for the treatment of colorectal cancer.

2. Use of the short hairpin RNA for knocking down MDFI as claimed in claim 1 in the preparation of a drug for treating colorectal cancer, characterized in that the sequence of the short hairpin RNA is as shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing.

3. Application of short hairpin RNA that knocks down MDFI in the preparation of drugs that enhance the sensitivity of colorectal cancer to chemotherapeutic drugs.

4. Use of the short hairpin RNA for knocking down MDFI as claimed in claim 3 in the preparation of a drug for enhancing the sensitivity of colorectal cancer to chemotherapeutic drugs, characterized in that the sequence of the short hairpin RNA is as shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing.

5. Use of the short hairpin RNA for knocking down MDFI as claimed in claim 4 in the preparation of a drug for enhancing the sensitivity of colorectal cancer to chemotherapeutic drugs, characterized in that the chemotherapeutic drugs are oxaliplatin and 5-fluorouracil.

6. Use of a combination of short hairpin RNA for knocking down MDFI and oxaliplatin in the preparation of drugs for treating colorectal cancer.

7. Use of the composition of the short hairpin RNA for knocking down MDFI and oxaliplatin as claimed in claim 6 in the preparation of a drug for treating colorectal cancer, wherein the sequence of the short hairpin RNA is as shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing.

8. Use of a combination of short hairpin RNA for knocking down MDFI and 5-fluorouracil in the preparation of drugs for treating colorectal cancer.

9. Use of the composition of the short hairpin RNA for knocking down MDFI and 5-fluorouracil as claimed in claim 8 in the preparation of a drug for treating colorectal cancer, wherein the sequence of the short hairpin RNA is as shown in the sequence listing SEQ ID NO: 1 and SEQ ID NO: 2.

Images