Inhibitory Effect Of Metformin On Proliferation And Invasion Of Bladder Cancer Cells And Its Mechanism

Objective:Bladder cancer is a common malignant tumor in the clinic.It ranks 4th and 12th in the most common malignant tumors of men and women,and ranks 8th among all tumor-related deaths in the whole body.According to statistics,in 2018,there were 81190 cases of newly diagnosed bladder cancer and 17240 cases of bladder cancer-specific deaths[1].According to the 2002 TNM staging method,bladder cancer can be divided into nonmuscle invasive bladder cancer(NMIBC)and muscle invasive bladder cancer(M]BC).Up to 70%of NMIBC patients relapse after surgery and progress to more severe muscle invasive bladder cancer(MIBC)[2].Even with radical cystectomy,only 50%of patients survive for 5 years[3].Unfortunately,although bladder cancer has such a large impact on the health of our people,no breakthrough treatment for bladder cancer has been found for more than two decades.Therefore,bladder cancer is considered to be one of the most expensive malignancies worldwide[4],and it is necessary to research and develop new methods for treating bladder cancer.As a commonly used drug for the treatment of type 2 diabetes,metformin has been confirmed by years of clinical practice for its safety,efficacy and cost.Studies have shown that metformin can also be used to treat polycystic ovary syndrome[5],nonalcoholic fatty liver disease[6]and premature pubertal response[7].More importantly,in recent years,there is evidence that metformin has the effect of reducing the risk of tumors and inhibiting the proliferation and invasion of tumor cells[8].However,its specific mode of action in tumor cells is still unknown.In summary,bladder cancer is a common tumor that jeopardizes the health of our people,but its treatment is currently limited and its effect is poor.Therefore,we urgently need to develop a new,safe and effective treatment.At present,there are many evidences from in vitro cell experiments and animal experiments that metformin can reduce the incidence of some malignant tumors and tumor-related mortality,but the specific mechanism of action is still unclear.As for whether our concern for metformin has an inhibitory effect on bladder tumor cells,it is still necessary to conduct studies in in vitro cell experiments and animal models.This study hopes to examine the effects of metformin on proliferation,invasion,apoptosis and cell cycle of bladder cancer cells by in vitro cell experiments,and to study the expression changes of downstream related proteins and genes,and to clarify some of themMethods:1.The effects of different concentrations of metformin on the proliferation of bladder cancer BIU-87 cells were detected by MTT proliferation assay;2.The effect of different concentrations of metformin on the colony forming ability of bladder cancer BIU-87 cells was detected by colony formation assay;3.The effect of different concentrations of metformin on the migration ability of bladder cancer BIU-87 cells was detected by cell scratch test.4.The effects of different concentrations of metformin on the invasion ability of bladder cancer BIU-87 cells were detected by Transwell in vitro cancer cell invasion assay.5.The effects of different concentrations of metformin on cell cycle and apoptosis of bladder cancer BIU-87 were detected by flow cytometry6.The expressions of genes and proteins related to cell cycle G0/G1 phase and apoptosis were detected by RT-PCR and Western blot in BIU-87 cells treated with different concentrations of metformin.Results:1.The results of MTT proliferation test showed that different concentrations of metformin acted on bladder cancer cells at different time(24,48,72 h),compared with the blank group(with equal volume of medium),with drug concentration and time of action.The increase in cell viability is gradually decreasing.2.The results of plate colony formation test showed that the cloning ability of the cells was significantly decreased after the different concentrations of metformin were applied to the bladder cancer cells,and the greater the concentration,the more obvious the decrease.3.The results of cell scratch test showed that different concentrations of metformin were added to the scratch-treated plate,and the stain healing ability of bladder cancer cells in the test group with metformin was weakened compared with the blank group,and the concentration was higher.The more obvious the decline in healing ability.4.The results of invasion experiment showed that the average number of cells in each field of metformin treatment group decreased after treatment with different concentrations of metformin for 24 hours,and the difference was statistically significant(P<0.05),and the different concentrations of metformin group were compared.There was also a statistically significant difference in the number of cells per field(P<0.05)5.Flow cytometry results showed that metformin could affect the percentage of apoptotic cells in bladder cancer cells compared with the blank group.The percentage of apoptotic cells was significantly different from that in the blank group(P<0.05)Metformin also affected.In the bladder cancer cell cycle,the metformin-treated group was able to inhibit bladder cancer cells in the G0/G1 phase compared with the blank group.6.The results of Rt-PCR showed that the expression of cyclinDl,CDK4,E2F1,Caspase-3 and MMP-9 gradually decreased with the increase of metformin concentration,while the expression of P21 gene gradually increased.7.Western blot analysis showed that the expression of cyclinDl related to cell cycle was significantly decreased after treatment with metformin.Compared with the blank group,the difference was statistically significant(P<0.05).The expression of P21 metformin concentration group and blank group The difference was statistically significant(P<0.05);the expression of CDK4 and E2F1 was not obvious,and the difference between the groups was not statistically significant(p>0.05).The expression of MMP-9 protein was significantly decreased compared with the migration group(P<0.05),but the difference between the groups was not statistically significant(p>0.05).Compared with the blank group,the expression of apoptosis-related protein Caspase-3 was lower in the 5Mm and 20mM metformin-treated groups,while the 10mM metformin-treated group was higher than the blank group(P<0.05).Conclusion:1.Metformin can inhibit the proliferation of bladder cancer cells and induce cell cycle arrest.The mechanism may be related to down-regulation of cyclinDl,CDK4 and E2F1 expression and up-regulation of p21 expression.2.Metformin can induce apoptosis in cells,and its mechanism may be related to down-regulation of caspase-3 expression3.Metformin can significantly inhibit the migration and invasion of bladder cancer cells,and its mechanism may be related to the decrease of MMP-9 expression.