| Objective: This study examined the expression of mi R-424-3p in bladder cancer tissues and analyzed its relationship with clinicopathological parameters of bladder cancer patients.At the same time,the effect of mi R-424-3p on cisplatin resistance in bladder cancer cells was investigated by in vitro cell experiments.And related mechanisms,aiming to clarify the specific mechanism of action of mi R-424-3p in cisplatin resistance of bladder cancer cells,and lay a theoretical and experimental basis for finding new molecular targets for the treatment of bladder cancer and reversing the resistance of bladder cancer to cisplatin.Methods: In this study,40 bladder cancer tissues and adjacent normal tissues were selected to detect the expression of mi R-424-3p,and the age,sex,tumor size,gross classification,histological classification and infiltration of bladder cancer patients were analyzed.Relationship between degree,TNM stage,lymph node metastasis,distant metastasis,recurrence,etc.Establish a bladder cancer cisplatin-resistant cell line(T24/DDP)by gradually increasing the concentration of cisplatin.The drug sensitivity of the drug-resistant cell line was detected by MTT assay,and the expression of mi R-424-3p in the cell line and the parental cell line(T24)was detected.The mi R-424-3p inhibitor was transfected into T24/DDP cells.The proliferation activity of the cells was detected by MTT assay.The apoptosis rate was detected by flow cytometry.The cell invasion ability and cell scratch were detected by transwell chamber method.The experiment detects the migration ability of the cells.Using the online Targetscan and Micro Cosm databases to find possible target genes of mi R-424-3p,mi R-424-3p was obtained by searching the mi RBase database.The 3' UTR sequence was obtained by searching the UCSC Genome Browser and using pubmed for blast alignment.The PTEN-MUT and PTEN-WT plasmids carrying the dual luciferase reporter gene were co-transfected into T24 cells with mi R-424-3p,and the activity of luciferase in each of the above groups was detected.The mi R-424-3p mimic was transfected into T24/DDP cells to detect the expression of PTEN m RNA and protein in the cells.T24 cells were randomly divided into control group;DDP group;DDP+mi R-424-3p group;DDP+mi R-424-3p+PTEN group.The IC50 of each group was detected by MTT assay.The apoptosis rate of each group was detected by flow cytometry.The expressions of PTEN,PI3 K,AKT and p-AKT protein in the above groups were detected by western blot.Results: Compared with normal tissues adjacent to the cancer,the expression of mi R-424-3p was significantly increased in bladder cancer tissues.The expression of mi R-424-3p was not associated with age and gender of bladder cancer patients,but was associated with tumor size,gross classification,histological type,degree of invasion,TNM stage,lymph node metastasis,distant metastasis,recurrence within 1 year.The larger the tumor and the higher the degree of malignancy,the higher the expression of mi R-424-3p and the higher the recurrence rate within 1 year.In this study,T24 cells were induced in vitro by increasing the concentration of cisplatin,and finally the resistant cell line T24/DDP stably growing under the action of 0.6 ?g/m L cisplatin was obtained.The MTT test showed that the resistance index(RI)of T24/DDP cells to cisplatin was 9.55.The expression level of mi R-424-3p in T24/DDP cells was significantly higher than that in T24 cells(P<0.05).Under the action of cisplatin,the proliferation activity of mi R-424-3p inhibitor cells was lower than that of control group,and the cell proliferation activity at 72 h and 96 h was significantly lower than that of control group(P<0.05);The apoptosis rate of cells in mi R-424-3p inhibitor group was significantly higher than that in control group(P<0.05),the number of invasive cells was significantly decreased(P<0.05),and the cell migration rate was significantly decreased(P<0.05).PTEN is a target gene of mi R-424-3p,and the position of mi R-424-3p bound to PTEN is located in the 3'UTR region.Compared with the control group,the luciferase activity of PTEN-WT mi R-424 cells was significantly decreased(P<0.05),while the luciferase activity of PTEN-MUT mi R-424 cells was not significantly changed(P>0.05).It is suggested that PTEN is a target gene of mi R-424-3p,and its expression is negatively regulated by mi R-424-3p.Compared with the control group,the expression levels of PTEN m RNA and protein in the mi R-424-3p group were significantly lower(P<0.05).Compared with the control group,the IC50 of DDP group decreased,but the difference was not significant(P>0.05);the IC50 of DDP mi R-424-3p group was significantly higher than that of DDP group(P<0.05);The IC50 of DDP mi R-424-3p PTEN cells was significantly lower(P<0.05).Compared with the control group,the apoptosis rate of DDP group was increased,but the difference was not significant(P>0.05).The apoptosis rate of DDP mi R-424-3p group was significantly lower than that of DDP group(P<0.05).Compared with the DDP group,the apoptosis rate of DDP mi R-424-3p PTEN group was significantly increased(P<0.01).Compared with the control group,the expression of PTEN,PI3 K,Akt and p-Akt in DDP group was not significantly different(P>0.05).Compared with DDP group,the expression of PTEN protein in DDP mi R-424-3p group The level of PI3 K and p-Akt protein was significantly increased(P<0.05),but the Akt protein was not significantly changed(P>0.05).Compared with DDP group,The expression of PTEN protein in DDP mi R-424-3p PTEN group was significantly increased(P<0.05),and the expression levels of PI3 K and p-Akt protein were significantly decreased(P<0.05),but Akt protein was not significantly changed(P>0.05).Conclusion: mi R-424-3p regulates the PI3K/Akt signaling pathway and inhibits the sensitivity of bladder cancer cells to cisplatin by inhibiting the expression of its target gene PTEN. |
PDF Full Text Request