ABSTRACT
Objective To investigate the activation effect of microRNA-1280 (miR-1280) on the expression of p21 gene in bladder cancer cell line BIU-87 and its effect on cell cycle and proliferation of bladder cancer cell line.Methods Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expressions of miR-1280 in bladder cancer cell lines T24,5637,J82,BIU-87 and normal bladder epithelial cells SV-HUC-1.miR-1280 mimics (experimental group) and miR-NC (control group) were transfected into the bladder cancer cells with the lowest expression of miR-1280.The expressions of miR-1280 and p21 mRNA were detected by qRT-PCR.Chromatin immunoprecipitation (ChIP) was used to verify the targeting effect of miR-1280 and p21 gene promoter.Western blotting was used to detect the expressions of p21,cell cycle-dependent kinase 1 (CDK1),Cyclin A2 mRNA and protein in the two groups.Cell cycle was detected by flow cytometry,and cell proliferation was detected by methyl thiazolyl tetrazclium (MTT) assay.Results The results of qRT-PCR indicated that the expression levels of miR-1280 in bladder cancer cell lines T24,5637,J82 and BIU-87 and normal urothelium cell line SV-HUC-1 were 0.503 ±0.094,0.611 ±0.054,0.567 ± 0.077,0.257 ± 0.032 and 1.014 ± 0.090 respectively,with a significant difference (F =1.880,P ＜0.001).Compared with bladder cancer cell lines T24,5637 and J82 cells,the expression of miR-1280 in BIU-87 cell was the lowest (P =0.026,P =0.003,P =0.008).Compared with the control group,the expression of miR-1280 in BIU-87 cell was significantly increased (1 041.000 ± 157.500 vs.1.023 ± 0.118,t =6.606,P ＜0.001),and the expression of p21 mRNA was also significantly increased (5.280 ± 0.660 vs.1.007 ± 0.070,t =6.440,P ＜ 0.001).Western blotting showed that p21 protein expression was up-regulated,CDK1 and Cyclin A2 protein expressions were down-regulated.ChIP experiments showed that compared with the miR-NC transfection group,the concentration of biotin modified miR-1280 in the p21 gene promoter region was significantly increased (1.246 ±0.171 vs.0.519 ± 0.087,t =3.787,P =0.009).The proportion of G0-G1 cells in the experimental group BIU-87 cells was significantly higher than that in the control group (68.360％ ±3.064％ vs.46.970％ ±3.971％,t =4.263,P =0.005).The results of MTT showed that compared with the control group,the cell proliferation ability of BIU-87 cells after being transfected miR-1280 was significantly decreased starting from day 3 (0.826 ± 0.099 vs.1.224 ± 0.057,t =3.505,P =0.013).Conclusion miR-1280 can activate the expression of p21 gene in bladder cancer cell line BIU-87 by binding the promoter region of p21 gene,blocking the progression of cell cycle and inhibiting cell proliferation,which provides a new direction for bladder cancer targeted therapy theory.
ABSTRACT
Objective To investigate the effect of miR-103b on the expression of P21 protein in renal cell carcinoma cell line 769-P and ACHN cells,and its effect on the growth of renal cell carcinoma.Methods Renal cancer cells were divided into two groups according to the transfected RNA,miR-103b (experimental group) and dsControl (control group),respectively.Real-time PCR and Western blotting were used to detect the expression of P21,cell cycle-dependent kinase 6,Cyclin D1 mRNA and protein expression.Flow cytometry was used to detect the cell cycle distribution.MTT assay was used to detect cell viability and colony formation assay was used to detect cell proliferation.Measurement data were represented as x ± s.Comparison between groups was analyed using t test.Results Real-time PCR results showed that the relative expression levels of P21,cell cycle-dependent kinase 6 and Cyclin D1 mRNA in 769-P and ACHN which belong to control group cells were 1.00 ±0.10 and 1.02 ±0.27,1.00 ±0.08 and 1.01 ±0.17,1.01 ±0.19 and 1.00 ±0.02.The experimental group was 2.36 ±0.51 and 2.03 ± 0.49,0.33 ± 0.20 and 0.58 ± 0.22,0.48 ± 0.11 and 0.60 ± 0.23,respectively,and the difference was statistically significant (P ＜ 0.05).Western blotting results were consistent with Real-time PCR results.Flow cytometry results showed that compared with the control group,the proportion of cells located in G0/G1 phase in the experimental group increased (P ＜ 0.05),suggesting that the cells were arrested in G0/G1 phase.MTT assay showed that the viability of 769-P and ACHN cells in the experimental group was significantly lower than that in the control group.Colony formation experiments showed that the number of colony formation in the experimental group was significantly less,suggesting that the cell proliferation capacity decreased.Conclusion miR-103b can inhibit the growth of renal cell carcinoma cells by activating the expression of P21 protein and blocking the progression of the renal cell cycle,which provides a theoretical basis for the molecular targeted therapy of renal cell carcinoma.
ABSTRACT
Objective To study the effects of a synthetic miR3619-5p mimics on bladder carcinoma cell lines of EJ and T24 in vitro.Methods EJ and T24 cells were cultured in vitro and treated with three different processing:negative control group(tinfection with dsControl),positive control group(infection with dsP21-322) and the experimental group(infection with miR-3619-5p)during October 2015 to March 2016.Real-time fluorescent quantitative PCR (qPCR) was performed to detect the expression of p21 mRNA,cell cycle protein D1 (CyclinD1) and cell cycle-dependent kinase (CDK4 and CDK6) mRNA.Western Blot method was conducted to evaluate the expression of p21,CyclinD1 and CDK4 and CDK6 proteins;the change of cell cycle was displayed by flow cytometric analysis.Colony formation assay was used to test the ability of single cancer cell clone proliferation.Cell proliferation assay(MTS) was implemented to observed the inhibitive effect of cell proliferative potential.Results qPCR results showed that miR-3619-5p upregulated p21 mRNA expression (P ＜ 0.05),while the expression of CyclinD1,CDK4 and CDK6 were a little lower(P ＜ 0.05) in EJ and T24cells,respectively.Western Blot analysis testified that the expressions of p21,CyclinD1,CDK4 and CDK6 were difference among groups.Flow cytometry displayed that,the G0/ G1 phase increased significantly after transfected with miR-3619-5p and dsP21-322,compared with dsControl group(P ＜ 0.05),indicating that the cell cycle block in G0/G1 phase.Cell colony formation assay certified that the colony formation rates were less in the groups of miR-3619-5p and dsP21-322 than in that of dsControl group(P ＜ 0.05).Cell proliferation assay demonstrated that,cell proliferation ability decreased obviously when transfected with miR-3619-5p and dsP21-322 (P ＜0.05),compared with dsControl group.Conclusions miR-3619-5p could up-regulate the expression of p21 by RNA activation pathway and remarkably induced cell cycle arrest in G0/G1 phase,inhibiting the proliferation of bladder cancer cells.