| Objective: To study the differential expressions of miR-455-3p.1 and PIK3R1 gene between clear cell renal cell carcinoma(ccRCC)tumor tissue and adjacent kidney tissue and their correlations;to study the impact of miR-455-3p.1 on proliferation,migration,invasion,cell cycle and apoptosis of ccRCC cells in ccRCC cell line in vitro;to study the functioning mechanism of miR-455-3p.1 in regulating proliferation,migration and invasion of ccRCC.Methods: Part one: 65 pairs of ccRCC tumor tissue of ccRCC and adjacent normal kidney tissues were sampled,reverse transcription polymerase chain reaction(RT-PCR)and quantificational real-time polymerase chain reaction(qRT-PCR)were used to test expression levels of miR-455-3p.1 and PIK3R1 mRNA and western blot was used to test expression of p85?,correlation between expression level of miR-455-3p.1 and PIK3R1 mRNA/p85? were analyzed.Part two: ccRCC cell lines ACHN,CAKI-1,786-O,769 P and renal tubular cell line HK2 were tested for expression difference of miR-455-3p.1 using RT-PCR and qRT-PCR.CcRCC Cell lines with lowest and highest expression level of miR-455-3p.1 were used for further study of up-regulation and down-regulation with miR-455-3p.1 mimic and inhibitor respectively,MTT assay was used to test the impact of miR-455-3p.1 on cell proliferation,transwell assay on cell migration and invasion and flow cytometry,on cell cycle and apoptosis of ccRCC cells.Part three: Western Blot was performed to test the effect of miR-455-3p.1 on the expression of signal pathway proteins including PIK3R1,PTEN,AKT,and p-AKT in miR-455-3p.1-mimictransfected 786-O cells and miR-455-3p.1 inhibitor-transfected ACHN cells;luciferase reporter assay was performed to identify if PIK3R1 was target gene of mi R-455-3p.1;MiR-455-3p.1 mimic+ PIK3R1 and miR-455-3p.1 mimic+ PIK3R1 NC were transfected into 786-O cells,respectively and miR-455-3p.1 inhibitor+si-PIK3R1 and si-PIK3R1 NC were transfected into ACHN cells,respectively,MTT assay was performed to test the impact of miR-455-3p.1 on proliferation of ccRCC cells by regulating PIK3R1;flow cytometry was performed to test the effect of miR-455-3p.1 on cell cycle and apoptosis of ccRCC cells by regulating PIK3R1;transwell assay was performed to test the effect of miR-455-3p.1 on the ability of migration and invasion of ccRCC cells by regulating PIK3R1.Results: Part one: MiR-455-3p.1was found to have higher expression level in tumor tissue than in adjacent kidney tissue with statistical significance,while expressions of PIK3R1 mRNA was significantly lower in tumor tissue than in adjacent kidney tissue and expression of p85?in most of tumor tissue was lower than in adjacent kidney tissue;there were negative correlations between expression levels of mi R-455-3p.1 and PIK3R1 mRNA and protein product p85? in ccRCC tumor tissue.Part two: Expression levels of miR-455-3p.1 were significantly higher in ccRCC cell lines than in renal tubular cell line HK2,with highest in ACHN cell and lowest in 786-O cell among ccRCC cell lines.786-O cell line were transfected with miR-455-3p.1 mimic for up-regulation and ACHN,miR-455-3p.1 inhibitor for down-regulation.MTT assay showed that up-regulation of miR-455-3p.1 promoted proliferation of 786-O cells while down-regulation of miR-455-3p.1 inhibited proliferation of ACHN cells;flow cytometry revealed that number of G2/M cells was significantly higher than control group in miR-455-3p.1 mimic-transfected 786-O cells while number of G2/M cells in miR-455-3p.1 inhibitor-transfected ACHN was significantly lower than control;number of G1 stage cells in miR-455-3p.1 mimictransfected 786-O cells was lower than control while higher in miR-455-3p.1 inhibitor-transfected ACHN cells than in control;transwell assay showed that overexpression of miR-455-3p.1 promoted migration and invasion of 786-O cells and downregulation of miR-455-3p.1 inhibited migration and invasion of ACHN cells than control.Part three: in miR-455-3p.1 mimic-transfected 786-O cells,the expression of PIK3R1 and PTEN protein were significantly lower than the control group,the expression of AKT and p-AKT protein were significantly higher than the control group,while in miR-455-3p.1 inhibitor-transfected ACHN cells,the expression PIK3R1 and PTEN protein were significantly higher than the control group,AKT and p-AKT protein expression were significantly lower than the control group.In luciferase report assay,relative luciferase activity of PIK3R1 in miR-455-3p.1 mimic-transfected 786-0 cells was significantly lower than in control groups,while luciferase activity in miR-455-3p.1 inhibitor-transfected ACHN cells was significantly higher than in control groups.According to functional rescue experiment,MTT assay showed that cell proliferation in the miR-455-3p.1 mimic+ PIK3R1 group was significantly lower than that of miR-455-3p.1+NC group,while cell proliferation in the miR-455-3p.1 inhibitor+si-PIK3R1 group was significantly higher than that of miR-455-3p.1 inhibitor+si-PIK3R1 NC group;apoptosis of miR-455-3p.1 mimic+ PIK3R1 group was significantly higher than that of miR-455-3p.1 mimic+ PIK3R1 NC group,while apoptosis of miR-455-3p.1 inhibitor+si-PIK3R1 group was significantly lower than that of miR-455-3p.1 inhibitor+si-PIK3R1 NC group;G2/M stage cells count in miR-455-3p.1 mimic+ PIK3R1 group was significantly lower than that in miR-455-3p.1 mimic+ PIK3R1 NC group,while was higher in miR-455-3p.1 inhibitor+si-PIK3R1 than that in miR-455-3p.1 inhibitor+si-PIK3R1 NC group;G1 stage cell count in miR-455-3p.1 mimic+ PIK3R1 group was significantly higher than that in miR-455-3p.1 mimic+ PIK3R1 NC group,while was lower in mi R-455-3p.1 inhibitor+siPIK3R1 than that in miR-455-3p.1 inhibitor+si-PIK3R1 NC group.Cell migration and invasion ability of miR-455-3p.1 mimic+ PIK3R1 group was significantly lower than that of miR-455-3p.1 mimic+ PIK3R1 NC group,while cell migration and invasion ability of mi R-455-3p.1 inhibitor+si-PIK3R1 group was significantly higher than that of miR-455-3p.1 inhibitor+si-PIK3R1 NC group.Conclusion: MiR-455-3p.1 was upregulated in ccRCC tissue while expression of PIK3R1 mRNA and its protein product p85 ? was down-regulated in ccRCC tissue and there were negative correlations between miR-455-3p.1 and PIK3R1 mRNA and its protein product p85?;miR-455-3p.1 can promote proliferation,migration and invasion,inhibit apoptosis and change cell cycle of ccRCC cells in vitro;miR-455-3p.1 inhibits expression of PIK3R1 and PTEN,and promoted expression of AKT and p-AKT;PIK3R1 is target of miR-455-3p.1,and miR-455-3p.1 promotes proliferation,migration and invasion and,inhibits apoptosis and regulates cell cycle of ccRCC cells by targeting PIK3R1.This provides new evidences for identifying new targets for managing of mccRCC. |
PDF Full Text Request