Screening Of MicroRNA Expression Profiles During Cisplatin-induced Apoptosis Of Human Renal Tubular Cells And The Effect Of HIPK2 On Apoptosis

Part I The establishment of cisplatin induced human renal tubular epithelial cell(HKC cells)apoptosis modelObjectiveTo establish the cisplatin-induced HKC cells apoptosis model and select the appropriate cisplatin concentration for the next step.MethodsThe culture medium concentration of 2,10 and 50μM cisplatin are builded by adding cisplatin to the logarithmic growth phase of HKC cell,while the control group is in the same conditions without cisplatin.Then the viability and apoptosis rate of HKC cells are detected.ResultsThe survival rate of HKC cells decreased with the increase of cisplatin concentration,and the apoptosis rate increased with the increase of cisplatin concentration.ConclusionThe cisplatin-induced HKC cell apoptosis model was successfully established and 10μM cisplatin was determined as the concentration required for the next experiment.Part II The expression of micoRNA in HKC cell apoptosis induced by cisplatin and its bioinformatics analysisObjectiveTo investigate the differential expression profile of microRNA and differential profile of mRNA induced by cisplatin-induced apoptosis in HKC cells.To screen the target genes and contract the miRNAs-GO-network and miRNAs-gene-network by bioinformatics.Then ensure the microRNAs that play important roles in cisplatin-induced apoptosis of HKC cells.MethodsThe differentially expressed microRNAs were detected by Affymetrix Gene Chip miRNA microarray.The differentially expressed mRNA was detected by AffymetrixU133 gene expression chip.The target gene databases of microRNA select the differential microRNA target gene.Further,the results combined with the gene expression chip results determine the microRNA target gene.Bioinformatics analysis suggests that what biological functions and mechanisms the differentially expressed microRNAs may have.Constructing Pathway-network,miRNAs-GO-network and miRNAs-Gene-network are through the bioinformatics method.miRNAs that play an important role in cisplatin-induced apoptosis in HKC cells are ensured Based on the above network.ResultsDifferential expression of miRNAs cluster analysis showed that the samples in each group were well consistent.Compared with the control group,the expression of microRNA in the experimental group was significantly different,and the microRNAs were screened out.3831 differentially expressed genes are screened by Affymetrix U133 gene chip.A total of 4760 target genes were screened out using the microRNA target gene database.1181 target genes were obtained by taking the intersection of this result and the result of gene chip,such as ARHGEF4 and BNC2.The miRNA-network and miRNAs-gene-network were further analyzed by bioinformatics,and further analysis shows the possible microRNAs that may play an important role in cisplatin-induced HKC cell apoptosis,t such as miR-9-3p and miR-371b-5p.ConclusionThe expression of microRNA in HKC cells induced by cisplatin was significantly different from that of the control group.MiR-9-3p,miR-371b-5p may play a role in cisplatin-induced apoptosis of HKC cells.Part III Effects of HIPK2 on cisplatin-induced apoptosis of HKCObjectiveTo investigate the effect of HIPK2 on apoptosis induced by cisplatin in HKC cells.MethodThe expression of HIPK2 in HKC cell apoptosis induced by different concentrations of cisplatin was detected by real-time fluorescence quantitative PCR and Western blot.Secondly,HIPK2 siRNA was designed and synthesized,and the cells were transfected into HKC cells by Lipofectamine.The expression of HIPK2 mRNA and protein was detected by real-time fluorescence quantitative PCR and Western blot respectively.The cells were treated with cisplatin Apoptosis was detected by Annexin V/PI.Finally,Western blot was used to detect the effect of HIPK2 on the expression of apoptosis-related protein bax.ResultsThe expression of HIPK2 mRNA and protein in HKC cells induced by cisplatin was significantly decreased(p<0.05),and the expression of HIPK2 decreased with the increase of cisplatin concentration.Transfection of small interfering RNA can significantly reduce the expression of HIPK2 mRNA and protein in HKC cells,and the decrease of HIPK2 expression will promote cisplatin-induced apoptosis of HKC cells.ConclusionHIPK2 inhibits cisplatin-induced apoptosis in HKC cells.