The Role And Mechanism Of LncRNA HCP5 In Renal Cell Carcinoma Cells With Sorafenib Resistance

Objective: To study the role of long non-coding RNA HCP5 and its potential mechanism in renal cell carcinoma(RCC)cells with sorafenib resistance.Methods: The gradient concentration method was used to detect the minimum killing concentration of sorafenib on RCC cells.Gene mutagenesis screening method was performed to construct a sorafenib-resistant RCC cell model.The differentially expressed Lnc RNAs(DELs)were detected and screened by RT-q PCR method.After lentiviral transfection,the expression of Lnc RNA HCP5,the most significant DEL,was detected and verified by RT-q PCR.CCK-8 assay was used to assess the tolerance of RCC cells with HCP5 over-expression to sorafenib.Clony formation was carried out to analyze the viability of RCC cells,while transwell assay was carried out to analyze cell invasion.The expression levels of PTEN/AKT/m TOR pathway related proteins were measured by Western Blotting.The online database was used to predict the mi RNA targets of HCP5.And the expressions of mi RNA targets were detected by RT-q PCR and dual luciferase reporter assay was used to verify the interaction of mi RNA targets and HCP5.Finally,bioinformatics method was applied to analyze the potential downstream target genes of mi RNA.Results: The results of CCK-8 assay and microscope observation of cell morphology and death showed that 30 μmol/L sorafenib could cause obvious morphological changes and death of more than 95% of the cells.Therefore,we chose 30 μmol/L as the minimum killing concentration of sorafenib on RCC cells.Subsequently,we successfully constructed sorafenib-resistant RCC cells(SR-769-P cells)with this concentration.RT-q PCR results showed that the level of Lnc RNA HCP5 in SR-769-P cells was obviously higher than that in the control group,and over-expression of HCP5 could increase the tolerance of RCC 769-P and 786-O cells to sorafenib.In addition,the results of cell clone formation and invasion assays showed that HCP5over-expression could promote the proliferation and invasion of RCC 769-P and786-O cells.Western blotting results showed that the expression of PTEN protein in drug-resistant cells was significantly lower than that in control cells and the expression of p AKT protein was significantly higher than that in control cells,while HCP5 over-expression could inhibit the expression of PTEN protein in RCC cells and promote the expressions of p AKT and m TOR proteins.Mi R-106b-5p was one of the predicted mi RNA targets of HCP5 and it was significantly decreased in RCC cells with sorafenib resistance and HCP5 overexpression when compared with the control group.The dual luciferase reporter result showed that HCP5 and mi R-106b-5p had a targeted regulatory relationship.Further bioinformatics analysis showed that INHBA,FAM45 A,NT5E,TMCC3 and LDLR were potential target genes of mi R-106b-5p in RCC cells with sorafenib resistance.Conclusions: HCP5 contributed to sorafenib resistance in RCC cells by regulating mi R-106b-5p and activating PTEN/AKT/m TOR signaling pathway.The target genes of mi R-106b-5p need further clarification.