| Objective: To further investigate the effect and mechanism of sorafenib resistance induced by LncRNA HCP5 in renal cell carcinoma(RCC)based on previous studies.Method:(1)The study was based on established sorafenib resistant RCC cells,which are 769-P and786-O renal clear cell cancer lines with stable over-expression of HCP5.We then identified the localization of HCP5 by means of fluorescence in situ hybridization(FISH).Flow cytometry was used to analyze the proportion of early apoptosis and cell cycle of RCC cells.Expressions of apoptotic protein Cleaved Caspase3 and EMT-process associated proteins were detected by Western Blotting.(2)On the previous studies,we found that HCP5 bound to miR-106b-5p.Potential downstream target genes of HCP5/miR-106b-5p including INHBA,FAM45 A,NT5E,TMCC3 and LDLR were screened out.Based on that,real-time quantitative PCR(q RT-PCR)was used to detect their levels,and si RNA was used to knock down the expression of TMCC3,q RT-PCR was then used to determinate the transfection efficiency.Flow cytometry was used to analyze the changes of early apoptosis ratio of RCC cells after TMCC3 knockout.Western Blotting was used to detect the expression level of target gene TMCC3 and the expression differences of PTEN/AKT/m TOR pathway,apoptotic protein Cleaved Caspase3 and EMT process related proteins after TMCC3 knockdown.The resistance of RCC cells to sorafenib after TMCC3 knockdown was also detected by CCK-8 assay.(3)The tumor volume and weight of subcutaneous tumor in nude mice were measured to verify the tumor formation of HCP5.The expression levels of HCP5 and TMCC3 were detected by q RT-PCR.The proliferative capacity of subcutaneous tumors was analyzed by immunohistochemical(IHC).Results:(1)Probes were used to detect the 769-P and 786-O renal cancer cells with overexpressed HCP5 by FISH,and it was showed that LncRNA HCP5 was mostly localized in the cytoplasm.Therefore,HCP5 basically plays biological functions in the cytoplasm.Flow cytometry assay idicated that the proportion of early apoptosis of RCC cells with overexpressed HCP5 was lower.In addition,Western Blotting showed that the apoptotic protein Cleaved Caspase3 was significantly lower in the experimental group than in the control group.Both of results indicated that overexpression of HCP5 inhibited apoptosis of RCC cells.Moreover,flow cytometry was also used to identify the changes of cell cycle.Results showed that the proportion of cells stuck in G1 phase decreased after over-expression of HCP5,while the proportion of S phase +G2 phase increased,which promoted the proliferation of RCC.It was convered that the epithelial biomarkers E-cadherin protein expression with overexpressed HCP5 in the experimental group was significantly decreased compared with that in the control group.At the same time,the expression levels of the mesenchymal biomarkers N-cadherin and Vimentin protein were significantly increased.These results indicated that the epithelial-mesenchymal transition(EMT)process could be promoted.(2)In order to verify the downstream target genes of HCP5/miR-106b-5p,q RT-PCR was used to detect the expressions of five previously screened m RNAs in RCC cells with overexpressed HCP5.Among them,expression of TMCC3 gene was the highest.Moreover,the expression of TMCC3 protein was also up-regulated,so TMCC3 was selected as the downstream m RNA target gene for subsequent experimental analysis.Then using three si RNA knockdowns on TMCC3 expression,the expressions of TMCC3 was decreased by q RT-PCR.Subsequently,si RNA was used to down-regulate TMCC3 expression to verify its effect on drug resistance.Western Blotting showed that the PTEN/AKT/m TOR pathway activated by HCP5 was effectively inhibited by si RNA-TMCC3.Flow cytometry showed that knockdown of TMCC3 reversed the early apoptosis induced by HCP5,and the proportion of early apoptosis increased significantly.Western Blotting also showed elevated level of Cleaved Caspase3.Down-regulation of TMCC3 reversed the expression of EMT related proteins induced by HCP5.CCK-8 results showed increased sensitivity of RCC cells to sorafenib after TMCC3 knockout.Therefore,we speculate that TMCC3 as a target gene can reverse the HCP5-induced resistance of RCC cells to sorafenib.(3)Subcutaneous tumor formation in nude mice suggested that overexpression of HCP5 promoted tumor growth,and the expression of TMCC3 was significantly increased in tumor tissues overexpression of HCP5 by q RT-PCR.Western Blotting showed that overexpression of HCP5 could activate the PTEN/AKT/m TOR pathway in nude mice.IHC results showed increased Ki-67 expression,indicating that HCP5 can promote tumor proliferation.Conclusion: Elevated HCP5 can lead to the resistance of RCC cells to sorafenib.The downstream target gene of HCP5/miR-106b-5p axis is TMCC3.Knockdown of TMCC3 can reverse HCP5-induced sorafenib resistance through inhibition of PTEN/AKT/m TOR signaling pathway.Overexpression of HCP5 promoted the progression of renal carcinoma subcutaneous tumor formation in nude mice. |
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