Preliminary Study Of Function And Molecular Mechanism Of Long Non-Coding RNA KUCG1 In Promoting Sunitinib Resistance In Clear Cell Renal Carcinoma

ObjectiveClear cell renal cell carcinoma(ccRCC)is often characterized by congenital or acquired resistance to molecular targeted drugs,which reduces the efficacy.The purpose of this study was to investigate the role of long non-coding RNA KUCG1 in sunitinib resistance in renal carcinoma and its possible molecular mechanism.MethodsCRISPR/Cas9 gene editing technology was used to to down-regulate the expression of 244 lncRNAs in 769-P renal cell carcinoma cells;and sunitinib was used to screen 769-P renal carcinoma cells with sunitinib resistance.In sunitinib-resistance renal cancer cells,244 lncRNAs were detected by quantitative real-time PCR(q PCR).The expression of KUCG1 in 769-P and 786-O renal cell carcinoma(RCC)cells was down-regulated by CRISPR/Cpf1 gene editing technique,and the down-regulation efficiency was detected by q PCR.KUCG1(-)-1 and KUCG1(-)-2 RCC cells with significantly down-regulated KUCG1 expression were obtained.Proliferation,invasion,apoptosis and drug resistance of KUCG1(-)-1 and KUCG1(-)-2 RCC cells were analyzed by CCK-8 assay,invasion assay and flow cytometry in vitro.The expression of PTEN/AKT/mTOR pathway in KUCG1(-)-1and KUCG1(-)-2 RCC cells was analyzed by western blot.The upstream and downstream adjacent genes of KUCG1 were acquired by STRING database;and the differences of KUCG1 adjacent coding genes in KUCG1(-)-1 and KUCG1(-)-2 RCC cells were detected by q PCR.The expression of related genes in KUCG1(-)-1 and KUCG1(-)-2 RCC cells was down-regulated by si RNA,and the influence of this gene on the biological behavior of RCC cells and PTEN/AKT/mTOR pathway was studied.ResultsQPCR data of 244 lncRNAs in senitinib-resistance 769-P RCC cells showed that the expression of KUCG1 was significantly down-regulated and most significantly decreased,suggesting that the decreased expression of KUCG1 was related to drug resistance of renal carcinoma.Compared with the control group,the expression of KUCG1(-)-1 and KUCG1(-)-2 RCC cells was significantly decreased.In vitro results showed that the proliferation,invasion and drug resistance of KUCG1(-)-1 and KUCG1(-)-2 RCC cells were significantly enhanced,and their apoptosis was inhibited.Moreover,compared with control cells,KUCG1(-)-1 and KUCG2(-)-1RCC cells showed no significant morphological changes,including loss of cell polarity,cell membrane contraction,and cytoplasmic vacuolization,48 h after sunitinib treatment.The results of western blot showed that down-regulation of KUCG1 could inhibit PTEN expression and activate AKT/mTOR pathway.By detecting the encoding genes in the upstream and downstream vicinity of KUCG1,we found that the expression of MAGEA11 in KUCG1(-)-1 and KUCG1(-)-2 renal cancer cells was significantly up-regulated compared with the control group.The expression of MAGEA11 in KUCG1(-)-1 and KUCG1(-)-2 RCC cells was down-regulated by Si RNA.Western blot showed that PTEN expression was increased and AKT/mTOR pathway was decreased.In addition,down-regulation of MAGEA11 inhibited KUCG1 in promoting proliferation,invasion and anti-apoptosis of RCC cells.ConclusionsThe activation of AKT signaling pathway after down-regulation of KUCG1 expression induces sunitinib resistance in renal clear cell carcinoma.MAGEA11 is the downstream regulatory site of KUCG1 and plays an important role in the activation of AKT signaling pathway.KUCG1 is expected to be a promising prognostic marker and potential therapeutic target for ccRCC molecular targeted drug resistance.