Long Non-coding RNA TUG1 Regulates Autophagy And Affects The Sensitivity Of Renal Cell Carcinoma To Sunitinib

Background and Objective:Kidney cancer is one of the common malignant tumors in the urinary system,the incidence is increasing year by year worldwide.Targeted therapy is the most commonly treatment for patients with renal cancer who have lost the opportunity to surgery.Resistance to targeted therapy usually leads to poor clinical benefit for patients.Sunitinib is the first-line treatment option for patients with advanced kidney cancer.However,approximately 10% of patients with advanced kidney cancer are not sensitive to sunitinib treatment at the time of initial treatment,while the rest are acquired the drug resistance after 6-15 months with Sunitinib.Thus,the targeted therapy of sunitinib unable to effectively prolong the survival time of patients with kidney cancer.Therefore,it is urgent and necessary to explore the biological mechanism of sunitinib resistance and to find molecular targets that enhance the sensitivity of sunitinib treatment in renal cell carcinoma.Non-coding RNA plays an important role in the occurrence and development of tumors,and the latest research shows that it also plays an important role in tumor drug resistance.TUG1 is a member of long-chain non-coding RNA,and its expression is significantly up-regulated in patient kidney cancer specimens and kidney cancer cell lines.Inhibiting the expression of TUG1 can effectively inhibit the proliferation and migration of kidney cancer cells.Studies have shown that TUG1 plays an important role in the chemotherapy resistance of breast cancer and lung cancer.However,there are few studies on the role and mechanism of TUG1 in sunitinib resistance in renal cell carcinoma.Autophagy plays an important role in sunitinib resistance in renal cancer.When tumor cells were stimulated by chemotherapeutic drugs,the autophagy signaling pathway is activated and the level of autophagy is increased,which means the drug molecules were decomposed and the sensitivity was decreased.The proliferation of renal cancer cell was increased.Autophagy-related gene HMGB1 is highly expressed in renal cancer tissues,and is related to the chemotherapy sensitivity of a variety of tumors,and related literature reported that HMGB1 is related to the resistance of targeted therapy for renal cancer which was possibly regulated by TUG1.The purpose of this study was to investigate the effect and mechanism of TUG1 on the sensitivity of Sunitinib to the treatment of renal cell carcinoma by regulating autophagy through HMGB1.Method:1.Fluorescence quantitative PCR was used to analyze the expression of TUG1 in kidney cancer tissues and cells.2.Customize sh-NC/sh-TUG1 lentivirus,construct renal cancer sh-NC/ACHN,sh-TUG1/ACHN and sh-NC/786-0,sh-TUG1/786-0 stably transfected cell lines.The IC50 of sunitinib in renal cancer cell lines ACHN and 786-0 was determined by cytotoxicity experiment.The CCK8 experiment was used to observe the proliferation ability of sh-NC/ACHN and sh-TUG1/ACHN cells under the intervention of sunitinib,then flow cytometry was used to detect the changes in the proportion of apoptosis,and Western blot was used to examine the expression of apoptosis proteins,to clarify the effect of down-regulation of TUG1 on the sensitivity of sunitinib for renal cell carcinoma.3.Western blot,cell immunofluorescence,and autophagy electron microscopy were used to detect the effect of down-regulating TUG1 on the autophagy level of renal cancer cells.The autophagy activator rapamycin was administered to down-regulated TUG1 renal cancer cells,and the effects on sunitinib sensitivity and apoptosis were verified through CCK8,flow cytometry,and Western blot.4.Fluorescence quantitative PCR and Western blot were used to detect the effect of down-regulation of TUG1 on the expression of HMGB1.In down-regulated TUG1 renal cancer cells,the expression of HMGB1 was increased,and the effects on sunitinib sensitivity and apoptosis were observed by CCK8,flow cytometry,and Western blot detection.Result:1.The results of fluorescence quantitative PCR confirmed that the expression level of lncRNA-TUG1 in kidney cancer tissues and kidney cancer cell lines was significantly increased(p <0.01).2.After down-regulating the expression of TUG1 in ACHN and 786-0 cells,given sunitinib for 48 hours,the CCK8 showed that its growth ability was weakened(p <0.01).The proportion of apoptosis was significantly increased(p <0.05).Western blot showed that the expression levels of apoptosis protein Bax and Caspase-3 protein were increased(p<0.05),and the expression level of apoptosis suppressor protein Bcl-2 was decreased(p<0.05).The results showed that down-regulation of TUG1 enhanced the sensitivity of kidney cancer cells to sunitinib.3.Western blot,cell immunofluorescence,and autophagy electron microscopy confirmed that down-regulating the expression of TUG1 in ACHN and 786-0 cells reduced the level of autophagy in renal cell carcinoma.The enhancement of autophagy by rapamycin can reverse the sensitivity of sunitinib to renal cancer treatment enhanced by the down-regulation of TUG1.4.After down-regulating the expression of TUG1 in ACHN and 786-0 cells,fluorescence quantitative PCR and Western blot results showed that the expression of HMGB1 was decreased.The overexpression of HMGB1 can enhance the level of autophagy and at the same time reverse the sensitivity of sunitinib to renal cancer treatment enhanced by the down-regulation of TUG1.Conclusion:lncRNA-TUG1 affects the treatment sensitivity of sunitinib in renal cell carcinoma through the regulation of autophagy by HMGB1.Our research results provided a theoretical basis for reversing the drug resistance of sunitinib in renal cell carcinoma,and explored the solutions for the targeted therapy of renal cell carcinoma.