Study On The Expression Of Autophagymarkers ATG5 And LC3 In Human Renal Carcinoma Tissue And Their Effecton Durg Resistance To Renal Carcinoma Cells

Renal cancer accounts for about 2% to 3% of adult malignancies and 80% to 90% of adult kidney cancer.It has ranked No.10 in the incidence of male malignancy by 2008 in China.In recent years,the morbidity of renal cancer has been increasing year by year,about 10 million people died of renal cancer in the world every year.Renal cancer is characterizedashigher distribution in urban areas than that in the rural areas,and malesare moresensitivity to cancer than females.Patients can be found in all ages,but those of 50 to 70 years old have higher incidence.Because of its concealedsymptoms in early s tage,there is no specific early diagnosis method.As the current rate of early detection of renal cancer is still low,most patients are diagnosed until late stage of cancer in China.Although the understanding of renal cancer has made great progress with more and more means of diagnosis and treatment,postoperative survival rate is still not ideal because of the development mechanism of renal cell carcinoma has notbeen clearly known.Besides,the diagnosis of renal cell carcinoma needs to be confirmed by laboratory assays,imaging and pathological examination.The purpose of laboratory assays is to evaluate the general condition,liver and kidney function and prognosis of patients.At present,there is no common tumor markers for clinical diagnosis of renal cell carcinoma.The clinical diagnosis of renal cell carcinoma mainly relys on imaging examination,however,only more than 5mm diameter of renal cell carcinoma can be found by imaging diagnosis due to the requirement of pathology.Therefore,exploring the specific molecular mechanisms of renal cell carcinoma is of great significance to the diagnosis and treatment of renal cell carcinoma.Autophagy is an important process in the eukaryotic cell organismduringthe evolution,especially during the cellular environment alteration.At present,autophagy is divided into macrophages（MA）,chaperone-mediated autophagy（CMA）and microautophagy.Autophagy indicates the process of bilayer membrane structure of the cytoplasm of soluble proteins and organelles encapsulating to form autophagic bubbles,lysosomal fusion degradation of proteins and organelles.It generally takes 4-6 hours to induce autophagy to reach the peak by serum starvation in vitro.Microautophagy means the lysosome directly phagocytoses soluble protein to degradation.Recent studies have found that autophagy can metabolize its own proteins and organelles to escape external stress in order to maintain cell homeostasis and promote cell survival.Recently evidence shows that tumor cells often enhance its resistance to anticancer drugs byregulation of autophagy,thus escaping from the killing effect of tumor drugs.The expression level ofautophagy-related gene 5（ATG5）,a key gene of autophagy,was significantly correlated with autophagy level.Microtubule-associated protein 1 light chain 3（LC3）,currently takenas an autophagic marker gene,is located in the pre-autophagic bubble and autophagic bubble membrane surface and involved in the formation of autophagosomes.At present,autophagy detecting methods include detecting autophagosome formation viaelectron microscopy and detecting LC3 via western blot.Some studies have shown that ATG5 expression in cancer tissue was significantly higher than that in the adjacent tissue.H₂O₂ stimulation assay also showed that renal cell growth was inhibited after ATG5 knockdown.These results suggest that autophagy may play an important role in the development of renal cell carcinomaSo far,the relationship between autophagy and renal cell carcinoma,especially its effect on drug resistance has not beenstudied.This study aimed at studying the role of autophagy in the proliferation and drug resistance of renal cell carcinoma and its molecular mechanism by using immunohistochemical staining,lentivirus infection gene silencing,Western Blot,MTT,flow cytometry and other experimental methods and techniques at the molecular and cellular levels,Main research contents and results:First,ATG5 and LC3 were found to be highly expressed in renal clear cell carcinoma and were correlated with classification and prognosisImmunohistochemistry was used to analyze 99 cases of renal cell carcinoma and 17 cases of adjacent tissues.The results showed that the expressions of ATG5 and LC3 in renal clear cell carcinoma were significantly higher than those in adjacent tissues.The statistical analysis of ATG5 and LC3 showed that there was no significant difference in age,se x and tumor location between ATG5 and LC3,but it was closely related with the degree of malignancy and TNM staging.Following the increase of pathology grade and TNM clinical stage,the expressions of ATG5 and LC3 increased in turn.Moreover,high ATG5-expression patients showedlower survival rate and poor prognosis.Second,autophagy promoted the proliferation of renal cell carcinoma cell line A-498 in the cell model1.Renal cell carcinoma cell line A-498 of ATG5 low expression was successfully constructed by lentivirus-mediated ATG5 shRNA expression vector and confirmed by Western Blot.The proliferation rate of A-498 cells low expression group and normal group was detected by flow cytometry.The 7-day MTT proliferation curve showed that knockdown of ATG5 in A-498 renal clear cell carcinoma could significantly reduce the proliferation of renal cell carcinoma cells.H₂O₂ stimulation assay showed that downregulation of ATG5 expression in A-498 renal cell carcinoma cell line could significantly reduce the survival rate of renal cell carcinoma under H₂O₂ stress.Third,inhibition of autophagy can reduce the resistance of renal cancer cells to sunitinibMTT assay showed that ATG5 knockdown increased sensitivity to sunitinib drugs in A-498 kidney cancer cells.Our study shows that autophagy could promote the proliferation of renal cell carcinoma,and its molecular mechanism is related with the inhibition of ATG5-mediated autophagy signal pathway.This study provides a new link among autophagy with drug resistance,proliferation and treatment of renal cell carcinoma,and provides new possible targets for the clinical diagnosis and treatment of renal cancer.