The Studies On The Mechanisms Of RIP3-regulated Autophagy And Its Relaptionship With Human Colon Cancer

Colorectal cancer(CRC)is one of the most common malignant tumors of digestive tract in clinic.In recent years,with the improvement of people's living standard and the change of eating habits,the incidence of colorectal cancer is increasing obviously,which is a serious threat to human health.Although the traditional mode of treatment was improved greatly,the treatment of advanced colon cancer was still unsatisfactory,and the overall survival rate was not significantly improved.Therefore,it is of great significance to find effective other therapies and even replace the surgical treatment to reduce the recurrence rate and mortality of colorectal cancer,to improve the cure rate and the quality of life of patients with colorectal cancer.Autophagy is a cellular degradation pathway that transports damaged,deformed,senescent or dysfunctional proteins and organelles to lysosomes for digestion and degradation.This process is necessary for cell homeostasis and a protective mechanism against pathogens.The relationship between autophagy and tumor is very complicated.It is thought that autophagy plays an inhibitory role in tumorigenesis,but in the process of tumor development,autophagy may provide the mechanism of anti-nutritional deficiency and hypoxia for tumor cells to promote the survival of tumor cells.Autophagy is the mechanism by which many tumors resist chemotherapeutic agents,but in some cases,autophagy plays a role in promoting cell death in cancer cells mediated by anti-cancer drugs.Promoting autophagic death is an effective method for the treatment of some tumors.With the deepening of autophagy research,autophagy has become a new cancer treatment target.We transfected RIP3 shRNA interference plasmid and overexpression plasmid into human colon cancer cell HT29,and induced autophagy with no glucose medium:It was found that the autophagy decreased after knocking down RIP3,but increased after over-expression of RIP3.We found for the first time that RIP3 promotes autophagy in human colon cancer HT29 cells.Then we further study the possible molecular mechanism of RIP3 regulating autophagy in human colon cancer cells.First,our Western Blotting results showed that glucose starvation induced autophagy of HT29 cells did not depend on the classical mTOR signaling pathway.We found that RIP3 interacts with G protein GNAI3 and G protein regulatory factor RGS19,which may be involved in autophagy of colon cancer,was further verified by co-immunoprecipitation method.We speculate that RIP3 may mediate autophagy of colon cancer cells through interacting with GNAI3 and RGS19.Although the expression of GNAI3 and RGS19 did not change after low or over expression of RIP3,we found that down-regulation of GNAI3 or RGS 19 expression inhibited autophagy of HT29 cells.Furthermore,the GTPase enzyme activity of GNAI3 can promote autophagy of HT29 cells.RIP3 is a serine/threonine kinase.We speculate that RIP3 may affect autophagy through its kinase activity,and we complement the RIP3 expression plasmid with kinase activity point mutation into the RIP3 knockdown cell lines.After inducing autophagy,it was found that the kinase activity of RIP3 could promote autophagy of HT29 cells,and its active center of kinase was K50.In vitro kinase activity analysis showed that RIP3 kinase could phosphorylate GNAI3 and RGS 19,respectively.Therefore,we speculate that RIP3 kinase may affect glucose starvation inducing autophagy of HT29 cells through phosphorylation of GNAI3 and RGS 19.We have screened some possible phosphorylation sites by co-immunoprecipitation and mass spectrometry.The sites at which GNAI3 may be phosphorylated are:S6,S164;the sites at which RGS 19 may be phosphorylated are:T12,S199,S213,S214.Besides,in order to search for the possible upstream and downstream proteins of RIP3 in HT29 cells autophagy induced by glucose starvation,two proteins,ACAA1 and DDX24 were screened by co-immunoprecipitation mass spectrometry except for GNAI3 and RGS 19.It has been reported that ACAA1 and DDX24 are closely related to tumor development,but there are no reports on whether they are involved in autophagy.This remains to be further studied.In order to investigate the effect of RIP3-regulated autophogy on the development and treatment of human colon cancer,we used cell growth curve,colony forming assay,MTT to detect drug resistance and the effect of RIP3-regulated autophagy on human colon cancer cells in nude mice.Our results showed that,after knocking down RIP3,HT29 cells grew faster,colony forming ability increased,cell proliferation inhibition rate decreased after inducing autophagy,and tumor volume increased in nude mice,but the growth of HT29 cells slowed down,the colony forming ability was weakened,and the volume of subcutaneous transplanted tumor was decreased in nude mice after knocking down RIP3.The inhibition rate of proliferation in low RIP3 of HT29 cells induced by no glucose medium was decreased,which indicated that the low expression of RIP3 could increase the survival rate of HT29 cells by decreasing the level of autophagy,while the inhibition rate of proliferation in low RIP3 of HT29 cells increased after adding the chemotherapeutic drug 5-FU.The results suggest that the low expression of RIP3 may increase the sensitivity of HT29 to the chemotherapy drug 5-FU by reducing the level of autophagy.We believe that the resistance of HT29 cells to 5-FU chemotherapeutic agents can be alleviated by reducing autophagy levels.The results in vivo and in vitro showed that RIP3 could inhibit the growth of HT29 by promoting autophagy,suggesting that RIP3 regulated autophagy was involved in the genesis and development of colon cancer.The results may provide potential application value for further understanding the pathogenesis of colon cancer and the clinical treatment of colon cancer.