Rev-erb Agonist SR9009 Inhibits The Proliferation Of Human Colon Cancer Cell HCT116 Through Reducing Autophagy

Colon cancer is one of the malignancies with high morbidity and mortality worldwide.In 2018,the global cancer statistics report reported about 1.8 million new cases of colorectal cancer,with a mortality rate of nearly 50%.Some studies believe that one of the reasons for the high death rate of colon cancer is the protective autophagy of cancer cells,that is,the rapid proliferation of cancer cells consume a large number of nutrients,leading to some of the cancer cells in a nutrient-deficient microenvironment.If this autophagy can be effectively inhibited,the proliferative activity of colon cancer cells can be restricted.However,currently,the means of clinical inhibition of autophagy are limited,so it is necessary to find a safe and effective program.Literatures have shown that metabolic activities,including autophagy,are regulated by the body's own biological clock.Among various clock genes,the rev-erb(Reverse erythroblastosis)gene,which regulates metabolic circadian rhythm,is closely related to autophagy of cancer cells.Rev-erb is a negative regulator of autophagy that inhibits autophagy.SR9009 is a small molecule agonist of rev-erb,which can improve the expression of rev-erb.Therefore,inhibition of autophagy by rev-erb may be a potential therapeutic strategy for colon cancer.In this study,the HCT116 human colon cancer cell line was used as the research object,and rev-erb agonist SR9009 was added to observe the cell proliferation with an inverted phase contrast microscope.Cck-8 kit was used to detect cell activity,and the appropriate concentration and action time of SR9009 were selected.The transcription changes of rev-erb promoter,rev-erb promoter and autophagy genes Beclin1,LC3 and p62 were detected by real-time PCR.Western blot was used to detect the protein expression changes of rev-erb,rev-erb,Beclin1,LC3 and p62.The results showed that rev-erb agonist SR9009 significantly inhibited the proliferation of HCT116 cells compared with the control group.The effective concentration of SR9009 was 20 ?mol/L and the duration of treatment was 24 hours.SR9009 significantly increased the expression of rev-erb promoter in HCT116 cells,and decreased the expression of autophagy genes Beclin1 and LC3 in HCT116 cells,causing the accumulation ofautophagy substrate p62.On the basis of SR9009 treatment,autophagy was inhibited with 3-methyl adenine,and the activity of HCT116 cells was decreased.Rapamycin was used to enhance autophagy and the viability of HCT116 cells was raised.Therefore,the small-molecule agonist SR9009 can activate the expression of the clock gene rev-erb and inhibit the proliferation of colon cancer cells by reducing autophagy.This provides a theoretical basis for exploring a chronobiological protocol for the treatment of colon cancer.Part I Rev-erb agonist SR9009 inhibits proliferation of HCT116 cellsObjective:To investigate the effect of Rev-erb agonist SR9009 on the proliferation,migration and activity of HCT116 colon cancer cells.Methods:(1)Microscopic observation: HCT116 colon cancer cells were cultured,a Rev-erb agonist SR9009 was added to the culture medium,and the cell proliferation was observed using an inverted phase contrast microscope.(2)CCK-8 method: CCK-8 kit was used to detect cell viability,and SR9009 was selected for the appropriate concentration and time.(3)Real-time PCR: After SR9009 treated HCT116 cells,the transcriptional changes of Rev-erb? and Rev-erb? were detected.(4)Western blot: After SR9009 treated HCT116 cells,the protein expressions of Rev-erb? and Rev-erb? were detected.Results:(1)SR9009 inhibits the proliferation and migration of HCT116 cells: Compared with the control group,Rev-erb agonist SR9009 significantly inhibits the proliferation of HCT116 cells.(2)Dose and duration of SR9009 inhibition of HCT116 cell proliferation: The effective concentration of SR9009 is 20 ?mol / L,and the duration of action is 24 h.(3)SR9009 increased Rev-erb expression in HCT116 cells: After the cells were treated with SR9009,the expressions of Rev-erb? and Rev-erb? in HCT116 cells were significantly increased by Real-time PCR and Western blot detection.Conclusion:Rev-erb agonist SR9009 inhibits the proliferation and migration of HCT116 cells.Part II SR9009 reduces autophagy in HCT116 cellsObjective:To investigate the effect of Rev-erb agonist SR9009 on the autophagy genes of HCT116 colon cancer cells.Methods:(1)Real-time PCR: After SR9009 treated HCT116 cells,the transcriptional changes of Beclin1,LC3,and p62 were detected.(2)Western blot: After SR9009 treatment of HCT116 cells,the protein expression changes of Beclin1,LC3,and p62 were detected.Results:(1)SR9009 reduces the transcription of autophagy-related genes Beclin1 and LC3 in HCT116 cells.(2)SR9009 decreased the expression of Beclin1 and LC3 in HCT116 cells,and increased the autophagy substrate P62.Conclusion:SR9009 reduces autophagy in HCT116 cells.Part III SR9009 inhibits HCT116 cell proliferation by reducing autophagyObjective:To investigate whether Rev-erb agonist SR9009 can inhibit the proliferation of HCT116 colon cancer cells by regulating autophagy.Methods:(1)Autophagy tool drugs: 3-MA inhibits autophagy,and rapamycin promotes autophagy.(2)CCK-8 method: CCK-8 kit is used to detect cell viability,and SR9009 is selected for the appropriate concentration and time.Results:(1)3-MA promotes the inhibition of HCT116 cell activity by SR9009.(2)rapamycin reversed the inhibition of HCT116 cell activity by SR9009.Conclusion:SR9009 inhibits the proliferation of HCT116 colon cancer cells by reducing autophagy.