The Mechanism Of Resveratrol In Treating Colorectal Cancer By Regulating Ferroptosis Via Down-regulating MiR-31

Objective:1.To study whether the mechanism of resveratrol in the treatment of colorectal cancer is related to ferroptosis.2.To study the role of miR-31 in resveratrol regulating ferroptosis.Methods:In vivo experiment: AOM+DSS induced mouse colon cancer model.28 male C57BL/6mice were adaptively fed for one week,during which the mice ate ordinary feed.The mice were randomly divided into four groups(n=7),Control group,AOM group,AOM+DSS group and AOM+DSS+Res group.The modeling cycle was 70 days.After modeling,each group was fed with high-speed iron feed.On the first day of modeling,AOM group,AOM+DSS group and AOM+DSS+Res group were intraperitoneally injected with AOM with a concentration of 10mg/kg.In the first week,all groups drank sterilized water.In the second week,AOM+DSS group and AOM+DSS+Res group drank 1% dextran sodium sulfate(DSS)water,and the other two groups still drank sterilized purified water.In the second week,AOM+DSS group and AOM+DSS+Res group drank 1% dextran sodium sulfate(DSS)water,and the other two groups still drank sterilized purified water.In the third week,AOM+DSS group and AOM+DSS+Res group drank sterilized water,and in the fourth week,drank water containing 1% DSS,and so on until the end of modeling.Other groups drank sterilized water throughout the modeling cycle.From the first week of modeling,AOM+DSS+Res group was given 0.1ml 10mg/ml Resveratrol by gavage.Resveratrol was given every day during the induction of colon cancer model.After modeling,the mice were killed,the colon specimens were taken and fixed,and the histological and pathological structure of the colon were observed by hematoxylin eosin(HE)staining;qRT-PCR was used to detect the expression of ACSL4,FTH,GPX4 and SLC7A11 genes in mouse colon;The expression levels of ACSL4,FTH,GPX4,xCT and p53 proteins in mouse colon were detected by immunohistochemistry;The protein expression levels of ACSL4,FTH,GPX4,xCT and p53 in mouse colon were detected by Western blot.In vitro experiments: resveratrol induces ferroptosis in HCT 116 cells,HCT 116 cells were divided into four resveratrol concentration gradient groups of Control/R4/R8/R16.After dosing 48 h,CCK-8 was used to detect cell proliferation activity;intracellular Fe content,MDA content,SOD content and GSH content were detected.The expression levels of ACSL4,FTH,GPX4 and xCT were detected by Western blot.The experiment of knockdown and overexpression of miR-31: HCT 116 cells were divided into control group,miR-31 inhibitor group,miR-31 inhibitor+Res group,miR-31 mimic group,miR-31mimic+Res group,and miR-31 inhibitor and miR-31 inhibitor+Res groups were transfected with miR-31 inhibitor,miR-31 mimic group and miR-31 mimic+Res group.Resveratrol was added to miR-31 inhibitor+Res group and miR-31 mimic+Res group 24 hours after transfection,and the same amount of DMSO was added to miR-31 inhibitor group and miR-31 mimic group.The cells were collected 48 hours after administration,and the expressions of ACSL4,FTH,GPX4 and xCT proteins were detected by Western blot.Results:1.The results of HE staining of mouse colon showed that the Control group had thin intestinal wall,uniform thickness,normal epithelial structure,normal recess structure,orderly arrangement of glands and no lesions.In AOM group,there were inflammatory cell infiltration in mucosal layer,destruction of gland structure and incomplete recess structure,but the glands were arranged regularly without atypia.In the AOM+DSS group,the intestinal wall was significantly thickened,the cancer cells were arranged in disorder,no glandular structure was formed,vacuoles were seen in the center,and the mucosa and epithelium were severely damaged.Compared with the AOM+DSS group,the AOM+DSS+Res group had thinner intestinal wall,more regular gland arrangement,and less mucosal and epithelial damage.2.Western Blot detected the protein expressions of xCT,GPX4,ACSL4,FT,and P53 in colon tissue of mice.Compared with the Control group,the AOM+DSS group had xCT(P<0.0001),FTH(P<0.05),GPX4(P<0.01)The protein expression increased,and the protein expression of P53(P<0.01)and ACSL4(P<0.05)decreased.In AOM group,the expressions of FTH protein(P<0.05)and GPX4(P<0.05)were decreased,and the expression of ACSL4 protein was increased(P<0.01).There was no significant difference in the expression of P53 and xCT proteins.Compared with the AOM+DSS group,the protein expressions of xCT(P<0.05),GPX4(P<0.01),and FTH(P<0.05)in the AOM+DSS+Res group decreased,P53(P<0.05),ACSL4(P<0.001))protein expression increased.3.IHC detected the protein expressions of xCT,GPX4,ACSL4,FTH and P53 in the colon tissue of mice.Compared with the control group,the expression of FTH protein in the colon tissue of the mice in the AOM group decreased(P<0.01),while the expressions of xCT,GPX4,ACSL4 and P53 were not.statistical difference.In the AOM+DSS group,the protein expressions of xCT(P<0.01),FTH(P<0.001),and GPX4(P<0.05)increased,while the protein expressions of P53(P<0.001)and ACSL4(P<0.0001)decreased.Compared with the AOM+DSS group,the protein expressions of xCT(P<0.01),GPX4(P<0.05),and FTH(P<0.05)in the AOM+DSS+Res group decreased,P53(P<0.01),ACSL4(P<0.001)protein expression increased.4.The expression of ferroptosis-related genes in mouse colon tissue was detected by qRT-PCR.The amplification results showed that compared with the Control group,the AOM group had an increase in the gene expression of ACSL4(P<0.01),a decrease in the gene expression of GPX4(P<0.01),and a decrease in the expression of SLC7A11 and SLC7A11 and There was no statistical difference in FTH expression(P>0.05).In AOM+DSS group,the gene expression of ACSL4 decreased(P<0.01),the expression of GPX4(P<0.01)and SLC7A11(P<0.01)increased,and the gene expression of FTH(P<0.0001)increased significantly.Compared with AOM+DSS group,ACSL4 gene expression in AOM+DSS+Res group increased(P<0.05),while GPX4,SLC7A11,and FTH gene expression decreased(P<0.05&P<0.05&P<0.05).5.Resveratrol can inhibit the proliferation activity of HCT 116 cells.The results of CCK-8 detection of HCT 116 cell proliferation activity showed that compared with the control group,resveratrol could significantly inhibit the proliferation of HCT 116 cells after acting on HCT 116 cells(P<0.05).6.The results of detecting intracellular MDA,SOD,GSH and Fe showed that compared with the control group,resveratrol could increase the content of intracellular MDA(P<0.01)and Fe(P<0.05),and decrease the activeity of SOD(P<0.05)and content of GSH(P<0.05),indicating that resveratrol promote ferroptosis in HCT 116 colon cancer cells by increasing intracellular iron content and oxidative stress.7.The results of Western Blot for changes in cellular protein levels showed that compared with the Control group,after administration of resveratrol,the protein expression of ACSL4(P<0.01)in HCT 116 cells increased,GPX4(P<0.05),FTH(P<0.01)),xCT(P<0.05)protein expression decreased.And the effect is dose-dependent.It shows that resveratrol can regulate the expression of key proteins of the ferroptosis pathway and promote ferroptosis.8.The experiments of miR-31 knockdown and overexpression showed that compared with the control group,the protein expression of ACSL4(P<0.01)in the miR-31 mimic group decreased,GPX4(P<0.01),xCT(P<0.05),FTH(P<0.05)protein expression increased.In the miR-31 inhibitor group,the protein expression of ACSL4(P<0.01)increased,while the protein expression of GPX4(P<0.01),xCT(P<0.01),and FTH(P<0.01)decreased.After administration of resveratrol,compared with the miR-31 mimic group,the protein expression of ACSL4(P<0.05)in the miR-31 mimic+Res group increased,GPX4(P<0.05),xCT(P<0.01),FTH(P<0.01)decreased protein expression.Compared with the Control group,the miR-31 mimic+Res group had no significant difference in the protein expressions of ACSL4,GPX4,xCT and FTH.It indicates that the mechanism of resveratrol regulating ferroptosis is related to the inhibition of miR-31 expression.Conclusion:Resveratrol can inhibit AOM+DSS-induced colon cancer in mice,and this anticancer effect can promote the production of ferroptosis in mouse colon cancer tissues,and its ferroptosis-promoting effect is related to down-regulation of miR-31 expression.