PTEN Regulates Invasion And Metastasis Of Colon Cancer By Regulating P66shc Expression

Part one:p66shc and PTEN gene expression in colon cancer and their relationshipObjective:To investigate the p66shc and PTEN expression in colon cancer and the effect of PTEN to the expression of p66shc.Method:(1)67 cases of colorectal Cancer tissues,25 cases of intraepithelial neoplasia tissues and 57 cases of paracarcinoma tissues were collected.The expression of p66shc was detected by immunohistochemistry in the collected specimens,and then an analysis of the collection between p66shc expression and clinical pathology features of 67 colon cancer patients was performed.(2)PTEN expression was detected by immunohistochemistry in 67 cases of colorectal Cancer tissues and 57 cases of paracarcinoma tissues,and the relationship of PTEN,and p66shc expression was analyzed(3)8 pairs of fresh colon cancer tissues and paracarcinoma specimens were collected,use Western blot to detect the expression of PTEN and p66shc.(4)PTEN gene was silenced in a lentiviral-mediated manner in the colonic cancer cell line HCT116,SW480,and mRNA and protein expression of PTEN and p66shc were detected by real-time quantitative PCR and Western blotResults:(1)the p66shc protein expression level gradually decreased in colon cancer,intraepithelial neoplasia and para-cancer tissue.In the colon cancer,there were no association between p66shc expression and clinicopathologic features such as age,sex,tumor size,tumor differentiation,T and M stages,but p66shc expression was positively correlated with tumor n stage,the higher the p66shc expressed,the greater the risk of lymphatic metastasis was.(2)PTEN protein expression was low expressed in colon cancer tissue compared with paracancerous tissues.In colon cancer and paracancerous tissues,p66shc and PTEN expression was inversely correlated.(3)Due to the knockdown of the PTEN,p66sch expression in mRNA and protein level increased in colon cancer cell lines.Conclusions:(1)p66shc may be implicated in the initiation and lymphatic metastasis of colon cancer;(2)PTEN could negatively regulate p66shc gene expression in mRNA and protein level.Part Two the research of PTEN gene regulating colon cancer cell invasion and metastasis by p66shcObjective:To investigate the effect and possible mechanism of PTEN on colon cancer metastasisMethod:(1)take colon cancer cell line HCT116 as object,use external cell invasion assay to detect the cancer cell invasiveness of blank control group,control lentivirus group(Control shRNA),PTEN gene knockdown group(PTEN shRNA)and PTEN gene knockdown combining with p66shc small interfering RNA group(PTENshRNA+p66siRNA).(2)HCT116 cells and PTEN knockdown HCT116 cells were implanted into the colon wall of nude mice to construct colon carcinoma in situ model of nude mice.After 7 weeks,observe the tumor formation rate and local and distant metastasis of nude mice in control group and PTEN gene knockdown group.(3)take the in situ cancer tissue of nude mice in control group and PTEN gene knockdown group,immunohistochemically detect PTEN and p66shc expression.AKT,p-AKT,GSK3P,p-GSK3βand p66shc protein expression was detected by Western Blot.Results:(1)colon cancer cell invasion ability improves through PTEN knockdown.PTEN gene negatively express colon cancer cell;the cancer cell invasion ability is suppressed after using small interference RNA to suppress p66shc.(2)PTEN knockdown enhances the colon in situ growing tumors and lymphatic metastasis.(3)in the in situ growing tumor tissues,the expression of PTEN and p66shc is opposite,the expression of PTEN is low while that of p66shc is high.Conclusion:(1)PTEN knockdown could facilitate colon cancer metastasis.(2)PTEN may suppress colon cancer metastasis by regulating p66shc expression.Part Three:The mechanism of PTEN regulating transcription of p66shcObjective:To investigate the mechanism of PTEN regulating p66shc gene transcriptionMethods:(1)Influence of PTEN gene knockdown,the PI3K inhibitor LY294002 treatment and GSK3 beta inhibitor treatment on p66shc expression in HCT116 cell line were detected by using Western blot;(2)Effect of PTEN gene knockdown,the PI3K inhibitor LY294002 treatment and GSK3 beta inhibitor treatment on AKT,p-AKT,GSK-3 beta,p-GSK-3 beta,p66shc expression and nucleus NFAT2 expression in HCT116 cell line were detected by using Western blot using;(3)The p66shc promoter and the NFAT2 binding sites were predicted by software,and the activity of NFAT2 combining with specific probes and the effect of PTEN knock-down on NFAT2 binding activity were investigated by using EMSA;(4)the activity of NFAT2 binding p66shc promoter-centered chromatin and the effect of PTEN knock-down on NFAT2 binding activity were evaluated by using CHIP;(5)p66shc promoter vector was designed and synthesized,NFAT2 combining with p66shc promoter were assessed by using the luciferase reporter gene assay;(6)According to the p66shc promoter sequences and NFAT2 binding site on promoter,truncated vector namely P-888,P-592,P-395 and P-348 were constructed.After vectors were transfected,the promoter activity was investigated by using the luciferase reporter gene assay.(7)The two NFAT2 binding sites in the promoter were mutated respectively,and named p66shc-M1,p66shc-M2,p66shc-M1-M2.After the vectors of mutation were transfected respectively,the promoter activity was detected by luciferase reporter gene assay.Results:(1)PTEN gene knock-down in HCT116 cell line promoted the expression of p66shc,and PI3K inhibitor LY294002 and GSK3 beta inhibitors could block the influence of PTEN knock-down on p66shc expression;(2)The nuclear NFAT2,p-AKT,p-GSK3 beta-protein expression increased after PTEN gene knock-down,While the expression of AKT and GSK3 beta did not change.In addition,PI3K inhibitors LY294002 and the GSK3 beta inhibitors could inhibit nuclear NFAT2,and p-AKT,and p-GSK3 beta protein expression;(3)NFAT2 could combine with specific biotin-labeled probe,but not NFAT2 mutation probe,PTEN gene knock-down could promote NFAT2 activity;(4)NFAT2 could combine with p66shc promoter-centered chromatin,and PTEN gene knock-down could enhance the activity of NFAT2 in conjunction with chromatin(5)PTEN gene knock-down could enhance promoter-luciferase activity of p66shc,however NFAT2siRNA transfection could suppress the impact of PTEN gene knock-down on p66shc promoter-luciferase activity.Additionally,Transfection of NFAT2 expression plasmid enhance promoter-luciferase activity of p66shc;(6)the vector P-395 missing the first NFAT2 binding site was transfected into HCT16 cell line,and the luciferase activity of the vector reduced compared with the vector P-888,which has no NFAT2 binding sites deletion.The vector P-348 missing two NFAT2 binding sites had the lowest luciferase activity after transfection.(7)Compared with the promoter without mutation,no matter how the two NFAT2 binding sites were mutated,the activity of promoter with mutation were reduced.Conclusions:(1)PTEN could negatively regulate p66shc gene expression via AKT/GSK3 β/NFAT2 pathway.(2)NFAT2 is a positive transcription factor of p66shc,and there are two NFAT2 binding sites on the p66shc promoter.