Estrogen Up-regulated The Expression Of Hmlh1in Colon Cancer Cell SW480and Embryonic Kydney Cell293T And Increased Microsatallite Stability

Objective:Numerous epidemiological have shown that prevalence of colon canceris significantly higher among male than female. Both incidence and death rates of rectalcancer can be reduced by hormone replacement therapy (HRT) among post-menopausefemales. Estrogen may has protective effects against colon cancer. The most commonfamilial form of colorectal cancer, hereditary nonpolyposis colorectal cancer,HNPCC,has been reported that it caused by mutations of mismatch repair geneshMLH1. Another10%-15%of sporadic colorectalcancers are caused by epigeneticsilencing of hMLH1gene. MMR dysfunction causes microsatellite instability (MSI). Ithas been reported that post-menopausal estrogen replacement.therapy can reduce therisk of MSI-positive colon cancer. These findings suggest that estrogen might preventCRC caused by MMR dysfunction.in the former study,we found that estrogen canup-regulate the expression of MMR and constructed the measurement of DNAmismatch repair activity in live cells.On the base of former study,we designed toexplore whether estrogen could increase the function of mismatch repair genes and theeffect of estrogen in keeping micro satallite instability.Methods:A eukaryotic expression vector targeting on mismatch repair genehMLH1was designed and constructed,and was named pGPU6/GFP/Neo-MLH1-homo-234.cultured cell line SW480and293T, then transfected plasmidpGPU6/GFP/Neo-MLH1-homo-234and nonsence control pGPU6/GFP/Neo-MLH1-NC into cells,using G418to select positive colonies.expressing level of hMLH1was assayed by western blot. Treat cells with estrogen to detected the difference ofexpressing level and microsatellite level of cells before and after the treatment ofestrogen. Results:Plasmid pGPU6/GFP/Neo-MLH1-homo-234was successfullyconstructed and transfected into cells. G418selected than picked single clone.westernblot showed that hMLH1gene were obviously knocked down(P<0.05).The expressionof hMLH1were up-regulated in knocked-down group after the treatment ofestrogen(P<0.05).The knock-down group was microsatellite instability conpared withcontrol group. But the microsatellite level changed to stability after the treatment ofestrogen.Conclusion:Koncked down the expression of hMLH1in cell line SW480and293T could cause microsatellite instability. But knocked-down groups with thetreatment of estrogen were showed to be microsatellite stability. The result verified outhypothesis that estrogen plays an important role in keeping microsatellite level throughup-regulate the expression of hMLH1.