Study On The Mechanism Of Estrogen Induction Of MLH1 Expression And Inhibition Of Colorectal Cancer Proliferation

Background and ObjectiveHuman DNA mismatch repair(MMR)system plays a key role in maintaining genomic stability.Microsatellite instability(MSI)resulted from a defective mismatch repair system is closely related to many malignancies,such as Lynch syndrome.Additionally,about 15% sporadic colorectal cancer(CRC)is concerned with MMR deficient.Epidemiological data demonstrated that hormone replace treatment has protective effect against colorectal cancer.Our previous studies showed that this effect of estrogen might be associated with DNA mismatch repair protein MLH1.MLH1 is one of the dispensable MMR genes.However,the mechanism of estrogen induction of MLH1 remains unclear.This study aims to investigate the mechanism of estrogen induction of MLH1,and the impact on colorectal tumor proliferation of MLH1 up-regulation by estrogen signal paythway.Part one Study on the mechanism of estrogen induction of MLH1BackgroundOur previous results demonstrated that serum estrogen level was positively correlated to MLH1 protein expression in normal colonic epithelia cells,but had little relationship with MSH2.And in vitro findings showed that estrogen enhanced MLH1 expression in colonic cells,but hardly affected MSH2.This study aims to investigate the mechanism of estrogen induction of MLH1 expression.Methods1.SW480,HT29 and LoVo cell lines were used to examine the regulation of MLH1 expression by over-expression of estrogen receptor-?(ER?)and estrogen receptor-?(ER?),under the treatment with 17?-estradiol or ?-Estradiol 6-(O-carboxy-methyl)oxime:BSA,followed by a real-time Q-PCR and Western Blot analysis.2.According to the UCSC(www.geome.ucsc.edu),MLH1 proximal promoter sequence(-1953/+53)were amplified from the genomic DNA of human by PCR and cloned into luciferase reporter vectors.Then luciferase reporter and chromatin immunoprecipitation assays were used to identify the estrogen response elements in the proximal promoter of MLH1 gene.Results1.Estrogen enhanced MLH1 gene expression significantly at mRNA level,in all the three cell lines,however,BSA-E2 showed very weak effect on the gene expression(P < 0.001).Furthermore,over expression of ER? synergistically increased the expression of MLH1 at mRNA level with estrogen treatment,compared with control(P < 0.001).2.We found estrogen responsive elements located in the proximal promoter of MLH1.Half-estrogen responsive element and AP1 binding sites were both critical for estrogen induced MLH1 expression.ConclusionEstrogen induces MLH1 expression mediated by ER?,through a transcriptional activation process.Part two ER? exerts an inhibitory effect on colorectal cancer growth in vitro and in vivoBackgroundIt's reported that ER?-specific agonists inhibited proliferation and induced apoptosis in vitro and in vivo.On the other side,postmenopausal hormone therapy reduced the risk of colorectal cancer with low level microsatellite instability or microsatellite stable.This study is designed to identify if ER?-mediated MLH1 up-regulation function as inhibitor on colorectal cancer cells growth in vitro and in vivo.Methods1.SW480 and Caco2 cells were transfected with siRNA-shMLH1 plasmid,with or without ER? over-expression,followed by incubation with vehicle,estrogen,or estrogen plus ER?-specific antagonist 4-[2-phenyl-5,7-bis(tri-fluoro-methyl)pyrazolo [1,5-a] pyrimidin-3-yl] phenol(PHTPP),respectively,and treated with 5-FU.Cells viability were estimated by Cell Counting Kit-8(CCK-8)assay.2.AOM/DSS induced colorectal cancer mice models were applied.Mice were ovariectomized before any intervention and divided into 2 groups randomly.There were five mice in each group.Each mouse in either group was subcutaneously injected with DPN(1 mg/kg per day),or the same volume of vehicle,respectively.All those mice were sacrificed on the 16 th week.Colons were dissected and weighed,and the average ratio of weight / length was compared between two groups.3.HT29 cells(5 x 106)were subcutaneously injected into the ovariectomized nude mice,which were divided into there groups randomly,with five mice in each group.Either group were subcutaneously injected with DPN(1 mg/kg per day),PHTPP(10-7 mol per day)or vehicle,respectively.Xenografts tumor size were monitored using the following formula: volume =(smallest diameter)2 ×(largest diameter)÷ 2.Finally,portion of tumor tissue was used to perform Western Blot analysis.Results1.Cells were insensitive to 5-FU when endogenous MLH1 expression were silenced by transfection with siRNA-shMLH1.However,cells sensitivity to 5-FU were enhanced through up-regulation of MLH1 by estrogen treatment plus ER? over expression.2.The average ratio of weight / length was decreased in DPN-injection group compared with vehicle-injection group.3.Xenograft tumor growth was inhibited in DPN-injection group,in which tumors average volume was decreased compared to PHTPP-injection group.At the same time,Western Blot assays indicated that MLH1 protein expression level was significantly higher in DPN-injection group than that of PHTPP-injection group.ConclusionER? sensitizes cells to 5-FU in vitro and exerts an inhibitory effect on CRC tumor growth throuth up-regulation of MLH1 expression.