The Primary Study About Mechanism Of Estradiol Up-regulating HMLH1Gene Expression

Background and ObjectiveHuman DNA mismatch repair （MMR） system plays a key role inmaintaining genomic stability. Microsatellite instability （MSI） resulted from adefective mismatch repair （MMR） system is closely related to the manymalignancies. A large proportion of hereditary nonpolyposis colorectal cancer（HNPCC） are caused by germline mutations in MMR genes. Additionally, somesporadic colorectal cancer （CRC） is related to a defection of MMR. hMLH1isone the most important MMR genes. Previous studies show that, in healthyindividuals, a positive correlation of E2level with hMLH1gene expression innormal colonic epithelia cell was observed. Similarly, the cell experimentsdemonstrate that E2increased the expression of hMLH1gene in COLO205cellline, and the up-regulating effect most probably occurs at the transcriptionallevel. However, the mechanism of E2up-regulating the hMLH1gene expressionhas not been fully understood yet. In this study, we aim to construct luciferasereporter gene vector containing human MLH1promoter and to assay thetranscriptional activity of hMLH1promoter induced by E2.Methods1.According to the UCSC （www.geome.ucsc.edu）, hMLH1promoter （-1953/+53）were amplified from the genomic DNA of human by PCR and cloned intoluciferase reporter gene vector, pGL3-Basic. Then the recombinatnt vector wasextracted and identified by double digestion and sequencing. The successfulrecombinant eukaryotic expression vector containing hMLH1promoter wasnamed pGL3-hMLH1-luc.2. The recombinant vector pGL3-Promoter-luc、pGL3-Basic and pGL3-Controlwere respectively transient co-transfected with pRL-SV40into HEK293andLoVo cells. The activity of the luciferase was measured after the cells were simulated by E2, E2-BSA and ICI182.780.3. Detection of expression of endogenous hMLH1in HEK293and LoVocells, transient transfected with pRST7-ERβ or not, simulated by E2or not,according to Western blot analysis.Results1. The results of restriction enzyme digestion and sequencing indicated thatthe insert sequence is in accord with the data of UCSC, and recombinant vectorpGL3-Promoter-luc was successfully constructed.2. The dosage and time dependence was observed. The activity of theluciferase was enhanced significantly when24h induced by10^-9mol/L E2（n=3,P<0.01）. The enhancement was suppressed by ICI182.780, the estrogen receptorinhibitor. Compared with E2, E2-BSA can not significantly up-regulate the expressionof reporter gene.3. It was shown that endogenous hMLH1expression in HEK293and LoVocells transient transfected with pRST7-ERβ and induced by E2was significantlyincreased compared with control according to Western blot analysis （n=3,P<0.01）.ConclusionsE2can enhance the activity of luciferase reporter vector containing hMLH1promoter, which indicates that hMLH1promoter contains the regulatorysequence associated with E2, and ERβ plays a important role in this process.