| Colorectal cancer ranks the second among the causes of cancer mortality in the Western world.Approximately～14,000 new cases are diagnosed and about～5,000 people die from it each year in the United States.Epidemiologic studies suggested that men are more likely than women to develop colon cancer at all ages.Hormone replacement therapy in postmenopausal women reduces the risk of colorectal cancer by 30%to 40%and also confers protection against the incidence of colorectal cancer. These reports suggest that estrogen plays a role in colorectal cancer prevention.The main biological function of estrogen is mediated by binding to its specific receptors.ERα(ERa66),which belongs to the nuclear receptor superfamily,was the first identified estrogen receptor.It can be subdivided into six distinct regions(A-F). The N-terminal domain A/B is responsible for the ligand-independent transactivation function(AF-1).Domain C is responsible for receptor dimerization and the binding to specific DNA sequences.The C-terminal domain E/F is responsible for the ligand-dependent transactivation(AF-2).Other estrogen receptors have also been identified thereafter,including ERβand several splice variants of ERαand ERβ.An exon-1 truncated ERαtranscript has recently been identified in human cell lines including MCF-7 cells,osteoblasts and vascular endothelial cells.In contrast to its full-length counterpart(ERα66),this truncated ERαlacks the transactivation domain AF-1 and encodes a protein with a predicted molecular weight of 46 kDa which was therefore designated as ERα46.ERα46 transcripts were found to be more abundant in non-reproductive organs than in reproductive tissues,and our preliminary work shows that ERα66 is not the only ERαisoforms in the colon mucosa,ERα46 also expressed at a high level,therefore ERα46 may be also play important physiological roles in colorectal tissues.In this study,we examined the expression of ERα46 messenger RNA in 32 normal colorectal tissues and matched cancer tissues,and investigated its function on cell growth by transfecting ERα46 into HT-29 colon cancer cells.The details are listed below.1.The expression of ERα46 in normal and malignant tissues of colorectal tractThe mRNA expression of ERα46 in normal and malignant tissues of colorectal tract was detected by real time fluorescent quantitative RT-PCR technique using glyceraldehydes-3-phophate dehydrogenase(GAPDH)as internal standard,and was evaluated for their correlations with clinicopathological parameters such as stage,age and histological differentiation.The expressions of ERα46 in colorectal cancer tissues were markedly lower than those in their adjacent normal tissues(P=0.008).Analysis of clinicopathological parameters demonstrated the differences of ERα46 expression were not significant between colorectal cancer tissues of different histological differentiation and invasive depth(P>0.05).2.Cloning of the full length coding region cDNA of ERα46To get the ERα46 cDNA with full length coding region for further study,a 1400 bp human ERα46 fragment was amplified by polymerase chain reaction(PCR)using pcDNA3.1- ERα66 as template and subsequently cloned into pGEM T-easy vector. The cDNA was verified by DNA sequencing and aligned with sequence data in GenBank database.ERα46 were acquired by restricted enzyme digestion with enzyme EcoRI and were ligated into prokaryotic expression vector pcDNA3.1(+). The transformants were screened by restricted enzyme digestion.These subcloned products were transfected into HT-29 cells and the expression of ERα46 protein was identified by Western blot.3.Effect of expression of ERα46 on the proliferation,migration and apoptosis of HT-29 cellsMTT assay was performed to evaluate the influence of ERα46 on the proliferation of HT-29 cells.The result showed that the growth rate of ERα46 transfected cells was significantly decreased than that of control cells in the presence of 10-8M 17β-oestradiol.However,no difference of viability between ERα46-transfected cells and the control cells was observed in the absence of 17β-oestradiol.In flow cytometric assay,an accumulation of cells in the G0/1phase and a reduced proportion of cells in G2/M phase were observed in ERα46 expressed cells.Trans-membrane migration assay showed inhibition of cell migration ability of ERα46 expressed in the presence of 17β-oestradiol(p<0.05).In DNA fragmentation assay,the yields of apoptotic DNA fragments in ERα46 transfectants were higher than the control group treated with 17β-oestradiol or the groups in the absence of 17β-oestradiol,and In TUNEL assay,there were more apoptotic cells in ERα46 transfected cells than that in control cells after treated with 1713-oestradiol(p<0.05)Conclusions1.The expressions of ERα46 in colorectal cancer tissues were markedly lower than those in their adjacent normal tissues;2.Exogenous ERα46 was stably expressed in HT-29 colon cancer cells;3.ERα46 can mediate growth and migration inhibition and induce apoptosis of HT-29 colon adenocarcinoma cells in the presence of 17β-oestradiol.
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