The Study On The Mechanism Of Tamoxifen Induced EMT By Downregulating MiR-200 In ECC-1 Cells

TAM as an antagonist to estradiol (E2) in estrogen receptor (ER) positive breast tumors can prevent breast cancer in high risk women. However, it shows partial estrogen like actions in other target tissues. The partial estrogen like actions make beneficial effect on bones in postmenopausal women, but the effect of TAM on uterus is well known to raise the incidences of endometrial cancer. Endometrial carcinoma is the most common gynaecological malignancy in which estradiol has been identified as a classic aetiological factor. Endometrial pathologies associated with TAM use include hyperplasia, polyps, carcinomas and sarcomas.MicroRNAs (miRNAs) are small noncoding RNAs ( 22n)that regulate gene expression by repressing target messenger RNAs (mRNAs) via specific base pairing interactions in 3'untranslated regions (3' UTRs). miRNAs repress gene expression by interfering with mRNA stability or protein translation. Recently miRNAs have been identified in regulating complex physiological processes such as embryogenesis and oncogenesis. miR 200 family has been found to play a central role in the regulation of the epithelial to mesenchymal transition (EMT) process during cancer progression and metastasis. The increase of the miR 200 family intracellular levels inhibits EMT by up regulation of E cadherin expression through direct targeting of its ZEB1 and ZEB2 transcriptional repressors. In this study, we tested the hypothesis that miR 200 family , an important miRNA during EMT process ,is regulated by Tamoxifen in ECC 1 endometrial cancer cells.We aimed to determine whether ECC 1 cells treated by TAM that have undergone an EMT ,which is an oestrogen responsive endometrial carcinoma cell line . We investigated the effect of Tamoxifen on the expression of this epithelial marker by RT PCR analysis when the ECC 1 cells after treating with TAM. The ECC 1 cells acquired a mesenchymal mRNA expression profile. We used wound healing assay and transwell assay to confirm that TAM induces EMT process in ECC 1 cells.We performed miRNAs microarray to investigate miRNAs expression profiles in ECC 1 cells and ECC 1 cells treated with TAM. The study showed significantly increased expression of four miRNAs and down regulation of nineteen miRNAs in ECC 1 cells treated with TAM compared with ECC 1 cells. Interestingly, miR 200b plays an important role in EMT, so we investigated if miR 200 is regulated by TAM in ECC 1 cells .The transcription factor ZEB2 has been suggested to be target of miR 200 family, According to TargetScan 4.1 (http://www.targetscan.org). We could show that overexpression of miR 200b led to reduced expression of ZEB2, Snail and N cadherin genes in ECC 1 cells,and increased expression of E cadherin simultaneously. As predicted, miR 200b had the strongest inhibitory effect on ZEB2 expression, as shown by mRNA and protein levels.Overexpression of miR 200b resulted in ~80% reduction in luciferase activity in ECC 1 cells transfected with wild type construct of human ZEB2 3' UTR . Furthermore, we evaluated the role of miR 200 suppression of ZEB2 expression on invasion in ECC 1 cells.Overexpression of miR 200b resulted in a significant derease in the number of invading cells which were treated with TAM.We investigated whether TAM regulates miR 200b 200a 429 gene expression through a region encompassing 1574 to +120 bp relative to the putative TSS region previously reported to function as a promoter of miR 200b 200a 429. Using TESS (http://www.cbil.upenn.edu/cgi bin/tess/tess) to analyze the 110/+19 promoter region, we found some important transcription factor binding sites such as c Myc, c Myb and Sp1. Previous study showed that extensive reprogramming of the miRNAs transcriptome by Myc contributes to tumorigenesis.So we supposed that c Myc may play an important role in regulating miR 200b 200a 429 promoter in ECC 1 cells.We found that c Myc inhibited the activity of the promoter in ECC 1 cells, we also observed that TAM can increase expression of c Myc in ECC 1 cells.In conclusion, we have identified miR 200 as an important factor that TAM mediates their carcinogenic roles in the uterus, our findings also illustrate the loss of miR 200 in regulating tumor cell EMT process. We have identified miR 200 that are regulated by tamxifen that by increasing c Myc expression in the endometrial cancer cells. Our study showed that TAM can induce EMT in ECC 1 cells, which revealed that c Myc directly regulates miR 200 expression . It is clear that miR200 regulates ZEB1/ZEB2/E Cadherin through TAM c Myc pathway.And our results provide new insights into molecular mechanism of TAM induced endometrial carcinoma.