Tamoxifen Regulates PAX2 Expression Via MiR-585 In Endometrial Cancer ECC-1 Cells

microRNAs(miRNAs) are a class of 21-25 nucleotides (nt) non-coding regulatory small RNA molecules. microRNAs are mainly transcripted by RNA polymerase II into primary molecules (pri-miRNAs). In the nucleus, RNase III Drosha cuts pri-miRNA into about 70 nucleotides hairpin miRNA precursor (pre-miRNA). Soon RAN-GTP dependent exportin 5 transfers pre-miRNA from nucleus into cytoplasm. Then RNase III Dicer cuts the hairpin into an imperfect complementary microRNA:microRNA* duplex, which is about 21-25 nucleotides in size. The mature microRNA is integrated into RISC(RNA-induced silencing complex) to form a miRISC complex to perform negative regulation. Perfect or near perfect base pairing of microRNA with its target mRNA promotes degradation of the target mRNA, while partially complementary leads to inhibition of transcription of the target mRNA.microRNA could bind to a complementary site in the 3'UTR as well as in the coding regions of the target mRNA. lin-4 is the first one of the microRNA family coming into our knowledge. Ambro and his colleagues found out that in the nematode C. elegans lin-4 could bind to a site in the 3'UTR of Lin-14 , and thus regulate Lin-14 protein expression. Up until now, tens of thousands of microRNAs have been found in virus, elegan, drosophila, mouse and human. These microRNAs participate in multi-process ranging from cell differentiation /proliferation/apoptosis to organ development, or even tumor genesis. In many kinds of tumors we could find variations of microRNAs'expression level. Many data shows microRNA could regulate up to about 60% of mammalian genes. A single microRNA could regulate several genes, and one gene could be regulated by several microRNAs.Tamoxifen(TAM) is a selective estrogen receptor modulator(SERM). Its function is tissue dependent: in breast tissue, TAM acts as estrogen antagonist, and could reduce high-risk women catching aggressive breast cancer by 49%; in endometrial tissue, it acts as partial estrogen agonist, and could increase the risk of endometrial cancer. Since Tamoxifen has therapeutic effect in breast tissue, it has been used in the treatment of ER positive breast cancer, post operation adjuvant treatment and prevention in high risk women. In 1985 Killackey reported patients receiving tamoxifen had a higher endometrial cancer incidence rate. Such report kept popping into scientic scope afterwards. Clinical statistics shows patients receiving tamoxifen are 2-3 times more likely to have endometrial cancer than normal population. Therefore in 1996 WHO listed tamoxifen as a carcinogen.PAX2(Paired box gene 2) is a transcription factor located in nucleus, which contains a DNA binding paired domain, a truncated homeodomain, an octapeptide region and a carboxyl-terminal transactivation domain. PAX2 regulates the expression of a series of genes involved in mediating cell growth, proliferation, migration, and anti-apoptosis. PAX2 gene plays a critital role in endometrial cell proliferation and carcinogenesis mediated by estrogen and tamoxifen. Estrogen and tamoxifen can activate PAX2 expression only in endometrial cancer cells, not in normal endometrial cells.Our research focused on the mechanism of tamoxifen regulatory effect on PAX2 expression in ECC-1 cells, and searching for microRNA involved in the process. First, we stimulated endometrial cancer ECC-1 cells with tamoxifen as test group, and ECC-1 cells with no stimulation as control group. Western Blot detected higher PAX2 expression level in the tamoxifen stimulated ECC-1 cells compared with control. We used Microcosm Targets (miRBase Sequence database) to predict PAX2 related microRNAs: hsa-miR-504, hsa-miR-362-5p, hsa-miR-186, hsa-miR-135b*, hsa-miR-99a*, hsa-miR-604, hsa-miR-937, hsa-miR-671-3p, hsa-miR-886-3p, hsa-miR-181c*, hsa-miR-585.In order to find out which of these predicted microRNAs could be the potential functional ones in this process, we used real-time quantitative PCR to detect the expression level of these microRNAs in both test group and control group. Results showed miR-135b*, miR-604, miR-585 and miR-181c* were significantly down regulated.The mimics of these 4 down-regulated microRNAs and negative control were synthesized for further study. We transfected these synthetic mimics into ECC-1 cells, and cultured for 48h. Western Blot detected down regulation of PAX2 expression in ECC-1 cells tranfected with miR-585 mimics,while RT-PCR detected no significant difference. The results showed that tamoxifen could moderately up regulate expression of PAX2 in ECC-1 cells not only by enhancing transcription, but also possibly by inhibition of mir-585 expression.Our research paved the way to better understanding of the mechanism of tamoxifen induced higher incidence rate of endometrial cancer, and maybe even to the future microRNA therapy. Yet how tamoxifen down regulates miR-585 still needs further research.