The Study On The Mechanism Of MiR-106b-93-25 Cluster Induced Malignant Phenotype In Endometrial Cancer Cells

Estrogen, a steroid hormone secreted by ovary exhibits a broad spectrum of physiological functions ranging from regulation of the menstrual cycle and reproduction to the modulation of bone density, brain function, and cholesterol mobilization via ligation with its receptor in the target tissues. However, estrogen is also associated with pathological complications particularly with the onset of gynaecological malignancies including breast cancer and endometrial cancer. Estrogen is now considered to be a classical etiological factor for breast cancer and endometrial cancer. Therefore, the endocrine therapy has been targeted to estradiol and its receptor. Estrogen receptors (ER) are classified as two subtypes, ERαand ERβ. They are members of the nuclear receptor (NR) superfamily of ligand-inducible transcription factors that share similar structures and modes of action. ERs are localized in the nucleus and cytoplasm. The overexpression of ER is related to breast cancer and some of gynaecological cancers. The ERs are transcription factors whose activities are dependent upon their ligand binding capacities. ERαis comprised of six regions, named A to F. The transactivation domain termed activation function(AF)-1 is contained within the amino-terminal A and B domains and exhibits a ligand-independent activation function. The C domain is composed of two zinc finger motifs and encodes the DNA binding domain that is responsible for binding to specific estrogen response elements (EREs) within the promoters of estrogen-responsive genes. The ER dimerization domain is discontinuous, is split between the C and E domains, and is required for the ERs to dimerize, allowing binding to the entire ERE site. The structural D domain contains the hinge region, part of the ligand-dependent, transactivation domain AF-2 and a portion of the ER nuclear localization signal. The carboxy-terminal E and F regions contain the ligand binding domain and the ligand-dependent AF-2 transactivation domain. AF-1 is hormone-independent whereas AF-2 is hormone-dependent. Their transcriptional regulation of target genes is a complex, multistep and tightly regulated process which is dependent on the interaction with other proteins. These ER-interacting proteins were termed ER coregulators which were divided into two groups, coactivators and corepressors. According to the classical model of ER action, in the absence of hormone, the receptor is sequestered in a multiprotein inhibitory complex in either the cytoplasm or nuclei of target cells. The binding of ligand induces an activating conformational change within the ER, promoting dimerization and high-affinity binding to specific estrogen-response elements (EREs) located within the regulatory regions of target genes where ER communicates with the general transcription apparatus to positively or negatively regulate gene transcription. Because of the important role of estrogen and its receptor in the development of gynaecological tumors, the study on the mechanism and therapy were focused on how to reverse the function of estrogen and/or induced the tumor cells to apoptosis. This is the purpose and significance of our research. PartⅠThe mechanism of miR-106b-93-25 cluster induced malignant phenotype in endometrial cancer cellsAs the first clinically available SERM tamoxifen was introduced in the 1970s for the treatment of advanced breast cancer in postmenopausal women. Now, it has been used in the treatment of all stages of hormone-responsive breast cancer. However, tamoxifen shows partial oestrogenic activity in the uterus and its use has been associated with an increased incidence of endometrial cancer. microRNAs (miRNAs) are noncoding RNAs of 22 nt that function as post-transcriptional regulators. By base-pairing with the complementary sites in the untranslated region (UTR) of mRNA, miRNAs can control the mRNA stability and the e?ciency of translation. Recent evidence has shown that miRNA mutations or mis-expression correlate with various human cancers and indicates that miRNAs can function as either tumour suppressors or oncogenic RNAs.After binding with their ligand, ER recruitments many coactivators/corepressors to form a complex and coregulate the expression of target genes. Some molecules have been shown to possess intrinsic histone acetyltransferase (HAT) activities capable of acetylating histones. Several reversible or irreversible HDACs inhibitors,such as TSA (trichostatin A), are able to upregulate gene expression via suppressing the histone deacetylation in the chromosomal context of the corresponding genes. Accumulating studies have shown that tamoxifen-bound ER might interact with corepressor complexes containing histone deacetylase (HDAC) activity, leading to chromatin condensation and gene silencing.Therefore, we treated ECC-1 cells with TAM or TSA. The proliferation and apoptosis and cell-cycle were detected by MTT assay and FCM. There are cell cycle arrest and increased apoptosis in ECC-1 cells treated with TSA, whereas increased proliferation in cells treated with TAM. Therefore, we treated ECC-1 cells with TAM alone or together with TSA. The microRNA array analysis indicated miR-106b-93-25 decreased significantly in cells after treatment with tamoxifen and TSA, but not in cells treated with tamoxifen alone, suggested that TSA plays a regulatory role in the expression this miRNA cluster. The result was verified by qRT-PCR.Synthesized RNA duplexes of miR mimics and miR inhibitors of miR-106b and miR-25 were transfected into ECC-1 cells. The cell-cycle progression were blocked at G1 after treated with miR-106b inhibitor whereas G1 population were reduced were observed in the cells treated with miR-106b. It indicated that miR106b-93-25 cluster suppress apoptosis and promote cell division. After cotransfected ECC-1 cells with miR-106b or miR-25 and p21 3'UTR or Bim 3'UTR, it shows miR-106b repress the expression of dual luciferase reporter containing the p21 3'UTR and miR-25 repress the expression of luciferase reporter containing Bim 3'UTR. It suggested p21 and Bim is the target of miR-106b and miR-25 respectively in the endometrial cancer cells. The miR-106b-93-25 cluster was downregulated in the endometrial cancer cells which were treated with TSA. The cell cycle arrest and apoptosis were observed as result from the upregulation of p21 and Bim which were negatively regulated by this cluster. It was verified by Western blot assay that TSA upregulated the expression of p21 and Bim via this cluster. c-Myc was identified bioinformatically as the regulator of this cluster. It was testified by western blot assay.To sum up, we observed that TSA regulated this miRNA cluster via v-myc myelocytomatosis vial oncogene homolog (MYC), and that miRNAs of this cluster regulated their target gene p21 and Bim which lead the cells to apoptosis. PartⅡ: Estradiol induced apoptosis of breast cancer cells modified with ERα-fused caspase-8 geneApoptosis is programmed and precisely regulated cell death involving the activation of a class of cysteine proteases termed caspases. At least 14 members of the caspase family have been identified from mammalian cells. Caspases are synthesized as inactive zymogens consisting of an amino-terminal prodomain, a large subunit, and a small subunit. Upon activation, the zymogen is proteolytically processed at specific caspase-processing sites, followed by association of the released large and small subunits to form a heterodimer. An"induced proximity model"of caspase activation demonstrated that caspase-8 could be activated after an inducible dimerization driven by a heterologous domain fused to the zymogen.In the presence of estrogen, ligand binding of ERαcauses its dissociation from an inactive complex of heat-shock proteins, resulting in dimerization of the receptor, and a consequent DNA binding and gene transcription.In this study, a fusion gene encoding the chimeric protein of caspase-8 and the LBD of ERαwas generated and introduced into ERαpositive and ERαnegative breast cancer cell lines. The expression of the fusion protein was detected by RT-PCR and western blot analysis. To examine the recruitment of the caspase-8 and ERαLBD chimeric proteins in the cells, the constructs of 6×His- and Flag-tagged fusion proteins were generated and introduced into MCF-7 cells. While indirect immunofluorescent staining revealed the expression and co-localization of 6×His- and Flag-tagged caspase8/ER, the association of the fusion proteins in the presence of E2 was confirmed by a co-immunoprecipitation assay using antibodies to the His and Flag tags. In a further detection of ER dimers in breast cancer cells, SKBr-3 cell lysates were separated on a non-denaturing polyacrylamide gel, transferred to the PVDF membrane and blotted with a caspase-8 antibody.Intratumoral administration of E2 caused dramatically suppressed tumor growth and prolonged mice survival in caspase-8 / ERαLBD-engineered breast cancer models, but not in those derived from unmodified MCF-7 or SKBr-3 cells. The tumoricidal capacity of estrogen and caspase-8 / ER was due to their apoptosis-inducing activity as revealed by a strong TUNEL staining specifically in E2-treated chimeric protein-expressing tumors. Thus, estrogen suppresses the in vivo development of caspase-8 and ERαLBD fusion gene-engineered tumors developed from human breast cancers which are originally either estrogenic hormone-dependent or -independent. The in vivo delivery of this chimeric gene has implications in developing novel approaches to treating ER-positive breast cancers.