The Effect Of Estrogen Receptor Alpha36Overexpression On Biological Behaviors Of Human Breast Cancer Cell MCF-7

Background:Breast cancer is the most common type of malignant cancer in women worldwide, which has fastest growing incidence, a maximum of X-ray examination and biopsy examination, and the most social spending. The global cancer statistics data made by the American Cancer Society (ACS) on February4,2011show that the breast cancer (accounting for23%of total cancers) is the most common type of female cancer, and its mortality rate (14%) is the first among the female cancers. In our country, the incidence of breast cancer has increased by3%every year in recent10years, the total incidence increased by37%than that10years ago, the mortality rate increased by38.9%, and the age of onset advanced15-20years than that in the developed countries. Breast cancer is one of cancers threatening the health of women in our country.Estrogen is an important steroid hormone, which involve in the regulation of physiological functions of reproductive system and non-reproductive system through estrogen receptor (ER), and is related with a variety of clinical diseases. Among these diseases, most attention has focused on breast cancer. The estrogen signaling in physiological state involves in proliferation and differentiation of mammary epithelial cells, and irregular estrogen signaling can stimulate cell proliferation, which increases the risk of breast cancer. The traditional estrogen receptor-a (ER-a) protein is called ER-a66which is composed of595amino acids, and its molecular weight is66kDa. A novel variant of estrogen receptor a, ER-a36has been identified and cloned in2005. Its molecular weight is35.7kDa, so it is named as ER-a36. Different from ER-a66, ER-a36lacks two transcription activation domains (AF-1and AF-2), but retains the DNA-binding domain and the partial dimerization and ligand-binding domain, followed by the unique27amino acid domain at the C-terminus instead of the last138amino acids of the ER-a66. The estrogen signaling pathway mediated by ER-a36is different from that of ER-a66, which may play more important role than that of ER-a66in the carcinogenesis and progression of breast cancer. At present, the mechanism of action of ER-a36has been identified as membrane.-initiated steroid signaling (MISS), namely membrane-initiated nongenomic action mode. In this mode of action, ER-a36can stimulate MAPK7ERK and PI3K/Akt signaling pathway in the way of ligand-dependent or non-ligand-dependent.Studies have shown that the expression level of ER-a36is closely linked with the progress,prognosis and the TAM innate drug resistance of breast cancer. In vitro studies indicate that TAM will not only fail to inhibit cell growth, but also stimulate cell proliferation in MCF-7cells which express ER-a36effectively. ER-a36and HER-2mediated non-genomic effects of crosstalk through positive feedback mechanism, that promote the survival of breast cancer cells and result in primary drug resistance and acquired resistance to tamoxifen (tamoxifen, TAM), at the same time, the high expression of this variant is also involved in endometrial cancer which caused by TAM side effects.ER-a36expressed not only in breast cancer, endometrial cancer, but also in the cells of gastric cancer, liver cancer, colon cancer and other malignant tumors, with the ever-depening understanding of the role and mechanism of ER-a36in disease.a growing number of research organizations (institutes) and medical institutions are concern about it.Expecially the treatment options for breast cancer, it is necessary to test ER-a36and ER-a66to determine whether patients can benefit from TAM treatment. ER-a36expression level can be further classified, that may contribute to the prognosis of endocrine therapy and apply to the estrogen-related diseases warning, the monitor and clinical evaluation of the occurrence of development. There is no business of the ER-a36antibody, in recent years, the Polyclonal Antibody of domestic research around the ER-a36protein are mostly donated by foreign research institutions as gifts or observe indirectly through induce expression the fusion protein of ER-a36and tag protein, There is no test kits of ER-a36approal international and domestic. This brought great difficulties to the identification of ER-a36in the intracellular localization, expression and disease diagnosis, treatment options. The same time, the clinical need a convenient, fast, efficient ER-α36and ER-a66union detection methods, and ER-a36the biological characteristics affection and mechanism of breast cancer has not yet been more comprehensive exposed.In view of this, the purpose of this study is to prepare purity,high activity and strong specificity sheep anti-human ER-a36polyclonal antibody, and explore its application system in the ER-a36immune histochemistry and protein blot,to provide follow-up study materials and immunological detection methods, we construct the eukaryotic expression vector and transfected it into estrogen receptor positive MCF-7cells, and thus obtain stable expression, explore from the genetic level to establish an efficient ER-α36and ER-a66union PCR detection methods in transfected cells, set up a more comprehensive discussion of the affection of ER-a36to estrogen receptor-positive human breast cancer MCF-7cells and its molecular mechanism of malignant behavior, as well as the role of testosterone on the ER-a36expression of ER-the a66positive breast cancer cells.Part I Preparation of the Anti-ER-a36Polyclonal Antibody and Its Preliminary ApplicationObjective:(1) Preparation of high purity high titers of anti-ER-a36polyclonal antibody.(2) Develop Western blot experiments and immunocytochemical detection methods.(3) The available experimental materials and methods will be used for further research.Methods:Synthetic modified37peptide which consists of ER-a36C-terminal27specific peptide and GGGGSGGGGS linker at the N-terminal acetylation modification, using the carbodiimide method37peptide and was coupling with OVA, BSA,named ER-a36-OVA and BSA, ER-a36-OVA respectively. Goats were immunized with ER-a36-OVA and BSA. The gained anti-ER-a36Serum was purified by affinity chromatography with ER-a36C-terminal27specific peptide.the Purity potency and specificity was identified by SDS-PAGE, ELISA, and Western blot France antibody titer and specificity for analysis and identification of and the access to multi-antibody was used to the initial application in immuno cytochemistry.Results:The ratio of ER-α3637peptide and OVA, BSA coupled mass was1:1; ERα36-BSA detection by16-fold dilution of goat antiserum after four immune goat still be seen clearly immunoprecipitation line; ELISA assay shows the antibody titer reached104or more, no cross-reaction with the ER-a46and ER-a66protein; and applied to the ER-a36immunohistochemistry cytochemistry and western blot detection. The antibody will be used to further study the function of ER-a36and application.Conclusions:The anti-ER-a36polyclonal antibody was of high purity, high titer, specificity, and can be used for the ER-a36Western blot experiments and immunohistochemical detection.Part Ⅱ Construction of the ER-a36Overexpression Breast Cancer Cell Line and its Sensitivity to Estrogen and TamoxifenObjective:To construct the eukaryotic expression vector of ER-a36and identification in MCF-7cells.Methods:As we know that ER-a36strong expression in human breast cancer MDA-MB-231cells to extract total RNA and to be template, after RT-PCR amplification of ER-a36gene sequence, recovery of the amplified fragment with EcoRI and Not I, respectively, fragments were linked with pcDNA3.1vector using T4DNA ligase, and then transformed the productions into DH5a competent cells, enzyme restriction electrophoresis and gene sequencing method were to be used for identification of positive clones, the positive clones were cultured enough to extract the recombinant DNA vector, and transfected MCF-7cells by liposome, screened G418-resistant single cell clones to expand cultivation. By fluorescence quantitative PCR and Western blot detection of ER-a36and ER-α66protein expression in MCF-7cells.Results:Construction of the ER-a36recombinant eukaryotic expression vector pcDNA3.1-ERa36; reorganization of the eukaryotic expression vector successfully transfected MCF-7cells stably expressing ER-a36gene and the protein is highly expressed in MCF-7cells.Conclusions:This study was successfully constructed the eukaryotic expression vector of the target gene transfection of eukaryotic cells, and high expression which provides a theoretical basis for the study of estrogen in breast cancer pathogenesis and treatment. Part Ⅲ The Effects of ER-α36Overexpression on Biological Behaviors in Human Breast Cancer Cell MCF-7.Objective:To explore the effect of ER-α36, a novel variant of estrogen receptor α, on the malignant behaviors such as the growth, proliferation, apoptosis, tumor formation in vitro, invasion and metastasis potential in the human estrogen receptor-positive breast cancer cell and its mechanism, and to observe the role of testosterone in the impact of ER-α36.Methods:Wild type MCF-7cell line (MCF-7/W), MCF-7cells transfected with pcDNA3.1empty vector (the MCF-7/pcDNA3), MCF-7cells transfected with the recombinant vector pcDNA3.1/ER-α36(the MCF-7/ER-α36) are the research objects. The cell growth was monitored with the MTT assay. The cell cycle and cell apoptosis index were detected by the flow cytometry. The tumorigenicity was observed by the colony formation test. The adhesion between tumor cells and extracellular matrix was observed by cell adhesion test. The cell invasion and metastasis was detected by Transwell chamber experiments. The expression levels of NF-κB p65, MMP-2, MMP-9, and TIMP-1were analysed by Western blot. MCF-7/ER-α36cells were treated with10-8mol/L testosterone to observe the impact of androgen on the above malignant behaviors and related protein expression.Results:There was no significant difference between the malignant behavior of the MCF-7/W cells and that of MCF-7/pcDNA3.1cells. Compared with MCF-7/W group, the growth curve of MCF-7/ER-α36cells was steep. Cell growth was ahead of1day into platform period and the populations of G0/G1phase cells decreased by12.33%, while the proportion of cells in S phase increased by39.96%. Apoptosis rate was reduced by56.84%. The colony formation rate after growing2weeks, the adhesion rate after growing2h and the number of penetrating cells after growing24h were increased by50.37%,42.31%and70.83%, respectively. The above changes had significant differences (P<0.05). The above trend was further increased and also had a significant difference (P<0.05) in MCF-7/ER-α36treated with10-8mol/L testosterone.The Western blot analysis results showed that the relative expression level of NF-κp65, MMP-2, MMP-9, TIMP-1and MMP-2/TIMP-1, MMP-9/TIMP-1in MCF-7/ER36group were1.91times,1.68times,1.70times,1.60times and1.62times,1.30times of that in MCF-7/W, respectively, except TIMP-1protein expression having no significant increase. The above trend was further increased and also had a significant difference (P<0.05) in MCF-7/ER-α36treated with10-8mol/L testosterone. Added the NF-κB p65inhibitor PDTC into the above groups, the above indicators restored the levels of the MCF-7/W, which reduced by about20%-80%than that of having no PDTC. But MMP-2and MMP-9protein expression is still a significant difference (P<0.05).Conclusions:(1) ER-α36is the molecular marker of the ER-α66-positive breast cancer cells differentiating to the high degree of malignancy. ER-α36can promote the ability of the cell growth, cell proliferation, cell tumorigenicity in vitro, cell invasion and metastasis potential in the ER-α66-positive breast cancer cells, and accelerat its cell cycle progression and reduced apoptosis. The mechanism is ralated with increasing the expression level of MMP-9and MMP-2through NF-κB pathway, and breaking the balance between this and TIMP-1.(2) Testosterone can promote that ER-α36increase the malignant behaviors of breast cancer cells through NF-κB pathway. It is unlikely that the ER-α36breast cancer patients benefit from testosterone treatment. NF-κB may become one of the treatment targeting molecules of the estrogen receptor positive and high expression ER-α36breast cancer.