The Gene Expression Of INHA In Breast Cancer And Its Preliminary Study On The Regulation Of Breast Cancer Cell Proliferation

Introduction:Breast cancer is a malignant tumor of serious harm to human health. Its incidence is showing a rising trend in China. May be related to the exception of several genes, including oncogenes, tumor suppressor genes and cancer susceptibility genes, these genes play an important role in the incidence of breast cancer development and progression of transforming growth factor β (transforming growth factor) family consists of a of class structure, function-related polypeptide growth factor subfamily, including inhibin (inhibin), activin Activin, bone morphogenetic protein (BMP), growth differentiation factor (GDF). TGF-β superfamily in the regulation of cell growth, differentiation, apoptosis, adhesion, extracellular matrix synthesis and deposition, embryogenesis and tissue repair, plays an important role in inflammation and fibrosis. Path of any one link changes can lead to abnormal signal transduction, may be associated with tumor development and tumor metastasis of inhibin (inhibin) is a hormone secreted by the gonads and placenta, belonging to the structure of the transforming growth factor beta (TGF-beta) family members. The study found that the inhibiting factors of pyrophosphate-like glycoprotein dimer composed of a and P subunits form of a disulfide bond (-S-S) coupling constitutes its main function is the inhibition of pituitary follicle stimulating hormone (the generation of FSH). The a subunit is encoded by the INHA gene, its gene is1101bp, encoding366amino acid protein of this gene may have and TGF-P family members in tumors similar biological characteristics, while literature reported in breast cancer research stays in the detection of clinical specimens and there is more controversy, therefore, the breast cancer study, the use of viral expression technology, RNA interference, quantitative proteomics and mass spectrometry modern experimental methods, the INHA its potential physiological functions and its role in breast cancer research.Methods:1.The INHA monoclonal antibody preparation Prepared using the antigen peptide synthesis of INHA N-terminal and C-terminal monoclonal antibodies and antibody specificity were identified.2.The expression of INHA in12kinds of tumors (cancer and paraneoplastic pairing) Using immunohistochemistry to detect the expression level differences in12kinds of tumor tissues.3.Breast cancer tissue, adjacent tissues and breast-related diseases in the INHA expression detection. The use of immunohistochemistry to detect breast cancer tissue and adjacent tissues and breast related diseases INHA protein expression level differences.4.The stable expression of INHA the construction of the cell lines.4.1PCR method was used in the target gene of INHA, after digestion with pmel target fragment with lentiviral expression vector pWPI-IRES connected recombinant plasmid pWPI-IRES/TNHA and sequencing for viral packaging and infection of breast cancer cells MDA-MB-231and of MCF-7. 4.2Transiently transfected293FT packaging viral particles and infection of the breast cancer cell line MDA-MB-231and of MCF-7.4.3Using flow cytometry cell sorting to isolate the fluorescence intensity of cell clones. The cloned hair strong fluorescence cryopreservation and identification, in order to obtain stable high expression cell clones of the target sequence.5.The construction of the stable interference INHA cell lines. Zimmer packaging specific interference INHA gene lentiviral particles infection of MDA-MB-231recombinant cell lines.6.Identification of stable expression and interfere with the INHA cell line6.1real-time fluorescent quantitative RT-PCR identification of stable cell interference6.2Western blot analysis and stable high expression of INHA and stable interference INHA cells7. The INHA infuences on breast cancer cell growth The CCK8France observed high expression of transfected and interference carrier cell proliferation and cell growth curve, and at the same time set the corresponding control cells.8.Interfere with the INHA gene in breast cancer cells subcutaneously into the tumor Interfere with the INHA gene and the control group, MCF-7breast cancer cell lines to nude mice experiments, and then collect data for analysis.9. Through2D/MALDI-TOF/TOF proteomics approach to analyze the differences of the cell MCF-7/NC and MCF-7/CD expressed proteins, and related proteins by Western Blot were identified.10.The INHA gene through the cell cycle protein targeting of the mechanism in breast cancer cells.10.1Through real-time quantitative PCR detection Cycle protein cyclin A, cyclin and cyclin D expression in INHA interfere with cell lines10.2By flow cytometry cell cycleResults:Successfully prepared, respectively, of INHA N-terminal and C-terminal monoclonal antibodies, immunofluorescence and Western blot identification of show we prepared antibodies specific.2INHA protein staining in tumor tissue.2.1The12kinds of tumor tissues INHA expression difference Immunohistochemical analysis revealed that, INHA expression in the esophagus, stomach, liver, rectum, lung, breast, cervical, ovarian and prostate cancer tissue was significantly lower than the adjacent tissues in the colon and kidney cancer organizations and Differential expression of adjacent tissues was not obvious, INHA in pancreas cancer tissues was higher than the adjacent tissues.2.2breast cancer organizations, as well as of INHA the adjacent tissues differentially expressedImmunohistochemical analysis showed that the two antibodies detect the INHA expression in adjacent normal tissue and cancer tissue were significantly different (t=12.61, p<0.001) and (t=12.82, p <0.001), poor values were respectively7.33and8.08, so the cancer adjacent tissue of INHA (C1and N2) expression were significantly higher in cancer tissue.2.3TheINHA expression difference in breast-related diseases Immunohistochemical analysis showed that the two antibodies were detected by INHA expression in different breast-related diseases, the next expression in the benign lesions and cancer were higher than all kinds of breast cancer tissue is found in different kinds of breast disease, two antibody test results are not consistent with carcinosarcoma. 3.Genewas cloned successfully constructed recombinant plasmid pWPI-IRES/INHA.4.293FT cells for virus packaging, and successfully build a high-expression and interfere with the INHA the of MCF-7, MDA-MB-231cell line.5.In vitro experiments showed that interference INHA inhibit breast cancer cell growthCCK8cell proliferation assay results showed that breast cancer cells transfected with siRNA expression vector, statistical analysis showed that MCF-7cell proliferation reduced ability to interfere with the INHA gene (P<0.05).6.1nterfere with the INHA gene in breast cancer cells subcutaneously into the tumor Data collection, analysis, interference reduced the INHA inhibition of MCF-7cells subcutaneously into nude mice tumorigenicity in nude mice tumor.7.2D/MALDI-TOF/TOF proteomics approach to analysis the cells MCF-7/NC MCF-7/CD differences expressed proteins, and related proteins by Western Blot were identified.The analysis of interference with the INHA the MCF-7cell protein expression profiles display interference the INHA make some tumor-promoting protein was decreased, may be related to cell cycle regulation.8. INHA gene in the mechanisms of breast cancer cells.8.1Through real-time quantitative PCR detection of the INHA interference cell lines cycle protein cyclin A, cyclin expression of cyclin D1expression shows interference the INHA can be significantly lowered Cyclin A, B, D1, mRNA levels, especially CyclinD land B.8.2By flow cytometry cell cycleThe test results showed that:interference INHA gene transfected breast cancer cells MCF-7breast cancer cell line MCF-7from G1phase to S phase of the process is significantly retarded the results to complete a cell cycle time required to extend the lead to cell proliferative ability decreased.Conclusion:Two antibody staining showed the expression of INHA gene in breast cancer tissue is a downward trend. Different types of breast disease showing strong expression of benign tissue and weak in malignant tissue. Sarcoma, the expression of the N-terminal antibody and C-terminal antibody is inconsistent, the C-terminal antibody staining was significantly increased. The expression of the intensity close to benign tissue.This shows that different splicing forms of the INHA may behave differently in different types of organizations. Over-expression or interfere with the INHA gene can inhibit breast cancer cell proliferation and cell cycle progression, suggesting that the INHA gene is a very complex genes, the functions in different cell types may be inconsistent. However, we still preliminary confirmed INHA gene can delay cell cycle progression by inhibiting the expression of the cyclin, and laid a good foundation for more study of the depth mechanism...