The Role And Mechanism Of Epigenetic Regulation In Breast Cancers

Part I The promoter status of RASSF6 and its function and potential mechanismObjective:1 Detection of RAS association domain family member 6(RalGDS/AF-6 domain family member 6, RASSF6) in human breast cancer cell lines and primary breast tissues. e2 Detection the promotr DNA hypermethylation of RASSF6 in human breast cancer cell lines and human breast cancers.3 Verify the function of RASSF6 on cell proliferation, cell cycle, apoptosis, cell migration and invasion in breast cancer cells.4 To investigate the mechanism of anti-tumor effect.Methods:1 the expression of RASSF6 was measured by RT-PCR, Western blot in human breast cancer cell lines, normal human tissues and normal breast tissues, and the expression of RASSF6 in breast cancer tissues and adjacent tissues were measured by real-time PCR, the expression of RASSF6 in normal breast tissues and breast cancers was analyzed by searching the Oncomine microarray database.2 the promoter DNA hypermethylation was measured by the Methylation-specific PCR(MSP) in human breast cancer cell lines and primary breast tissues, and the promoter DNA hypermethylation was measured by BGS in#115samples.3 the proliferation was measured by colony formation assay, soft agar assay, CCK-8 assay and EdU incorporation assay in vitro.4 the proliferation was measured by xenografts in nude mice in vivo.5 the cell cycle status was measured by flow cytometry, and the expression of p21 and p27 was measured by Western blot.6 the apoptosis was analyzed by AO/EB staining and flow cytometry in breast cancer cells, also, the expression of cleaved-caspase3, cleaved-caspase7, cleaved-caspase9 and cleaved-PARP was measured by Western blot.7 the migration was measured by wound-healing assay and Transwell assay, and the invasion was measured by Transwell assay with Matrigel, also, the expression of S100A4 and MMP2 tested by Western blot.8 the Akt signal pathway was determined by Western blot.Results:1 the expression of RASSF6 was widely expressed in human normal tissues and normal breast tissues; and in human breast cancer cell lines, several cells were down-regulated or silenced, including MDA-MB-231 and BT549, also, compared with adjacent tissues, the expression of RASSF6 is down-regulated in breast cancer tissues significantly.2 RASSF6 is methylated in MDA-MB-231 and BT549 cell lines and in primary breast cancers.3 RASSF6 inhibits the colony formation and the proliferation ability in vitro, and also suppresses the growth of xenografts in vivo.4 RASSF6 inhibits G0/G1 to S phase transition by increasing p21 significantly; and RASSF6 induces cell apoptosis by cleaving caspase3, caspase7, caspase9, PARR5 RASSF6 inhibits cell migration and invasion by upregulating S100A4 and MMP2 significantly.6 RASSF6 significantly suppresses the levels of phosphorylated Akt, and also the downstream target gene S100A4 and MMP2.Conclusion:1 the expression of RASSF6 is downregulated due to its promoter hypermethylation in breast cancers.2 RASSF6 as a tumor suppressor gene by targeting Akt signal pathway.Part Ⅱ miR-7-5p suppresses cell proliferation by targeting REGγ in breast cancersObjective:1 measure the anti-proliferation effect of miR-7-5p by suppressing the expression of miR-7-5p.2 to make sure that miR-7-5p suppresses cell proliferation by targeting REGy.3 to test the anti-proliferation effect of miR-7-5p in vivo.Methods:1 miR-7-5p inhibitor and its negative control are transfected transiently, and proliferation is measured by CCK-8, the expression of REGy is determined by Western blot, also the cell cycle is measured by flow cytometry.2 co-transfection miR-7-5p and REGγ transiently, proliferation and cell cycle are measured by CCK-8 and flow cytometry, respectively.3 the anti-proliferation effect of miR-7-5p is measured in xenografts, including the weight and the size of tumors, also the Ki-67 and REGy are measured by IHC.4 the correlation between miR-7-5p and REGy are analyzed by extracting the data from Oncomine microarray database.Results:1 Knockdown miR-7-5p promote the proliferation of breast cancer cells, and the transformation of G0/G1 to S phase, also the protein level of REGy upregulated significantly.2 REGy can reverse the anti-proliferation effect of miR-7-5p, measured by CCK-8 assay and flow cytometry.3 miR-7-5p inhibits the growth of the xenografts, also the expression of Ki-67 and REGy。Conclusion:1 miR-7-5p inhibits the proliferation of breast cancer cells by suppressing its downstream target gene REGy.