FTO And GEN1in Mammary Gland Development And Tumorigenesis

Research background:Breast cancer is the first common tumor malignant among the worldwidewomen. Each year about1500000women are diagnosed with breast cancer, inwhich500000people died. In recent years, the incidence of breast cancer increasedyear by year, and present the trend of younger age, bring the great pressure anddamage to the female body and mind. The mechanism of breast cancer was quite acomplex pathological process, lots of research showed that early menarche, latemenopause, obesity, hormone inappropriate ingestion are predisposing factors forbreast cancer, In the last two decades，there is little progression on treatment ofbreast cancer. It has been a major research subjects to improve the therapeutic effectsof breast cancer. Molecular targeting therapy have been successful in many kinds oftumor treatment，but failed in breast cancer according that the mechanism of diseaseis unclear. Therefore，the molecular mechanism of breast cancer is need to confirmfurther which should proceed with analyzing of abnormal expression of oncogeneproteins and exploring more appropriate molecular targets， which may bringbreakthroughs to treatment of breast cancer.Genomewide association studies (GWAS) have indicated that SNPs on achromosome16locus encompassing FTO，430KB，containing9exons，are robustlyassociated with human obesity，FTO was susceptible to disease in type2diabetes，cardiovascular disease，but its relationship with breast cancer and mammary glanddevelopment have not been clear. Studies confirm that FTO exons rs9939609polymorphism and abnormal glucose levels and adipose accumulation relatedproduct index (LAP)，LAP is alternative sign of diabetes and cardiovascular diseasein postmenopausal women; The crowd in Europe and Asia several studies haveconfirmed that the FTO gene rs9939609are closely associated with increased risk ofmetabolic syndrome (MS). Hormone levels at the same time testing results show that the FTO gene variant associated with free testosterone and insulin increasedsignificantly; Clinical studies have shown that the FTO gene exon1of4SNPs areassociated with breast cancer，and provide effective classification identificationpredict the risk of breast cancer，these studies indicate that FTO mutation andabnormal expression may be closely related to the development of breast cancer，however，the molecular mechanisms involved are still remains to be elucidated.DNA damage repair gene mutation and abnormal expression of thought is closelyassociated with the occurrence of tumor. The central intermediate of HR is Hollidayjunc-tions (HJs)，which are resolved by a class of structure-specifc nucleases. As acrucial HJ resolvase，GEN1has proven to be a centrosome-associated proteinresponsible for centrosome integrity，and the depletion of GEN1results in aberrantcentrosome amplifcation，multinuclei formation，DNA damage，and increasedapoptosis. GENl as new members of the family of XPG，holliday connecting withdecomposition，ensuring that the DNA repair function smoothly，in GEN1found inpatients with breast cancer mutations，For GEN1in breast cancer specific biologicalfunction，especially the gene molecular mechanism is unclear. For GNE1involved inregulating the development mechanism of tumor to clarify depends on theparticipation in the regulation of signaling pathways and downstream target genes ofrecognition，but at present，little is known，there is an urgent need to explore it.Research objectives:1. Screen the expression pattern of FTO，GEN1in breast tumor.2. Analyze the correlation between the expression level of FTO,GEN1andclinical data.3. Complete assess the function of Gen1,FTO in generation and development ofbreast cancer. Discuss the mechanism of FTO,GEN1promotes cancer，clarify thesignal pathway and target genes regulated by GEN1and FTO.Research method:1.Separation female mice (C57BL/6) at different stages (puberty，pregnancy，lactation，from milk period) of the breast skin cells， the dynamic role of theGEN1,FTO expression pattern was investigated during the normal mammary glanddevelopmentin various physiological stage of correlation; 2.The protein expression levels of FTO，GEN1were detect by western blot.3.The subcellular localization of GEN1and FTO were analyzized byimmuno-fluorescence in primary culture of mouse mammary epithelial cells.4.Application of RNAi technology, instantaneous interference of FTO in breastcancer cells by immunofluorescence analysis, explore the FTO’s relationship withthe udder cell histone methylation5.Using DNA damage model, this paper discusses the influence of Gen1onbreast x7890ancer cell DNA damage repair.Research result:1.FTO and GEN1showed specificity of change in different stages of thedevelopment of mammary gland, in adolescence FTO expression basically stable,into the expression of FTO began to decline after pregnancy, lactation FTOexpression quantity further decrease L18days express the lowest, after entering fromthe breast feeding FTO expression quantity increased, reached FTO linked to fatmetabolism, presumably FTO and adjust the breastfeeding and breast milk and fadephysiological activity plays an important role; Adolescence basically stable, thenumber of GEN1expression after entering pregnancy began to rise in the number ofGEN1expression appeared a peak, the number of GEN1breastfeeding expressionquantity decrease L18days express the lowest, after entering from the breast feedingGEN1expression gradually stabilized, the changes in volatility may be associatedwith the activity of breast tissue DNA replication2. Cellular localization analysis showed that the mammary gland epithelial cellsof split phase, the expression of FTO mainly located in nucleus and cytoplasm lessquantity distribution; Rare mitosis meta phase, the end of the FTO mainly locate inthe spindle fiber (alpha-tuberculin) and cytoplasm, expressing quantity increased,suggesting FTO function may be the udder cell mitosis has close relationship;Combined with the FTO MCF-7in breast cancer cells and MDA-MB-231differentchanges of cell cycle, we hypothesized that FTO influence on mammary gland cellcycle changes, related to its Cellular localization not its expression quantity.3.FTO expression level is negatively related to the degree of H3K4me3lamentation, also prompted us FTO in the occurrence and development of breast cancer also has the key value.4.The Cellular location of GEN1was close to the central body, which is directlyrelated to the function and its involved in DNA damage repair.5.Breast cancer cell DNA damage experiment results show that the GEN1inbreast cancer cells in the process of DNA damage, may has important protectiveeffect, its expression quantity increase is directly related to DNA damage repairactivity enhancement.Research conclusions:This study completed the research on gene function of FTO and GEN1at thecellular level，molecular level and animal level，and proved that FTO，GEN1forbreast cell growth and function of breast cancer research，made clear and the Wehave found that the elevated expression of FTO and GEN1was associated withvirgin proliferating mammary，The Cellular localization of FTO and GEN1showedclosely related to the rest of the mammary gland development; In breast cancer，theexpression of FTO can promote tumor cell proliferation，cell clone formation andinhibition of apotheosis，and GEN1expression inhibition of cell proliferation，cellclone formation and promote apotheosis.the effects on tumor in breast cancer cellproliferation is closely related to the cancer cell DNA damage repair. New molecularmechanism stud may give little help to understand the occurrence and developmentof breast cancer，breast cancer for the study of DNA damage repair and the relevanceof research. In addition，this study also for the development of FTO and GEN1astargets for breast cancer molecular targeted therapy provides a theoretical basis andexperimental basis.