Study Of The Demethylase FTO Expression In Human Breast Cancer And Its Effects On Biological Behaviors Of Breast Cancer Cell Lines

Obesity is one of the important risk factors for breast cancer.Fat mass and obesity associated gene(FTO)has been reported to be associated with several tumors,including breast cancer.On the other hand,recent studies found that FTO was a member of the ALKB(bacterial DNA repair enzyme)protein family.The main substrate of FTO is 6-methyladenine(m~6A),involving in the modification of m~6 A demethylation.Current studies have shown that the m~6 A methylation is involved in the occurrence and development of multiple tumors.So far,the expression of FTO in breast cancer,particularly in the different molecular subtypes of breast cancer,and its role of demethylation on the development of breast cancer are still unclear.The purpose of this paper is:1)to detect the FTO expression in the normal breast epithelial tissue,the primary lesion and matched metastases of breast cancer,collect the detailed clinical data and follow-up data,and analyze the differences of FTO expression among the normal breast epithelial tissue,the primary lesion and matched metastases of breast cancer,and its relationships with clinical parameters and survival data;2)to establish the human breast cancer cell lines of wild-type FTO and FTO R96 Q missense mutation body over-expression,down-regulate the expression of FTO using small interference RNA,and explore the effects of FTO expression and its demethylation on the biological behaviors,including m~6 A levels in RNA,proliferation,invasion,apoptosis and so on.Further to explore its initial mechanism by testing the levels of Vimentin\TGF-β1\C-myc and so on.Chapter Ⅰ Expression of FTO protein in human breast cancer tissues and their relationships with clinical parameters and survival dataObjectiveTo test the expression of FTO protein in normal breast epithelial tissues,breast cancer primary tumors and their matched metastases,and to analyze the differences of FTO protein expression among the normal breast epithelial tissue,the primary lesion and matched metastases of breast cancer,and their relationships with clinical parameters and survival data.MethodsArchival formalin fixed,paraffin embedded specimens from 108 consecutive primary breast cancer patients in Cancer Hospital of Guangxi Medical University between January 2009 to June 2014 were collected,including 63 recurrent cases.Forty-six patients without recurrence were followed for at least 3 years,and 41 normal breast tissues were also collected.All tissues were collected before receiving any cancer treatment.All patients were pathologically diagnosed as invasive carcinoma.FTO protein expression in the normal breast epithelial tissue,the primary lesion and matched metastases of breast cancer was detected by using immunohistochemical assay.The differences of FTO protein expression among the normal breast epithelial tissue,the primary lesion and matched metastases of breast cancer,and their relationships with clinical parameters,survival data were analyzed.Results1.FTO protein was expressed in both mammary epithelial and breast cancer tissues.FTO protein expression in breast cancer was significantly higher than that in normal tissue(46.5% vs 29.3%,P=0.03).Stratified analysis showed that in the BMI≥24 group,the high expression rate of FTO protein in breast cancer tissues was higher than that in normal breast epithelial tissues(51.5%vs 25.0%,P=0.03);2.In Luminal B subtype,the high expression rate of FTO protein in the primary cancer of relapsed group was 60%,which was significantly higher than that of non-relapsed homotypic breast cancer patients(60.0% vs 26.1%,P=0.027).3.There were no significant associations between FTO protein expression and age,BMI,T stage,N stage,clinical stage,ER status,PR status,HER-2status and Ki-67 index in breast cancer(all P﹥0.05).4.There was no correlation between the expression level of FTO protein in primary cancer tissues and the recurrence time of breast cancer patients.5.In 63 matched breast cancer primary and metastatic lesions,the total rate of change in FTO protein levels between the primary and metastatic breast cancers was 49.2%,and the high expression rate of FTO in the primary tissue and metastatic tissue was 44.4 % and 49.2%,respectively.The expression of FTO protein was not significantly different between primary lesions and metastatic lesions(P=0.590).Further analysis of the expression of FTO protein in primary and metastatic lesions in different subtypes of breast cancer also found no significant difference,but the change rates of FTO protein in luminal B,HER-2 over-expressing,and base-like subtype were:52.5%,50%,and 50%,respectively.Conclusion1.The expression of FTO protein in breast cancer tissues was higher than that in normal breast epithelial tissues.2.In the Luminal B subtype,the high expression rate of FTO protein was significantly higher in the relapsed breast cancer group than in the relapse-free patients.3.There were no significant associations between FTO protein expression and age,BMI,T stage,N stage,clinical stage,ER status,PR status,HER-2 status and Ki-67 index in breast cancer.4.There were significant changes in the expression of FTO in primary lesion and metastatic foci of breast cancer.Chapter Ⅱ Construction and identification of breast cancer cell lines with different FTO expressionObjective:The construction of different expression levels of FTO and FTO R96 Q missense mutation in human breast cancer cells MCF-7 and MDA-MB-231 cells lay the foundation for exploring the effect of FTO expression on the biological behavior of breast cancer cells and the molecular mechanism.Method:Construction of lentivirus vectors expressing different FTO expression levels and FTO R96 Q missense mutants,sequencing and verifying the correctness of the plasmid,and infecting human breast cancer cell lines,q PCR and Western blot to determine the infection effect of FTO after lentivirus vector transfection.Result:1.FTO overexpression plasmid and FTO R96 Q missense mutation plasmid were constructed and sequenced successfully,and FTO sh RNA knockdown plasmids were also constructed.2.After transfection of different FTO plasmid,the FTO mRNA of MDA-MB-231 cells in the groups of wild-type FTO expression plasmid,FTO R96 Q mutant expression plasmid were 7.66±0.398,3.50± 0.431,respectively;which were significant higher than that in no-load plasmid group(1.64±0.036)(P all < 0.05).The relative expression of FTO m RNA in MDA-MB-231 cells after transfection of si RNA was 0.21± 0.013,which was significantly lower than that of the no-load plasmid group(P < 0.05).3.The change of FTO protein expression in MDA-MB-231 cells with different plasmids were consistent with that of FTO m RNA,and the expression of FTO was increased in the wild-type FTO overexpression plasmid group and the FTO R96 Q mutant overexpression plasmid group.The expression of FTO protein in the Si RNA interference group decreased.4.After transfection of different FTO lentiviral vectors into MCF-7 breast cancer cells,FTO did not change significantly at m RNA and protein levels.Conclusion:We successfully established MDA-MB-231 cell lines with different expression of FTO gene.Chapter Ⅲ Experimental study on the effects of FTO gene expression level and FTO-R96 Q missense mutant on the biological behavior of MDA-MB-231 cellObjectiveTo explore the effects of FTO expression and its demethylation on the biological behaviors,including proliferation,invasion,apoptosis and so on.Further to explore its initial mechanism by testing the levels of Vimentin 、TGF-β1、C-myc and so on.Methods1.The Epi Quik? m~6 A RNA Methylation Quantification Kit(Colorimetric)is used to detect N6-methyladenosine(m~6A)RNA methylation status directly using total RNA.2.MTT assay was used to detect the changes of cell growth and proliferation,and to draw the cell growth curve.2.The migration and invasion of breast cancer cells were determined by Transwell assay.3.Flow cytometry was used to detect the changes of cell cycle and apoptosis after transfection.4.Real time quantitative PCR technique was used to detect the expression of Vimentin、TGF-β1、C-myc m RNA in MDA-MB-231 after transfection.Results1.FTO overexpression decreased by 15% the levels of m~6 A,FTO depletion enhanced 79% levels of m~6 A.After transfection of FTO R96 Q mutant plasmid,m~6 A levels in RNA of MDA-MB-231 cells increased by 30%.2.Results determined by MTT showed that the absorbance of wild-type FTO overexpression cell lines in 24,48,72,96 hours were:0.335±0.01、0.60±0.07、0.867±0.147 、 1.216±0.006,respectively,which were not different from the empty plasmid group(0.305±0.004、0.452±0.182、0.615±0.015、0.882±0.012).The absorbance of FTO R96 Q mutant overexpressed cells was 0.542±0.009、0.860±0.01、1.13±0.048、1.238±0.010,respectively,and was significantly higher than that in the control plasmid group.2.The results of Transwell showed that the number of transmembrane cells in the wild-type FTO overexpression group was 167.4±10.2,more than that of the control plasmid group(41.6±6.3)(P < 0.01).The number of transmembrane cells in the Si RNA interference group was 32.5±4.8,slightly less than the number of transmembrane cells in the control plasmid group,and the difference was not statistically significant.The number of transmembrane cells in FTO R96 Q mutant overexpression plasmid group was 78±7,more than that of the control plasmid,but significantly less than that in the wild-type FTO overexpression group.3.Cell cycle results showed that there was no significant difference in the cell cycle composition of among the wild-type FTO overexpression group,FTO R96 Q mutant overexpression plasmid group and Si RNA interference group(P=﹥0.05).4.The study of apoptosis showed that after interfering with FTO expression,the total apoptosis rate of MDA-MB-231 cells was 18.12%,significantly higher than that of the control plasmid group(7.01%).The apoptosis rates of the wild-type FTO overexpression group was 5.96%,which were statistically significant lower compared with that of the control plasmid group.The apoptosis rates of FTO R96 Q mutant overexpression plasmid group were 9.6%significantly higher than that of the control plasmid group.5.For tumor migration related factors,Vimentin,TGF-β1 mRNA levels in the wild-type FTO overexpression group increased,Vimentin m RNA levels in the FTO R96 Q mutant overexpression plasmid group obviously decreased,and after interfering with FTO expression,Vimentin m RNA level decrease,and TGF-β1 m RNA levels obviously increased.For tumor proliferation related factors,there was no obvious change of Cyclin D1 m RNA level in wild type FTO express group,and Cyclin D1 and C-myc m RNA level obviously decreased in the FTO R96 Q mutant overexpression plasmid group.After interfere with the FTO expression of MDA-MB-231 cells,the Cyclin D1 m RNA decreased and C-myc m RNA increased.Conclusion1.FTO expression and m~6 A levels of in MDA-MB-231 cell RNA are inversely correlated.Level of N6-methyladenosine(m~6A)is decreased in cells over-expressing FTO.The FTO-sh RNA in stable lines exhibit a noticeable increase in m~6 A level compared with the control lines.Overexpression of R96 Q,a FTO missense mutant lack of demethylase activity,also increase cellular m~6 A.2.The proliferation of MDA-MB-231 cells enhanced after FTO R96 Q missense mutation plasmid transfected.There were no significant differences of the proliferation ability in MDA-MB-231 cells among the wild-type FTO overexpression group,FTO Si RNA group and the control group.3.The migration ability of MDA-MB-231 cells obviously reduced after interfering with FTO expression.The migration ability of MDA-MB-231 cells enhanced in the wild-type FTO overexpression group,and obviously decreasedin the FTO R96 Q mutant overexpression plasmid group.3.There were no effects of FTO expression on the cell cycle of MDA-MB-231 cells.4.The apoptosis rate of MDA-MB-231 cells increased after interfering with FTO expression and the FTO R96 Q mutant overexpression plasmid group.The apoptosis rate of MDA-MB-231 cells significantly decreased in the wild-type FTO overexpression group.5.In MDA-MB-231 cells,Vimentin,TGF-β1 m RNA levels in the wild-type FTO overexpression group increased,Vimentin m RNA levels in the FTO R96 Q mutant overexpression plasmid group and the FTO si RNA group decreased,suggesting that FTO gene may affects cell migration through TGF-β1 pathway,this effect may be related to its demethylation.