Associations Between Breast Cancer And The Polymorphism Of FGFR-2,a Potential Target Gene Of MicroRNA-494

BackgroundBreast cancer(BC)is the most common cancer affecting women worldwide.BC is a complex heterogeneous disease that can be divided into hormone receptor-positive,human epidermal growth factor receptor-2 overexpression(HER2+),and triple-negative breast cancer(TNBC)according to histologic characteristics.There are various factors inducing BC,such as induced abortion,induction of ovulation with clomiphene citrate and gonadotropins,ionizing radiation,alcohol abuse,and cigarette smoking.Due to shortcomings in breast self-examination and clinical examination,BC is difficult to be detect in the early stage,thus delaying the timely treatment of patients.Therefore,improving the prognosis and survival rate of BC through early examination is still a top priority.It is therefore of great significance for the prevention and treatment of BC to determine the risk factors and internal pathogenesis of BC.ObjectiveThis study aimed to determine the effects of miR-494 on the proliferation,apoptosis,migration,and invasion of BC cells by mediating fibroblast growth factor receptor-2(FGFR-2).We first explored the significance of FGFR-2 and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha(PI3KCA)gene variation in BC and the relationship with prognosis.Subsequently,by studying cell proliferation,apoptosis,migration,and invasion,the effect of miR-494 overexpression or silencing on the behavior of BC cells(MDA-MB-453 and MCF-7)was examined.Based on the regulatory relationship between miR-494 and FGFR-2,the inhibitory effect of miR-494 on proliferation,migration,and invasion of BC cells,the promoting effect of miR-494 on apoptosis,and the inhibitory effect of miR-494 on the RAS/RAF/mitogen-activated protein kinase(MEK)/extracellular regulated protein kinase(ERK)and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)signaling pathways were determined.We attempted to explain the molecular mechanism underlying miR-494 regulation BC through FGFR-2 to provide strong theoretical support and strategies for the treatment of BC.MethodsChapter 1:Relationship between FGFR-2 and PI3KCA gene mutations and BC risk in a Chinese populationThree hundred twenty-eight BC patients and 389 healthy controls were genotyped.Subsequently,we evaluated the association between FGFR-2 rs 1219648 and PI3KCA rs6443 624,and BC susceptibility and clinicopathologic features,including age,tumor size,clinical stage,grade,lymph node infiltration,estrogen receptor(ER),progesterone receptor(PR),and HER2 gene status.The log-rank test Kaplan-Meier curve was used to evaluate the correlation between FGFR-2 rs1219648 and PI3KCA rs6443624 with prognostic indicators to explain the relationship between these genes and the risk and survival rate of female Chinese patients with BC.Genomic DNA was extracted using a DNA extraction kit and genotyped.Chapter 2:Effect of miR-494 overexpression or silencing on the behavior of BC cells(MDA-MB-453 and MCF-7)The expression of miR-494 in MCF-10A,SK-BR-3,MDA-MB-231,MDA-MB-453,BT-549,MCF-7,and other BC cell lines and BC tissues was detected.Based on the level of expression,MDA-MB-453 and MCF-7 cells were selected for subsequent experiments.Then,we overexpressed or silenced miR-494 in the selected cells,detected cell proliferation by cell viability and the expression of cyclin D1,cyclin-dependent kinase(CDK)4,and CDK.6.The apoptosis rate and protein expression of apoptosis-related factors,such as pro-caspase 3,cleaved-caspase 3,and Bax,were used to detect cell apoptosis.The cell migration rate,invasion rate,and levels of protein expression of related factors,such as matrix metallopeptidase 9(MMP-9)and vimentin,were used to detect cell migration and invasion.In this study qRT-PCR was used to detect the expression of miR-494 in BC cell lines.A Cell Counting Kit-8(CCK-8)was used to detect cell viability.Lipofectamine 3000 reagent was used for cell transfection.Propidium iodide(PI)and fluorescein isothiocyanate(FITC),coupled with Annexin V staining,were used to detect the apoptosis rate.The dual-chamber migration method was used for cell migration and invasion.Western blot analysis was used to detect the expression of cyclin D1,CDK4,CDK6,pro-caspase 3,cleaved-caspase 3,Bax,MMP-9,vimentin,and ?-actin expression.Chapter 3:miR-494 inhibits the behavior of MDA-MB-453 and MCF-7 cells by targeting the FGFR-2 geneMiR-494 mimics or negative control(NC)mimics were first transfected into MDA-MB-453 and MCF-7 cells,and the expression of FGFR-2 was detected 48 h after transfection.A dual-luciferase reporting experiment was used to detect the targeting regulation of miR-494 on FGFR-2.NC mimic and pEX-2,miR-494 mimic and pEX-2,and miR-494 mimic and pEX-FGFR-2 were co-transfected into MDA-MB-453 and MCF-7 cells to detect cell viability,the apoptosis rate,the cell migration rate,the cell invasion rate,the changes in cell cycle-related factor expression,such as cyclin D1,CDK4,and CDK6,apoptosis-related factor expression,such as pro-caspase 3,cleaved-caspase 3,and Bax,and cell migration and invasion-related factor expression,such as MMP-9 and vimentin,to determine the effect of miR-494 on proliferation,apoptosis,migration,and invasion of BC cells by regulating FGFR-2 expression.Finally,the protein expression of core factors in the RAS/RAF/MEK/ERK(RAS,RAF,p/t-MEK.and p/t-ERK)and PI3K/AKT signaling pathways(p/t-PI3K and p/t-AKT)were detected to determine the molecular mechanism underlying the miR-494 effect on proliferation,apoptosis,migration,and invasion of BC cells by regulating FGFR-2 expression.The dual-luciferase reporting experiment was used to detect whether FGFR-2 is the target gene of miR-494.The CCK-8 method was used to detect cell viability.Lipofectamine 3000 reagent was used for cell transfection.A FITC-Annexin V/PI detection kit was used to detect the apoptosis rate.The dual-chamber migration method was used for cell migration and invasion.Western blot analysis was used to detect the expression of FGFR-2,cyclin D1,CDK4,CDK6,pro-caspase 3,cleaved-caspase 3,Bax,MMP-9,vimentin,and?-actin protein.ResultsChapter 1:Relationship between FGFR-2 and PI3KCA gene mutations and BC risk in a Chinese populationWe showed that FGFR-2 rs1219648 and AG alleles were significantly correlated with clinical stage and grade in BC patients,and the GG allele was significantly correlated with grade in BC patients.At the same time,the FGFR-2 rs1219648 dominance model also showed a significant correlation with clinical grade in BC patients.For pi3kca rs6443624,the AC allele was significantly associated with clinical stage and the AA allele was significantly associated with grade.In addition,the PI3KCA rs6443624 dominance model was significantly correlated with clinical stage.The prognostic impact of FGFR-2 rs1219648 or PI3KCA rs6443624 in BC patients showed that patients with AG and GG alleles had shorter survival times than patients with the AA allele of FGFR-2 rs1219648.There was no difference between each allele in PI3KCA rs6443624 among the patients.In addition,we used the stepwise Cox regression model for multivariate analysis,and found that clinical stage,and ER and PR status may be important prognostic indicators.Chapter 2:Effect of miR-494 overexpression or silencing on the behavior of BC cells(MDA-MB-453 and MCF-7)We showed that miR-494 had low expression in MCF-10A,SK-BR-3,MDA-MB-231,MDA-MB-453,BT-549,MCF-7,and other BC cell lines and tissues.Because the expression of miR-494 in MDA-MB-453 and MCF-7 cells was lower than other cells,MDA-MB-453 and MCF-7 cells were selected for subsequent experiments.We then overexpressed miR-494 in the selected cells and found that miR-494 overexpression reduced the vitality and proliferation of MDA-MB-453 and MCF-7 cells,reduced the expression of cyclin DI,CDK4,and CDK6,increased the apoptosis rate and promoted apoptosis,increased the expression of apoptosis-related factors(cleaved-caspase 3 and Bax)protein,reduced the cell migration and invasion rates,and reduced the expression of cell migration and invasion-related factors(MMP-9 and vimentin)protein.In addition,the results showed that miR-494 silencing improved the viability and proliferation of MDA-MB-453 and MCF-7 cells,promoted the expression of cyclin D1,CDK4,and CDK6,had no obvious effect on apoptosis,promoted cell migration,the invasion rate,and expression of invasion-related factors(MMP-9 and vimentin)protein.Chapter 3:miR-494 inhibits the behavior of MDA-MB-453 and MCF-7 cells by targeting FGFR-2 geneThe results showed that overexpression of miR-494 inhibited the expression of FGFR-2 in MDA-MB-453 and MCF-7 cells.The results of the dual-luciferase report experiment confirmed that FGFR-2 is a target gene of miR-494.FGFR-2 overexpression promoted the vitality,cell migration,and invasion rate of MDA-MB-453 and MCF-7 cells,up-regulated the expression of cell cycle-related(cyclin D1,CDK4,and CDK6)and cell migration and invasion-related factor(MMP-9 and vimentin)protein,reduced the apoptosis rate,and down-regulated the expression of apoptosis-related factors(c/p-caspase 3 and Bax).In addition,miR-494 overexpression down-regulated the expression of core factors of the RAS/RAF/MEK/ERK(RAS,RAF,p/t-MEK,and p/t-ERK)and PI3K/AKT signaling pathway(p/t-PI3Kand p/t-AKT)protein in MDA-MB-453 and MCF-7 cells,while FGFR-2 overexpression reversed this trend.ConclusionsIn this study,we systematically studied the effect and molecular mechanism underlying miR-494 on the proliferation,migration,invasion,and apoptosis of BC cells by regulating FGFR-2 expression.(1)In a Chinese population,FGFR-2 rs1219648 and PI3KCA rs6443624 were shown to be associated with BC risk.Single nucleotide polymorphisms of FGFR-2 rs1219648 and PI3KCA rs6443624 may contribute to the identification of high-risk BC patients and may be a potential prognostic factor for BC in a Chinese population.(2)MiR-494 overexpression inhibited the proliferation,migration,and invasion of MDA-MB-453 and MCF-7 cells,and promoted apoptosis.MiR-494 silencing improved the proliferation,migration,and invasion of MDA-MB-453 and MCF-7 cells,but had no obvious effect on apoptosis.(3)Mir-494 inhibited FGFR-2 expression and FGFR-2 was a target gene of miR-494.(4)MiR-494 overexpression inhibited the proliferation,migration,and invasion of BC cells and promoted apoptosis by regulating FGFR-2.(5)Mir-494 inhibited RAS/RAF/MEK/ERK and PI3K/AKT signaling pathways by regulating FGFR-2 expression.