FOXL2基因对乳腺癌细胞MCF-7生长的影响
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摘要
目的检测FOXL2基因的过表达及沉默对乳腺癌细胞系MCF-7细胞生长状况的影响。方法培养乳腺正常细胞系HBL-100和乳腺癌细胞系MCF-7细胞,Trizol法提取两者总RNA,利用逆转录聚合酶链式反应(RT-PCR)技术分别检测FOXL2基因在这两种细胞中的表达水平。用FOXL2-pEGFP质粒和FOXL2-shRNA质粒转染MCF-7细胞,分别使其过表达或沉默FOXL2,并建立分组为过表达FOXL2(A组)及沉默FOXL2(B组)以及未作转染处理的MCF-7细胞(C组),Western blot检测3组FOXL2蛋白表达量,验证转染是否成功,MTT法测转染后3组细胞的增殖情况。结果 RT-PCR结果显示HBL-100和MCF-7细胞均可转录FOXL2的mRNA,且2~(-ΔΔCt)值> 2,证明FOXL2在MCF-7细胞中表现为上调,且差异有统计学意义(P <0.05)。成功转染MCF-7细胞后,Western blot结果与C组相比,A组细胞中FOXL2蛋白表达量明显增加,B组细胞中FOXL2蛋白表达量明显减少,差异均有统计学意义(P <0.05),可见模型构建成功。MTT法检测3组MCF-7细胞的OD值及细胞增殖率,A组FOXL2基因过表达,MCF-7细胞的增殖能力明显被抑制,而B组的细胞增殖能力增加,差异均有统计学意义(P <0.05)。结论 FOXL2基因在乳腺癌中高表达,且可以抑制乳腺癌细胞的增殖,可能为一个新的肿瘤抑制因子,但仍需要做进一步的研究。
Objective To investigate the effect of over-expressed and silenced FOXL2 gene on the growthof breast cancer cell line MCF-7. Methods HBL-100 and MCF-7 cells were cultured,and the total RNA wasextracted by Trizol reagent. We investigated the mRNA level of FOXL2 in HBL-100 and MCF-7 cells by reversetranscription polymerase chain reaction(RT-PCR). We overexpressed FOXL2 by FOXL2-pEGFP plasmid andsilenced it by FOXL2-shRNA plasmid transfection in MCF-7 cells and verified using Western blot. Finally MTTassay was used to measure the proliferation of these groups. Results The RT-PCR results demonstrated thatHBL-100 and MCF-7 cells could transcribe the FOXL2 mRNA,and the result of 2~(-ΔΔCt)was greater than two,which showed that FOXL2 is upregulated in MCF-7 cells(P < 0.05). After the transfection of MCF-7 cells,theWestern blot results showed that the expression of FOXL2 protein in group A was significantly up-regulated,andthe expression of FOXL2 protein in group B was significantly down-regulated compared with the blank control group(P < 0.05). MTT assay demonstrated that group A can obviously inhibit MCF-7 proliferation,and group B canobviously promote MCF-7 proliferation(P < 0.05). Conclusions FOXL2 was highly expressed in breast cancer,and can inhibit the breast cancer cell proliferation. It may be a novel tumor suppressor.
Objective To investigate the effect of over-expressed and silenced FOXL2 gene on the growthof breast cancer cell line MCF-7. Methods HBL-100 and MCF-7 cells were cultured,and the total RNA wasextracted by Trizol reagent. We investigated the mRNA level of FOXL2 in HBL-100 and MCF-7 cells by reversetranscription polymerase chain reaction(RT-PCR). We overexpressed FOXL2 by FOXL2-pEGFP plasmid andsilenced it by FOXL2-shRNA plasmid transfection in MCF-7 cells and verified using Western blot. Finally MTTassay was used to measure the proliferation of these groups. Results The RT-PCR results demonstrated thatHBL-100 and MCF-7 cells could transcribe the FOXL2 mRNA,and the result of 2~(-ΔΔCt)was greater than two,which showed that FOXL2 is upregulated in MCF-7 cells(P < 0.05). After the transfection of MCF-7 cells,theWestern blot results showed that the expression of FOXL2 protein in group A was significantly up-regulated,andthe expression of FOXL2 protein in group B was significantly down-regulated compared with the blank control group(P < 0.05). MTT assay demonstrated that group A can obviously inhibit MCF-7 proliferation,and group B canobviously promote MCF-7 proliferation(P < 0.05). Conclusions FOXL2 was highly expressed in breast cancer,and can inhibit the breast cancer cell proliferation. It may be a novel tumor suppressor.
引文
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[7]LEO F,BARTELS S,MAGEL L,et al.Prognostic factors in the myoepithelial-like spindle cell type of metaplastic breast cancer[J].Virchows Arch,2016,469(2):191-201.
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[12]YANG Y,YANG C,ZHU Y,et al.Intragenic and extragenic disruptions of FOXL2 mapped by whole genomelow-coverage sequencing in two BPES families with chromosome reciprocal translocation[J].Genomics,2014,104(3):170-176.
[13]GULATI,VERDIN,HALANAIK D,et al.Co-occurrence of congenital hydronephrosis and FOXL2-associated blepharophimosis,ptosis,epicanthus inversus syndrome(BPES)[J].Eur JMed Genet,2014,57(10):576-578.
[14]AO L,LIU Y.Clinical diagnose and significance of congenital sensorineural hearing loss combined with BPES[J].Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi,2015,29(18):1660-1663.
[15]GARCIA-ORTIZ J E,PELOSI E,OMARI S,et al.FOXL2functions in sex determination and histogenesis throughout mouse ovary development[J].BMC Dev Biol,2009,9:36.
[16]CHENG L,LI L,WANG L.A random forest classifier predicts recurrence risk in patients with ovarian cancer[J].Mol Med Rep,2018,18(3):3289-3297.
[17]GARDNER L,ANDERSON T,PLACE A R,et al.Sex change strategy and the aromatase genes[J].J Steroid Biochem Mol Biol,2005,94(5):395-404.
[18]CHOI Y J,HO J,LEE A.Disparate genomic characteristics of concurrent endometrial adenocarcinoma and ovarian granulosa cell tumor,revealed by targeted next-generation sequencing[J].Pathol Res Pract,2018,214(8):1231-1233.
[2]NOLAN A,JOSEPH N M,SANGOI A R.FOXL2 mutation status in granulosa theca cell tumors of the ovary[J].Int J Gynecol Pathol,2017,36(6):568-574.
[3]OHSHIMA K,HATAKEYAMA K,NAGASHIMA T,et al.Integrated analysis of gene expression and copy number identified potential cancer driver genes with amplification-dependent overexpression in 1,454 solid tumors[J].Sci Rep,2017,7(1):641.
[4]SCHUEBEL K E,CHEN W,COPE L,et al.Comparing the DNA hypermethylome with gene mutations in human colorectal cancer[J].PLoS Genet,2007,3(9):1709-1723.
[5]WEGMAN P,GOTHLIN A,LINDLOF A,et al.Expression of the forkhead transcription factor FOXL2 correlates with good prognosis in breast cancer patients treated with tamoxifen[J].Int J Oncol,2011,38(4):1145-1151.
[6]WANG T,LI F,TANG S.MiR-30a upregulates BCL2A1,IER3 and cyclin D2 expression by targetingFOXL2[J].Oncol Lett,2015,9(2):967-971.
[7]LEO F,BARTELS S,MAGEL L,et al.Prognostic factors in the myoepithelial-like spindle cell type of metaplastic breast cancer[J].Virchows Arch,2016,469(2):191-201.
[8]ZHOU X Y,SUN J F,HE Y H,et al.Correlation of the methylation status of CpG islands in the promoter region of 10 genes with the 5-Fu chemosensitivity in 3 breast cancer cell lines[J].Zhonghua Zhong Liu Za Zhi,2010,32(5):328-333.
[9]SCHRADER K A,GORBATCHEVA B,SENZ J,et al.The specificity of the FOXL2 c.402C>G somatic mutation:a survey of solid tumors[J].PLoS One,2009,4(11):e7988.
[10]SHAH S P,KOBEL M,SENZ J,et al.Mutation of FOXL2 in granulosa-cell tumors of the ovary[J].N Engl J Med,2009,360(26):2719-2729.
[11]CRISPONI L,DEIANA M,LOI A,et al.The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome[J].Nat Genet,2001,27(2):159-166.
[12]YANG Y,YANG C,ZHU Y,et al.Intragenic and extragenic disruptions of FOXL2 mapped by whole genomelow-coverage sequencing in two BPES families with chromosome reciprocal translocation[J].Genomics,2014,104(3):170-176.
[13]GULATI,VERDIN,HALANAIK D,et al.Co-occurrence of congenital hydronephrosis and FOXL2-associated blepharophimosis,ptosis,epicanthus inversus syndrome(BPES)[J].Eur JMed Genet,2014,57(10):576-578.
[14]AO L,LIU Y.Clinical diagnose and significance of congenital sensorineural hearing loss combined with BPES[J].Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi,2015,29(18):1660-1663.
[15]GARCIA-ORTIZ J E,PELOSI E,OMARI S,et al.FOXL2functions in sex determination and histogenesis throughout mouse ovary development[J].BMC Dev Biol,2009,9:36.
[16]CHENG L,LI L,WANG L.A random forest classifier predicts recurrence risk in patients with ovarian cancer[J].Mol Med Rep,2018,18(3):3289-3297.
[17]GARDNER L,ANDERSON T,PLACE A R,et al.Sex change strategy and the aromatase genes[J].J Steroid Biochem Mol Biol,2005,94(5):395-404.
[18]CHOI Y J,HO J,LEE A.Disparate genomic characteristics of concurrent endometrial adenocarcinoma and ovarian granulosa cell tumor,revealed by targeted next-generation sequencing[J].Pathol Res Pract,2018,214(8):1231-1233.