miR-429对乳癌细胞增殖和迁移影响及其靶基因初步鉴定
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摘要
目的探讨miR-429在人乳癌细胞系中的表达及其对细胞增殖和迁移的影响,初步鉴定miR-429的靶基因。方法采用实时荧光定量PCR(qPCR)方法检测miR-429在人乳癌细胞系中的表达。通过MTT实验和划痕实验确定miR-429对人乳癌细胞系MDA-MB-231细胞增殖和迁移的影响。采用生物信息学分析、qPCR和蛋白质印迹方法鉴定miR-429的靶基因。结果 miR-429在人乳癌细胞系MDA-MB-231和MCF-7中低表达。上调miR-429表达后,MDA-MB-231细胞增殖能力被显著抑制(F=33.106,P<0.05),迁移能力明显减弱(F=57.59,P<0.05)。过表达和抑制miR-429后,预测靶基因金属蛋白酶2组织抑制剂(TIMP2)、环磷腺苷效应元件结合蛋白1(CREB1)mRNA表达水平均无明显变化;然而,过表达miR-429后,TIMP2蛋白的相对表达量显著降低(F=33.74,P<0.05)。结论在MDA-MB-231细胞中,过表达miR-429可抑制细胞增殖和迁移。miR-429的过表达可能通过转录后翻译抑制TIMP2蛋白在MDA-MB-231细胞中的表达,初步鉴定TIMP2是miR-429的靶基因。
Objective To investigate the expression of microRNA-429(miR-429)in human breast cancer cell line and its effect on the proliferation and migration of human breast cancer cell,and to preliminarily identify the target genes of miR-429.Methods Quantitative real-time PCR(qPCR)was used to measure the expression of miR-429 in human breast cancer cell line.MTT assay and wound healing assay were used to investigate the effect of miR-429 on the proliferation and migration of human breast cancer MDA-MB-231 cell line.bioinformatics analysis,qPCR,and Western blot were used to identify the target genes of miR-429. Results MiR-429 showed low expression in human breast cancer MDA-MB-231 and MCF-7 cell lines.After upregulation of miR-429 expression,MDA-MB-231 cells had significant reductions in proliferative capacity(F=33.106,P<0.05)and migration ability(F=57.59,P<0.05).There were no significant changes in the mRNA expression of the predicted target genes TIMP2 and CREB1 after miR-429 overexpression or inhibition;however,there was a significant reduction in the relative expression of TIMP2 protein after miR-429 overexpression(F=33.74,P<0.05). Conclusion Overexpression of miR-429 may inhibit the proliferation and migration of MDA-MB-231 cell line and reduce the expression of TIMP2 protein in MDA-MB-231 cell line through post-transcriptional translation,and therefore,TIMP2 is preliminarily identified as the target gene of miR-429.
Objective To investigate the expression of microRNA-429(miR-429)in human breast cancer cell line and its effect on the proliferation and migration of human breast cancer cell,and to preliminarily identify the target genes of miR-429.Methods Quantitative real-time PCR(qPCR)was used to measure the expression of miR-429 in human breast cancer cell line.MTT assay and wound healing assay were used to investigate the effect of miR-429 on the proliferation and migration of human breast cancer MDA-MB-231 cell line.bioinformatics analysis,qPCR,and Western blot were used to identify the target genes of miR-429. Results MiR-429 showed low expression in human breast cancer MDA-MB-231 and MCF-7 cell lines.After upregulation of miR-429 expression,MDA-MB-231 cells had significant reductions in proliferative capacity(F=33.106,P<0.05)and migration ability(F=57.59,P<0.05).There were no significant changes in the mRNA expression of the predicted target genes TIMP2 and CREB1 after miR-429 overexpression or inhibition;however,there was a significant reduction in the relative expression of TIMP2 protein after miR-429 overexpression(F=33.74,P<0.05). Conclusion Overexpression of miR-429 may inhibit the proliferation and migration of MDA-MB-231 cell line and reduce the expression of TIMP2 protein in MDA-MB-231 cell line through post-transcriptional translation,and therefore,TIMP2 is preliminarily identified as the target gene of miR-429.
引文
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[21]CHEN Hong,XIA Bairong,LIU Tianbo,et al.KIAA0101,a target gene of miR-429,enhances migration and chemoresistance of epithelial ovarian cancer cells[J].Cancer Cell International,2016,16:74.
[2]CROCE C M.Causes and consequences of microRNA dysregulation in cancer[J].Nature Reviews Genetics,2009,10(10):704-714.
[3]秦科宇,李恒宇,许鹿,等.微RNA-429调控人乳腺癌Bcap37细胞系的增殖和迁移能力[J].中华乳腺病杂志(电子版),2015,9(2):89-95.
[4]LI Dengfeng,WANG Hong,SONG Hongming,et al.The microRNAs miR-200b-3p and miR-429-5p target the LIMK1/CFL1pathway to inhibit growth and motility of breast cancer cells[J].Oncotarget,2017,8(49):85276-85289.
[5]UHLMANN S,ZHANG J D,SCHWGER A,et al.miR-200bc/429cluster targets PLCgamma1and differentially regulates proliferation and EGF-driven invasion than miR-200a/141in breast cancer[J].Oncogene,2010,29(30):4297-4306.
[6]YE Zhibin,MA Gang,ZHAO Yahui,et al.miR-429inhibits migration and invasion of breast cancer cells in vitro[J].International Journal of Oncology,2015,46(2):531-538.
[7]LANG Yaoguo,XU Shidong,MA Jianqun,et al.MicroRNA-429induces tumorigenesis of human non-small cell lung cancer cells and targets multiple tumor suppressor genes[J].Biochemical and Biophysical Research Communications,2014,450(1):154-159.
[8]BAJRACHARYA D,SHRESTHA B,KAMATH A A,et al.Immunohistochemical correlation of matrix metalloproteinase-2and tissue inhibitors of metalloproteinase-2in tobacco associated epithelial dysplasia[J].Disease Markers,2014,2014(3):197813.
[9]ELLINA M I,BOURIS P,ALETRAS A J,et al.EGFR and HER2exert distinct roles on colon cancer cell functional properties and expression of matrix macromolecules[J].Biochimica et Biophysica Acta,2014,1840(8):2651-2661.
[10]GURGEL D C,VALENCA-JUNIOR J T,DORNELAS C A,et al.Immunoexpression of metalloproteinases 2and 14and TIMP-2inhibitor in main types of primary gastric carcinomas and lymph node metastasis[J].Pathology&Oncology Research,2015,21(1):73-81.
[11]NAGASE H,VISSE R,MURPHY G.Structure and function of matrix metalloproteinases and TIMPs[J].Cardiovascular Research,2006,69(3):562-573.
[12]BEN NEJIMA D,BEN ZARKOUNA Y,GAMMOUDI A,et al.Prognostic impact of polymorphism of matrix metalloproteinase-2and metalloproteinase tissue inhibitor-2promoters in breast cancer in Tunisia:case-control study[J].Tumor Biology,2015,36(5):3815-3822.
[13]ZHANG Ming,TENG Xiaodan,GUO Xinxin,et al.Expression of tissue levels of matrix metalloproteinases and their inhibitors in breast cancer[J].Breast,2013,22(3):330-334.
[14]HAOA,NOWAK-MARKWITZ E,DONIZY P,et al.Enhanced immunoreactivity of TIMP-2in the stromal compartment of tumor as a marker of favorable prognosis in ovarian cancer patients[J].The Journal of Histochemistry and Cytochemistry,2012,60(7):491-501.
[15]ZHU Lin,YU Hong,LIU Shiyuan,et al.Prognostic value of tissue inhibitor of metalloproteinase-2expression in patients with non-small cell lung cancer:a systematic review and metaanalysis[J].PLoS One,2015,10(4):e0124230.
[16]CHOWDHURY A,BRINSON R,WEI B A.Tissue inhibitor of metalloprotease-2(TIMP-2):bioprocess development,physicochemical,biochemical,and biological characterization of highly expressed recombinant protein[J].Biochemistry,2017,56(49):6423-6433.
[17]STETLER-STEVENSON W G,SEO D W.TIMP-2:an endogenous inhibitor of angiogenesis[J].Trends in Molecular Medicine,2005,11(3):97-103.
[18]BAKER A H,EDWARDS D R,MURPHY G.Metalloproteinase inhibitors:biological actions and therapeutic opportunities[J].Journal of Cell Science,2002,115(Pt 19):3719-3727.
[19]WANG Peng,CAO Jia,LIU Shihai,et al.Upregulated microRNA-429inhibits the migration of HCC cells by targeting TRAF6through the NF-κB pathway[J].Oncology Reports,2017,37(5):2883-2890.
[20]TANG Jing,LI Liang,HUANG Wentao,et al.MiR-429increases the metastatic capability of HCC via regulating classic Wnt pathway rather than epithelial-mesenchymal transition[J].Cancer Letters,2015,364(1):33-43.
[21]CHEN Hong,XIA Bairong,LIU Tianbo,et al.KIAA0101,a target gene of miR-429,enhances migration and chemoresistance of epithelial ovarian cancer cells[J].Cancer Cell International,2016,16:74.