miR-519d靶向CDKN1A/p21调控宫颈癌细胞增殖的初步研究
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摘要
目的探讨miR-519d对人宫颈癌细胞中CDKN1A/p21基因的靶向调控作用以及对细胞增殖、周期的影响。方法通过荧光定量PCR技术检测miR-519d抑制物对其活性的调控作用。在宫颈癌He La和Si Ha细胞中下调miR-519d表达,利用MTT法检测细胞增殖能力,通过流式细胞术检测细胞周期的分布情况。将miR-519d mimic转染He La和Si Ha细胞,用荧光定量PCR、Western Blot分别检测p21 m RNA和蛋白水平的表达。利用双荧光素酶报告基因系统确认miR-519d与p21的靶向关系。结果 miR-519d抑制物能有效下调宫颈癌He La和Si Ha细胞内miR-519d的表达。MTT实验和细胞周期分析结果提示,下调细胞内miR-519d的表达能够显著降低细胞增殖活力,并使细胞周期阻滞于G1期。进一步通过双荧光素酶报告基因系统鉴定miR-519d能够结合p21 m RNA3′UTR有效抑制其表达。q RT-PCR和Western blot检测结果表明,过表达miR-519d能在m RNA和蛋白水平上抑制p21的表达。结论 p21是miR-519d的直接靶基因,miR-519d可能通过靶向p21调控宫颈癌细胞增殖。
Objective To investigate the role of mi R-519 d in the targeted regulation of CDKN1 A/p21 gene in human cervical cancer cells and its effect on cell proliferation and cell cycle. Methods The effect of mi R-519 d inhibitor on its activity was detected by fluorescence quantitative PCR. The mi R-519 d expressions in cervical cancer cell He La and Si Ha were down-regulated. The cell proliferation abilitywas detected by MTT assay, and cell cycle distribution was detected by flow cytometry. Mi R-519 d mimic was transfected into He La and Si Ha cells, and the expression of p21 m RNA and protein was detected by fluorescence quantitative PCR and Western Blot, respectively. A dual luciferase reporter gene system was used to confirm the targeting relationship between mi R-519 d and p21. Results The mi R-519 d inhibitor can effectively down-regulate the expression of mi R-519 d in He La and Si Ha cells of cervical cancer. The results of MTT assay and cell cycle analysis suggested that the down-regulation of mi R-519 d expression in cells could significantly reduce cell proliferation and arrest cell cycle at G1 phase. Further the dual luciferase reporter system identified that mi R-519 d was able to in combination with p21 m RNA 3'UTR and effectively inhibit the expression of p21.The results of q RT-PCR and Western blot showed that overexpression of mi R-519 d inhibited the expression of p21 at m RNA and protein levels. Conclusion p21 is a direct target gene of mi R-519 d, and mi R-519 d may regulate the proliferation of cervical cancer cells by targeting p21.
Objective To investigate the role of mi R-519 d in the targeted regulation of CDKN1 A/p21 gene in human cervical cancer cells and its effect on cell proliferation and cell cycle. Methods The effect of mi R-519 d inhibitor on its activity was detected by fluorescence quantitative PCR. The mi R-519 d expressions in cervical cancer cell He La and Si Ha were down-regulated. The cell proliferation abilitywas detected by MTT assay, and cell cycle distribution was detected by flow cytometry. Mi R-519 d mimic was transfected into He La and Si Ha cells, and the expression of p21 m RNA and protein was detected by fluorescence quantitative PCR and Western Blot, respectively. A dual luciferase reporter gene system was used to confirm the targeting relationship between mi R-519 d and p21. Results The mi R-519 d inhibitor can effectively down-regulate the expression of mi R-519 d in He La and Si Ha cells of cervical cancer. The results of MTT assay and cell cycle analysis suggested that the down-regulation of mi R-519 d expression in cells could significantly reduce cell proliferation and arrest cell cycle at G1 phase. Further the dual luciferase reporter system identified that mi R-519 d was able to in combination with p21 m RNA 3'UTR and effectively inhibit the expression of p21.The results of q RT-PCR and Western blot showed that overexpression of mi R-519 d inhibited the expression of p21 at m RNA and protein levels. Conclusion p21 is a direct target gene of mi R-519 d, and mi R-519 d may regulate the proliferation of cervical cancer cells by targeting p21.
引文
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[14]Yang X,Hu Y,Liu Y,et al.C14orf28 downregulated by mi R-519d contributes to oncogenicity and regulates apoptosis and EMT in colorectal cancer[J].Mol Cell Biochem,2017,434(1-2):197-208.
[15]Xie Q,Wang S,Zhao Y,et al.Mi R-519d impedes cisplatin-resistance in breast cancer stem cells by downregulating the expression of MCL-1[J].Oncotarget,2017,8(13):22003-22013.
[16]Bai Y,Lu C,Zhang G,et al.Overexpression of mi R-519d in lung adenocarcinoma inhibits cell proliferation and invasion via the association of e IF4H[J].Tumour Biol,2017,39(3):1010428317694566.
[17]Hua KT,Hong JB,Sheen YS,et al.mi R-519d promotes melanoma progression by downregulating Eph A4[J].Cancer Res,2018,78(1):216-229.
[2]Martinelli R,Nardelli C,Pilone V,et al.mi R-519d overexpression is associated with human obesity[J].Obesity(Silver Spring),2010,18(11):2170-2176.
[3]Lehmann U,Streichert T,Otto B,et al.Identification of differentially expressed micro RNAs in human male breast cancer[J].BMC Cancer,2010,10:109.
[4]Hou YY,Cao WW,Li L,et al.Micro RNA-519d targets MKi67 and suppresses cell growth in the hepa tocellular carcinoma cell line QGY-7703[J].Cancer Lett,2011,307(2):182-190.
[5]Fornari F,Milazzo M,Chieco P,et al.In hepatocellular carcinoma mi R-519d is up-regulated by p53 and DNA hypomethylation and targets CDKN1A/p21,PTEN,AKT3and TIMP2[J].J Pathol,2012,227(3):275-285.
[6]Gennarino VA,D'Angelo G,Dharmalingam G,et al.Identification of micro RNA-regulated gene networks by expression analysis of target genes[J].Genome Res,2012,22(6):1163-1172.
[7]Pang Y,Mao H,Shen L,et al.Mi R-519d represses ovarian cancer cell proliferation and enhances cisplatin-mediated cytotoxicity in vitro by targeting XIAP[J].Onco Targets Ther,2014,7:587-597.
[8]Martin CM,Astbury K,O'Leary JJ.Molecular profiling of cervical neoplasia[J].Expert Rev Mol Diagn,2006,6(2):217-229.
[9]Ivanovska I,Ball AS,Diaz RL,et al.Micro RNAs in the mi R-106b family regulate p21/CDKN1A and promote cell cycle progression[J].Mol Cell Biol,2008,28(7):2167-2174.
[10]Wu S,Huang S,Ding J,et al.Multiple micro RNAs modulate p21Cip1/Waf1 expression by directly targeting its3'untranslated region[J].Oncogene,2010,29(15):2302-2308.
[11]Deng X,Zhao Y,Wang B.mi R-519d-mediated downregulation of STAT3 suppresses breast cancer progression[J].Oncol Rep,2015,34(4):2188-2194.
[12]Fornari F,Ferracin M,TrerèD,et al.Circulating micro RNAs,mi R-939,mi R-595,mi R-519d and mi R-494,Identify Cirrhotic Patients with HCC[J].PLo S One,2015,10(10):e0141448.
[13]Yue H,Tang B,Zhao Y,et al.MIR-519d suppresses the gastric cancer epithelial-mesenchymal transition via Twist1and inhibits Wnt/β-catenin signaling pathway[J].Am J Transl Res,2017,9(8):3654-3664.
[14]Yang X,Hu Y,Liu Y,et al.C14orf28 downregulated by mi R-519d contributes to oncogenicity and regulates apoptosis and EMT in colorectal cancer[J].Mol Cell Biochem,2017,434(1-2):197-208.
[15]Xie Q,Wang S,Zhao Y,et al.Mi R-519d impedes cisplatin-resistance in breast cancer stem cells by downregulating the expression of MCL-1[J].Oncotarget,2017,8(13):22003-22013.
[16]Bai Y,Lu C,Zhang G,et al.Overexpression of mi R-519d in lung adenocarcinoma inhibits cell proliferation and invasion via the association of e IF4H[J].Tumour Biol,2017,39(3):1010428317694566.
[17]Hua KT,Hong JB,Sheen YS,et al.mi R-519d promotes melanoma progression by downregulating Eph A4[J].Cancer Res,2018,78(1):216-229.