miR-135b-5p靶向KLF4基因调控胃癌SGC-7901细胞生物学行为的研究
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摘要
目的 研究miR-135b-5p通过靶向KLF4基因对人胃癌SGC-7901细胞生物学行为的影响。方法 通过RT-PCR检测人正常胃黏膜上皮细胞株GES-1及胃癌细胞株SGC-7901、MKN-45、BGC-823、AGS中miR-135b-5p表达水平,将miR-135b-5p inhibitor转染至胃癌SGC-7901细胞中,RT-PCR和Western blotting分别检测转染后细胞中miR-135b-5p和KLF4蛋白表达水平,MTT法、流式细胞术、Transwell实验分别检测转染对细胞增殖、凋亡、侵袭和迁移能力的影响。生物信息学软件预测及双荧光素酶报告基因实验验证KLF4是否为miR-135b-5p的靶基因。结果 胃癌细胞SGC-7901、MKN-45、BGC-823、AGS中miR-135b-5p表达水平显著高于人正常胃黏膜上皮细胞GES-1,且胃癌SGC-7901细胞中miR-135b-5p表达水平最高。在SGC-7901细胞中转染miR-135b-5p inhibitor 48 h后,miR-135b-5p的表达水平显著降低,KLF4 mRNA和蛋白表达水平明显升高。下调miR-135b-5p后SGC-7901细胞增殖能力下降,凋亡率升高,侵袭和迁移能力减弱。双荧光素酶报告基因实验结果显示,KLF4是miR-135b-5p靶基因。结论 miR-135b-5p能够通过靶向KLF4抑制胃癌SGC-7901细胞增殖、侵袭和迁移,诱导细胞凋亡。
Objective To investigate the effect of miR-135 b-5 p on the biological behavior of human gastric cancer SGC-7901 cells by targeting KLF4 gene. Methods The expression of miR-135 b-5 p in human normal gastric mucosal epithelial cell line GES-1 and gastric cancer cell lines SGC-7901, MKN-45, BGC-823 and AGS was detected by RT-PCR. The expressions of miR-135 b-5 p and KLF4 proteins in transfected cells were detected by RT-PCR and Western blotting, respectively. MTT assay, flow cytometry and Transwell assay were used to detect the proliferation of the transfected cells, the effects of apoptosis, invasion and migration. Bioinformatics software prediction and dual luciferase reporter gene experiments verified whether KLF4 was a target gene of miR-135 b-5 p. Results The expressions of miR-135 b-5 p in gastric cancer cells SGC-7901, MKN-45, BGC-823 and AGS were significantly higher than shose in human gastric epithelial cells GES-1, and the expression of miR-135 b-5 p in gastric cancer SGC-7901 cells was the highest. After transfection of miR-135 b-5 p inhibitor for 48 hours in SGC-7901 cells, the expression of miR-135 b-5 p was significantly decreased, and the expressions of KLF4 mRNA and protein were significantly increased. After down-regulating miR-135 b-5 p, the proliferation of SGC-7901 cells was decreased, the apoptotic rate was increased, and the invasion and migration ability were decreased. The results of the dual luciferase reporter gene assay showed that KLF4 was a miR-135 b-5 p target gene. Conclusion miR-135 b-5 p can inhibit the proliferation, invasion and migration of gastric cancer SGC-7901 cells by targeting KLF4 and can induce apoptosis.
Objective To investigate the effect of miR-135 b-5 p on the biological behavior of human gastric cancer SGC-7901 cells by targeting KLF4 gene. Methods The expression of miR-135 b-5 p in human normal gastric mucosal epithelial cell line GES-1 and gastric cancer cell lines SGC-7901, MKN-45, BGC-823 and AGS was detected by RT-PCR. The expressions of miR-135 b-5 p and KLF4 proteins in transfected cells were detected by RT-PCR and Western blotting, respectively. MTT assay, flow cytometry and Transwell assay were used to detect the proliferation of the transfected cells, the effects of apoptosis, invasion and migration. Bioinformatics software prediction and dual luciferase reporter gene experiments verified whether KLF4 was a target gene of miR-135 b-5 p. Results The expressions of miR-135 b-5 p in gastric cancer cells SGC-7901, MKN-45, BGC-823 and AGS were significantly higher than shose in human gastric epithelial cells GES-1, and the expression of miR-135 b-5 p in gastric cancer SGC-7901 cells was the highest. After transfection of miR-135 b-5 p inhibitor for 48 hours in SGC-7901 cells, the expression of miR-135 b-5 p was significantly decreased, and the expressions of KLF4 mRNA and protein were significantly increased. After down-regulating miR-135 b-5 p, the proliferation of SGC-7901 cells was decreased, the apoptotic rate was increased, and the invasion and migration ability were decreased. The results of the dual luciferase reporter gene assay showed that KLF4 was a miR-135 b-5 p target gene. Conclusion miR-135 b-5 p can inhibit the proliferation, invasion and migration of gastric cancer SGC-7901 cells by targeting KLF4 and can induce apoptosis.
引文
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[2] TOMASELLO G,GHIDINI M,LIGUIGLI W,et al.Targeted therapies in gastric cancer treatment:where we are and where we are going [J].Invest New Drugs,2016,34(3):378-393.DOI:10.1007/s10637-016-0330-2.
[3] LI J Q,RONG Z H,CHEN X,et al.MCMDA:matrix completion for miRNA-disease association prediction [J].Oncotarget,2017,8(13):21187-21199.DOI:10.18632/oncotarget.15061.
[4] GIULIETTI M,OCCHIPINTI G,PRINCIPATO G,et al.Identification of candidate miRNA biomarkers for pancreatic ductal adenocarcinoma by weighted gene co-expression network analysis [J].Cell Oncol (Dordr),2017,40(2):181-192.DOI:10.1007/s13402-017-0315-y.
[5] HUANG K H,LAN Y T,FANG W L,et al.The correlation between miRNA and lymph node metastasis in gastric cancer [J].Biomed Res Int,2015,2015:543163.DOI:10.1155/2015/543163.
[6] HAO N B,HE Y F,LI X Q,et al.The role of miRNA and lncRNA in gastric cancer [J].Oncotarget,2017,8(46):81572-81582.DOI:10.18632/oncotarget.19197.
[7] LI C Y,LIANG G Y,YAO W Z,et al.Identification and functional characterization of microRNAs reveal a potential role in gastric cancer progression [J].Clin Transl Oncol,2017,19(2):162-172.DOI:10.1007/s12094-016-1516-y.
[8] LU M,HUANG Y,SUN W,et al.miR-135b-5p promotes gastric cancer progression by targeting CMTM3 [J].Int J Oncol,2018,52(2):589-598.DOI:10.3892/ijo.2017.4222.
[9] CHEN M B,LIU Y Y,CHENG L B,et al.AMPKα phosphatase Ppm1E upregulation in human gastric cancer is required for cell proliferation [J].Oncotarget,2017,8(19):31288-31296.DOI:10.18632/oncotarget.16126.
[10] HASHIMOTO I,NAGATA T,SEKINE S,et al.Prognostic significance of KLF4 expression in gastric cancer [J].Oncol Lett,2017,13(2):819-826.DOI:10.3892/ol.2016.5499.
[11] CAO M X,JIANG Y P,TANG Y L,et al.The crosstalk between lncRNA and microRNA in cancer metastasis:orchestrating the epithelial-mesenchymal plasticity [J].Oncotarget,2017,8(7):12472-12483.DOI:10.18632/oncotarget.13957.
[12] LV Z D,XIN H N,YANG Z C,et al.miR-135b promotes proliferation and metastasis by targeting APC in triple-negative breast cancer [J].J Cell Physiol,2019,234(7):10819-10826.DOI:10.1002/jcp.27906.
[13] AAKULA A,LEIVONEN S K,HINTSANEN P,et al.MicroRNA-135b regulates ERα,AR and HIF1AN and affects breast and prostate cancer cell growth [J].Mol Oncol,2015,9(7):1287-1300.DOI:10.1016/j.molonc.2015.03.001.
[14] JUNG H S,SEO Y R,YANG Y M,et al.Gα12gep oncogene inhibits FOXO1 in hepatocellular carcinoma as a consequence of miR-135b and miR-194 dysregulation [J].Cell Signal,2014,26(7):1456-1465.DOI:10.1016/j.cellsig.2014.02.022.
[15] HUA K,JIN J,ZHAO J,et al.miR-135b,upregulated in breast cancer,promotes cell growth and disrupts the cell cycle by regulating LATS2 [J].Int J Oncol,2016,48(5):1997-2006.DOI:10.3892/ijo.2016.3405.
[16] XUE Y,NI T,JIANG Y,et al.Long noncoding RNA GAS5 inhibits tumorigenesis and enhances radiosensitivity by suppressing miR-135b expression in non-small cell lung cancer [J].Oncol Res,2017,25(8):1305-1316.DOI:10.3727/096504017X14850182723737.
[17] PEI H,JIN Z,CHEN S,et al.MiR-135b promotes proliferation and invasion of osteosarcoma cells via targeting FOXO1 [J].Mol Cell Biochem,2015,400(1-2):245-252.DOI:10.1007/s11010-014-2281-2.
[18] HAN X,SAIYIN H,ZHAO J,et al.Overexpression of miR-135b-5p promotes unfavorable clinical characteristics and poor prognosis via the repression of SFRP4 in pancreatic cancer [J].Oncotarget,2017,8(37):62195-62207.DOI:10.18632/oncotarget.19150.
[19] 王霖沛,马筱秋,蔡建春.微小RNA-135b在胃癌发生发展中的临床病理意义及其作用[J].中华医学杂志,2012,92(46):3269-3273.DOI:10.3760/cma.j.issn.0376-2491.2012.46.009.WANG L P,MA X Q,CAI J C.Clinicopathological significance and function of miR-135b in the occurrence and development of gastric cancer [J].Natl Med J China,2012,92(46):3269-3273.DOI:10.3760/cma.j.issn.0376-2491.2012.46.009.
[20] 阳圣,张雯,杨燕青,等.胃癌组织miRNA差异表达的初步分析[J].上海交通大学学报(医学版),2010,30(11):1317-1323.DOI:10.3969/j.issn.1674-8115.2010.11.001.YANG S,ZHANG W,YANG Y Q,et al.Preliminary analysis of miRNA expression profile of gastric cancer tissues [J].Journal of Shanghai Jiaotong University (Medical Science),2010,30(11):1317-1323.DOI:10.3969/j.issn.1674-8115.2010.11.001.
[21] HU W,JIA Y,XIAO X,et al.KLF4 downregulates hTERT expression and telomerase activity to inhibit lung carcinoma growth [J].Oncotarget,2016,7(33):52870-52887.DOI:10.18632/oncotarget.9141.
[22] MARTINS-NEVES S R,CORVER W E,PAIVA-OLIVEIRA D I,et al.Osteosarcoma stem cells have active Wnt/β-catenin and overexpress SOX2 and KLF4 [J].J Cell Physiol,2016,231(4):876-886.DOI:10.1002/jcp.25179.
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[25] ZHAO L,HAN T,LI Y,et al.The lncRNA SNHG5/miR-32 axis regulates gastric cancer cell proliferation and migration by targeting KLF4 [J].FASEB J,2017,31(3):893-903.DOI:10.1096/fj.201600994R.