基因芯片技术检测NOB1对骨肉瘤细胞相关基因的调控作用
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摘要
目的:探讨应用基因芯片技术筛选出可能受NOB1调控的基因,阐明NOB1对骨肉瘤细胞相关基因表达的调控作用。方法:应用慢病毒介导的短发夹RNA(shRNA)干扰技术建立抑制NOB1 mRNA表达的Lv-shNOB1-U2OS骨肉瘤细胞株,实验分为2组,分别为对照病毒液感染U2OS骨肉瘤细胞(Lv-shCon-U2OS)组和含NOB1干扰质粒病毒感染U2OS骨肉瘤细胞(Lv-shNOB1-U2OS)组。利用mRNA表达谱芯片对LvshCon-U2OS组和Lv-shNOB1-U2OS组细胞分别行表达谱分析,并利用生物信息学数据库肿瘤基因(GO)和京都基因与基因组百科全书(KEGG)预测miRNA参与调控的生物学过程、细胞组分、分子功能和信号通路。结果:mRNA表达谱芯片数据显示,NOB1干扰后共有792个基因mRNA表达水平上调,1 059个基因mRNA表达水平下调,总共差异变化基因为1 851个。GO分析,从细胞位置条目的富集程度来看,差异基因编码产物蛋白主要分布于细胞质膜上,占总差异基因的56.9%,分布于细胞外区域的差异基因编码产物蛋白占总差异基因的39.4%,分布于细胞外空间占总差异基因的20%;从分子功能条目的富集程度来看,差异基因编码产物蛋白主要功能为钙离子结合相关功能(占总差异基因的22%),其次功能为转运子活性(占总差异基因的9.2%),第3位功能为肌动蛋白结合活性,(占总差异基因的8.7%);从生化过程条目的富集程度来看,差异基因编码产物蛋白主要参与过程为信号转导(占总差异基因的18.7%),其次参与过程为多种细胞器的生成(占总差异基因的15.6%),第3位过程为细胞黏附过程(占总差异基因的10.2%);KEGG分析,差异分子所编码的蛋白涉及领域包括细胞质膜成分、钙离子结合活性以及信号转导过程。结论:敲除NOB1可以对骨肉瘤细胞相关基因表达产生全方位的影响。
Objective:To screen the genes may be regulated by NOB1 by using gene microarray technique,and to clarify the regulatory effect of NOB1 on the expression of osteosarcoma cell-related genes.Methods:The U2 OS cells were treated with lentivirus-mediated RNA interference and to establish the osteosarcoma cells Lv-shNOB1-U2 OS.Lv-shCon-U2 OS group and Lv-shNOB1-U2 OS group were set up.The mRNA expressions of those cells were detected using expression pattern analysis.Gene Oncology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were used to predict the global functions of NOB1,including biological processes,cellular components,molecular functions,and signaling pathways.Results:After NOB1 interference in the U2 OS cells,there were 792 genes with up-regulated mRNA expression level and 1 059 genes with down-regulated mRNA expression level,with a total variation of 1 851 genes.The GO analysis results showed that from the enrichment degree of cell location entries,the differentially encoded product proteins were mainly distributed on the cell membrane as 56.9% of the total difference genes,39.4% of the totally differential genes distributed in the extracellular region,and 20% of the totally differential genes in the extracellular space;from the enrichment degrees of the molecular function items,the main function of the differentially encoded product proteins was calciu mion binding-related function(22% of the totally differential genes),the second function was transporter activity(9.2% of the totally differenital genes),and the third function was actin binding activity(8.7% of the totally differential genes).In terms of the enrichment of biochemical process entries,the main participation process of differentially encoded product proteins was 18.7% of the totally differential genes of signal transduction,the second involved process was 15.6% of the totally differential genes produced by multiple organelles,and the third process was the cell adhesion process accounted for 10.2% of the totally differential genes.The KEGG analysis results showed that their encoding proteins were involved in plasma membrane,calcium ion binding activity and signal transduction.Conclusion:Knockout of NOB1 can affect the expressions of osteosarcoma cell-related genes in an all-round way.
Objective:To screen the genes may be regulated by NOB1 by using gene microarray technique,and to clarify the regulatory effect of NOB1 on the expression of osteosarcoma cell-related genes.Methods:The U2 OS cells were treated with lentivirus-mediated RNA interference and to establish the osteosarcoma cells Lv-shNOB1-U2 OS.Lv-shCon-U2 OS group and Lv-shNOB1-U2 OS group were set up.The mRNA expressions of those cells were detected using expression pattern analysis.Gene Oncology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were used to predict the global functions of NOB1,including biological processes,cellular components,molecular functions,and signaling pathways.Results:After NOB1 interference in the U2 OS cells,there were 792 genes with up-regulated mRNA expression level and 1 059 genes with down-regulated mRNA expression level,with a total variation of 1 851 genes.The GO analysis results showed that from the enrichment degree of cell location entries,the differentially encoded product proteins were mainly distributed on the cell membrane as 56.9% of the total difference genes,39.4% of the totally differential genes distributed in the extracellular region,and 20% of the totally differential genes in the extracellular space;from the enrichment degrees of the molecular function items,the main function of the differentially encoded product proteins was calciu mion binding-related function(22% of the totally differential genes),the second function was transporter activity(9.2% of the totally differenital genes),and the third function was actin binding activity(8.7% of the totally differential genes).In terms of the enrichment of biochemical process entries,the main participation process of differentially encoded product proteins was 18.7% of the totally differential genes of signal transduction,the second involved process was 15.6% of the totally differential genes produced by multiple organelles,and the third process was the cell adhesion process accounted for 10.2% of the totally differential genes.The KEGG analysis results showed that their encoding proteins were involved in plasma membrane,calcium ion binding activity and signal transduction.Conclusion:Knockout of NOB1 can affect the expressions of osteosarcoma cell-related genes in an all-round way.
引文
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[22]Ghosh G,Wang VY,Huang DB,et al.NF-κB regulation:lessons from structures[J].Immunol Rev,2012,246(1):36-58.
[2]PosthumaDeBoer J,Witlox MA,Kaspers GJ,et al.Molecular alterations as target for therapy in metastatic osteosarcoma:a review of literature[J].Clin Exp Metastasis,2011,28(5):493-503.
[3]Kim C,Shin E,Hong S,et al.Clinical value of ezrin expression in primary osteosarcoma[J].Cancer Res Treat,2009,41(3):138-144.
[4]Zhao Q,Wang C,Zhu J,et al.RNAi-mediated knockdown of cyclooxygenase2inhibits the growth,invasion and migration of SaOS2human osteosarcoma cells:a case control study[J].J Exp Clin Cancer Res,2011,30:26.
[5]Hughes DP.How the NOTCH pathway contributes to the ability of osteosarcoma cells to metastasize[J].Cancer Treat Res,2009,152:479-496.
[6]Dai H,Hou K,Cai Z,et al.Low-level miR-646in colorectal cancer inhibits cell proliferation and migration by targeting NOB1expression[J].Oncol Lett,2017,14(6):6708-6714.
[7]Lin Y,Xu T,Zhou S,et al.MicroRNA-363inhibits ovarian cancer progression by inhibiting NOB1[J].Oncotarget,2017,8(60):101649-101658.
[8]Kong R,Liu W,Guo Y,et al.Inhibition of NOB1 by microRNA-330-5p overexpression represses cell growth of nonsmall cell lung cancer[J].Oncol Rep,2017,38(4):2572-2580.
[9]Liu K,Chen H,You Q,et al.miR-145inhibits human nonsmall-cell lung cancer growth by dual-targeting RIOK2and NOB1[J].Int J Oncol,2018,3.doi:10.3892/ijo.2018.4393.
[10]Lin Y,Xu T,Teng H,et al.Anticancer activity of NOB1-targeted shRNA combination with TRAIL in epithelial ovarian cancer cells[J].Int J Clin Exp Pathol,2015,8(9):10061-10071.
[11]Gao X,Wang J,Bai W,et al.NOB1silencing inhibits the growth and metastasis of laryngeal cancer cells through the regulation of JNK signaling pathway[J].Oncol Rep,2016,35(6):3313-3320.
[12]Niinaka Y,Harada K,Fujimuro M,et al.Silencing of autocrine motility factor induces mesenchymal-to-epithelial transition and suppression of osteosarcoma pulmonary metastasis[J].Cancer Res,2010,70(22):9483-9493.
[13]Luo L,Wang Y,Yin Y,et al.Effects of NOB1 on the pathogenesis of osteosarcoma and its expression on the chemosensitivity to cisplatin[J].Oncol Lett,2018,15(3):3548-3551.
[14]Huang P,Xi J,Liu S.MiR-139-3p induces cell apoptosis and inhibits metastasis of cervical cancer by targeting NOB1[J].Biomed Pharmacother,2016,83:850-856.
[15]Ma Y,Zhou W,Hong L,et al.Establishment of a novel monoclonal antibody L6 specific to NOB1[J].Monoclon Antib Immunodiagn Immunother,2016,35(2):100-103.
[16]Lingwood D,Binnington B,Róg T,et al.Cholesterol modulates glycolipid conformation and receptor activity[J].Nat Chem Biol,2011,7(5):260-262.
[17]Teschendorff AE,Severini S.Increased entropy of signal transduction in the cancer metastasis phenotype[J].BMC Syst Biol,2010,4:104.
[18]Darnell JE Jr.The JAK-STAT pathway at 20[J].JAKSTAT,2012,1(1):2.doi:10.4161/jkst.18726.
[19]张朝亚,赵相轩,卢再鸣,等.索拉非尼诱导肝癌细胞凋亡信号通路的研究进展[J].临床肝胆病杂志,2016,32(4):816-820.
[20]Xie C,Kang J,Ferguson ME,et al.Blueberries reduce proinflammatory cytokine TNF-αand IL-6production in mouse macrophages by inhibiting NF-κB activation and the MAPK pathway[J].Mol Nutr Food Res,2011,55(10):1587-1591.
[21]Quintás-Cardama A,Verstovsek S.Molecular pathways:Jak/STAT pathway:mutations,inhibitors,and resistance[J].Clin Cancer Res,2013,19(8):1933-1940.
[22]Ghosh G,Wang VY,Huang DB,et al.NF-κB regulation:lessons from structures[J].Immunol Rev,2012,246(1):36-58.
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