胃癌中miRNA-340的体内外增殖能力检测及生物信息分析
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摘要
目的了解miRNA-340在胃癌发生中的可能机制,在线预测与其相互作用的circRNAs及其下游靶基因和参与的信号通路。方法利用芯片筛选胃癌细胞株中差异表达miRNA;利用Real-time RT-PCR,检测miRNA-340在21对胃癌组织与癌旁正常组织中的表达水平;利用MTT及EdU细胞增殖实验,检测miRNA-340对正常胃上皮细胞HFE145与胃癌细胞BGC-823的体外增殖能力;利用裸鼠成瘤实验检测miRNA-340在体内成瘤的能力。随后,通过在线软件Targetscan、David数据库及Starbase分别对miRNA-340的下游的靶基因、相关信号通路及相互作用的circRNAs进行生物信息分析。结果与正常胃上皮细胞HFE145相比较,miRNA-340在胃癌细胞株中的表达水平显著下调;Real-time RT-PCR结果显示,miRNA-340在胃癌组织中的表达明显低于正常粘膜组织(P<0.048);体内体外实验表明miRNA-340对细胞的体外增殖能力具有一定的抑制作用;生物信息分析显示,预测的miRNA-340相互作用的下游靶基因中,筛选出21个具有3个及以上的保守结合部位的靶基因,其中多个靶基因均参与肿瘤的发生及侵袭;相互作用的circRNAs中筛选出分值前10个circRNAs作为与miRNA-340可能作用最强的sponge circRNAs。结论 miRNA-340在肿瘤发生发展中可能起到了抑癌基因的作用,对胃癌细胞的增殖具有一定的抑制作用;miRNA-340与多个circRNAs可能存在相互作用,共同调控下游靶基因参与胃癌的发生发展。
Objective To investigate the mechanism of miRNA-340 for regulating the proliferation of gastric cancer(GC) cells and predict its interacting circular RNAs(circRNAs), its downstream target genes and the involved signaling pathways.Methods The differentially expressed miRNAs in GC cell lines were analyzed and screened using miRNA microarrays. The expression level of miRNA-340 in 21 pairs of GC tissues and adjacent normal tissues was detected using real-time PCR. MTT and EdU assays were performed to examine the effect of miRNA-340 on the proliferation ability of HFE145 and BGC-823 cells.We also tested the effect of miRNA-340 inhibition on subcutaneous tumorigenesis of GC cells in a nude mouse model. The downstream target genes of miRNA-340 and the probable signal pathways were predicted online using Targetscan and DAVID database, respectively. The interacting circRNAs of miRNA-340 were analyzed using starBase platform. Results Among the differentially expressed miRNAs, miRNA-340 was significantly down-regulated in GC cell lines. Real-time PCR results showed that the expression of miRNA-340 was significantly lower in GC tissues than in the adjacent tissues(P<0.05). MTT and EdU cell proliferation assays showed that miRNA-340 overexpression inhibited the proliferation of GC cells in vitro. In the nude mouse models, the proliferation of GC cells transfected with miRNA-340 inhibitor was obviously enhanced.Bioinformatics analysis suggested that miRNA-340 had 21 target genes with 3 or more conserved sites, and these genes were involved in tumorigenesis and invasion. The top 10 circRNAs were selected as the most powerful sponge circRNAs interacting with miRNA-340. Conclusion miRNA-340 may play the role of a tumor suppressor in tumorigenesis and progression.Overexpression of miRNA-340 suppress the proliferation of GC cells, suggesting its involvement in the development of GC along with multiple circRNAs.
Objective To investigate the mechanism of miRNA-340 for regulating the proliferation of gastric cancer(GC) cells and predict its interacting circular RNAs(circRNAs), its downstream target genes and the involved signaling pathways.Methods The differentially expressed miRNAs in GC cell lines were analyzed and screened using miRNA microarrays. The expression level of miRNA-340 in 21 pairs of GC tissues and adjacent normal tissues was detected using real-time PCR. MTT and EdU assays were performed to examine the effect of miRNA-340 on the proliferation ability of HFE145 and BGC-823 cells.We also tested the effect of miRNA-340 inhibition on subcutaneous tumorigenesis of GC cells in a nude mouse model. The downstream target genes of miRNA-340 and the probable signal pathways were predicted online using Targetscan and DAVID database, respectively. The interacting circRNAs of miRNA-340 were analyzed using starBase platform. Results Among the differentially expressed miRNAs, miRNA-340 was significantly down-regulated in GC cell lines. Real-time PCR results showed that the expression of miRNA-340 was significantly lower in GC tissues than in the adjacent tissues(P<0.05). MTT and EdU cell proliferation assays showed that miRNA-340 overexpression inhibited the proliferation of GC cells in vitro. In the nude mouse models, the proliferation of GC cells transfected with miRNA-340 inhibitor was obviously enhanced.Bioinformatics analysis suggested that miRNA-340 had 21 target genes with 3 or more conserved sites, and these genes were involved in tumorigenesis and invasion. The top 10 circRNAs were selected as the most powerful sponge circRNAs interacting with miRNA-340. Conclusion miRNA-340 may play the role of a tumor suppressor in tumorigenesis and progression.Overexpression of miRNA-340 suppress the proliferation of GC cells, suggesting its involvement in the development of GC along with multiple circRNAs.
引文
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[18]Wu ZS,Wu Q,Wang CQ,et al.miR-340 inhibition of breast cancer cell migration and invasion through targeting of oncoprotein c-Met[J].Cancer,2011,117(13):2842-52.
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[20]Huang K,Tang Y,He L,et al.MicroRNA-340 inhibits prostate cancer cell proliferation and metastasis by targeting the MDM2-p53pathway[J].Oncol Rep,2016,35(2):887-95.
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[24]Fernandez S,Risolino M,Mandia N,et al.miR-340 inhibits tumor cell proliferation and induces apoptosis by targeting multiple negative regulators of p27 in non-small cell lung cancer[J].Oncogene,2015,34(25):3240-50.
[25]Takeyama H,Yamamoto H,Yamashita S,et al.Decreased miR-340expression in bone marrow is associated with liver metastasis of colorectal cancer[J].Mol Cancer Ther,2014,13(4):976-85.
[26]Memczak S,Jens M,Elefsinioti A,et al.Circular RNAs are a large class of animal RNAs with regulatory potency[J].Nature,2013,495(7441):333-8.
[27]Capel B,Swain A,Nicolis S,et al.Circular transcripts of the testisdetermining gene Sry in adult mouse testis[J].Cell,1993,73(5):1019-30.
[28]Pearce MB,Jayaraman A,Pappas C,et al.Pathogenesis and transmission of swine origin A(H3N2)v influenza viruses in ferrets[J].Proc NatlAcad Sci USA,2012,109(10):3944-9.
[29]Hsu MT,Coca-Prados M.Electron microscopic evidence for the circular form of RNA in the cytoplasm of eukaryotic cells[J].Nature,1979,280(5720):339-40.
[30]Xue J,Liu Y,Luo F,et al.Circl 00284,via miR-217 regulation of EZH2,is involved in the arsenite-accelerated cell cycle of human keratinocytes in carcinogenesis[J].Biochim Biophys Acta Mol Basis Dis,2017,1863(3):753-63.
[31]Khan I,Kerwin J,Owen K,et al.Correction:Registered report:Acoding-independent function of gene and pseudogene mRNAs regulates tumour biology[J].Elife,2015,4:e13015.
[2]Chen WQ,Zheng RS,Zeng HM,et al.The updated incidences and mortalities of major cancers in China,2011[J].Chin J Cancer,2015,34(3):502-7.
[3]Chen W,Zheng R,Baade PD,et al.Cancer statistics in China,2015[J].CACancer J Clin,2016,66(2):115-32.
[4]Bray F,Ferlay J,Soerjomataram I,et al.Global cancer statistics2018:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J].CA Cancer J Clin,2018,68(6):394-424.
[5]Liang L,Li X,Zhang X,et al.MicroRNA-137,an HMGA1 target,suppresses colorectal cancer cell invasion and metastasis in mice by directly targeting FMNL2[J].Gastroenterology,2013,144(3):624-635.e4.
[6]Jin Z,Selaru FM,Cheng Y,et al.MicroRNA-192 and-215 are upregulated in human gastric cancer in vivo and suppress ALCAMexpression in vitro[J].Oncogene,2011,30(13):1577-85.
[7]Zhang XJ,Peng Y,Huang Y,et al.Inhibition of the miR-192/215-Rab11-FIP2 axis suppresses human gastric cancer progression[J].Cell Death Dis,2018,9(7):778.
[8]Zhang XJ,Peng Y,Huang Y,et al.SMG-1 inhibition by miR-192/-215 causes epithelial-mesenchymal transition in gastric carcinogenesis via activation of Wnt signaling[J].Cancer Med,2018,7(1):146-56.
[9]Kurozumi A,Goto Y,Matsushita R,et al.Tumor-suppressive microRNA-223 inhibits cancer cell migration and invasion by targeting ITGA3/ITGB1 signaling in prostate cancer[J].Cancer Sci,2016,107(1):84-94.
[10]YangWY,Lan X,Li DM,et al.MiR-223 targeting MAFB suppresses proliferation and migration of nasopharyngeal carcinoma cells[J].BMC Cancer,2015,15:461.
[11]Maskey N,Li DF,Xu H,et al.MicroRNA-340 inhibits invasion and metastasis by downregulating ROCK1 in breast cancer cells[J].Oncol Lett,2017,14(2):2261-7.
[12]Su P,Mu S,Wang Z.Long noncoding RNA SNHG16 promotes osteosarcoma cells migration and invasion via sponging miRNA-340[J].DNACell Biol,2019,38(2):170-5.
[13]Yang DY,Li YT,Zhao DQ.Curcumin induces apoptotic cell death in human pancreatic cancer cells via the miR-340/XIAP signaling pathway[J].Oncol Lett,2017,14(2):1811-6.
[14]Xie W,Qin W,Kang YL,et al.MicroRNA-340 inhibits tumor cell proliferation and induces apoptosis in endometrial carcinoma cell line RL 95-2[J].Med SciMonit,2016,22(5):1540-6.
[15]Fernandez S,Risolino M,Verde P.A novel miRNA-mediated STOPsign in lung cancer:miR-340 inhibits the proliferation of lung cancer cells through p27(KIP1)[J].Mol Cell Oncol,2015,2(2):e977147.
[16]Xie L,Chen Z,Liu H,et al.Effects of miR-340 on hepatocellular carcinoma by targeting the DcR3 gene[J].Dig Liver Dis,2018,50(3):291-6.
[17]Xiao HM,Yu LJ,Li FD,et al.MiR-340 suppresses the metastasis by targeting EphA3 in cervical cancer[J].Cell Biol Int,2018,42(9):1115-23.
[18]Wu ZS,Wu Q,Wang CQ,et al.miR-340 inhibition of breast cancer cell migration and invasion through targeting of oncoprotein c-Met[J].Cancer,2011,117(13):2842-52.
[19]Chen CP,Sun ZL,Lu X,et al.miR-340 suppresses cell migration and invasion by targeting MYO10 in breast cancer[J].Oncol Rep,2016,35(2):709-16.
[20]Huang K,Tang Y,He L,et al.MicroRNA-340 inhibits prostate cancer cell proliferation and metastasis by targeting the MDM2-p53pathway[J].Oncol Rep,2016,35(2):887-95.
[21]Presneau N,Eskandarpour M,Shemais T,et al.MicroRNA profiling of peripheral nerve sheath tumours identifies miR-29c as a tumour suppressor gene involved in tumour progression[J].Br J Cancer,2013,108(4):964-72.
[22]Li X,Gong X,Chen J,et al.miR-340 inhibits glioblastoma cell proliferation by suppressing CDK6,cyclin-D1 and cyclin-D2[J].Biochem Biophys Res Commun,2015,460(3):670-7.
[23]Das S,Bryan K,Buckley PG,et al.Modulation of neuroblastoma disease pathogenesis by an extensive network of epigenetically regulated microRNAs[J].Oncogene,2013,32(24):2927-36.
[24]Fernandez S,Risolino M,Mandia N,et al.miR-340 inhibits tumor cell proliferation and induces apoptosis by targeting multiple negative regulators of p27 in non-small cell lung cancer[J].Oncogene,2015,34(25):3240-50.
[25]Takeyama H,Yamamoto H,Yamashita S,et al.Decreased miR-340expression in bone marrow is associated with liver metastasis of colorectal cancer[J].Mol Cancer Ther,2014,13(4):976-85.
[26]Memczak S,Jens M,Elefsinioti A,et al.Circular RNAs are a large class of animal RNAs with regulatory potency[J].Nature,2013,495(7441):333-8.
[27]Capel B,Swain A,Nicolis S,et al.Circular transcripts of the testisdetermining gene Sry in adult mouse testis[J].Cell,1993,73(5):1019-30.
[28]Pearce MB,Jayaraman A,Pappas C,et al.Pathogenesis and transmission of swine origin A(H3N2)v influenza viruses in ferrets[J].Proc NatlAcad Sci USA,2012,109(10):3944-9.
[29]Hsu MT,Coca-Prados M.Electron microscopic evidence for the circular form of RNA in the cytoplasm of eukaryotic cells[J].Nature,1979,280(5720):339-40.
[30]Xue J,Liu Y,Luo F,et al.Circl 00284,via miR-217 regulation of EZH2,is involved in the arsenite-accelerated cell cycle of human keratinocytes in carcinogenesis[J].Biochim Biophys Acta Mol Basis Dis,2017,1863(3):753-63.
[31]Khan I,Kerwin J,Owen K,et al.Correction:Registered report:Acoding-independent function of gene and pseudogene mRNAs regulates tumour biology[J].Elife,2015,4:e13015.