Drosha通过调控微RNA-195-5P促进胃癌细胞的迁移和侵袭
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摘要
目的 :探讨Drosha对胃癌MGC-803和SGC-7901细胞迁移和侵袭能力的影响及其可能的作用机制。方法:将携带有Drosha-shRNA的重组慢病毒感染胃癌MGC-803和SGC-7901细胞,以感染携带有阴性对照(negative control,NC)-shRNA的重组慢病毒作为对照。采用实时荧光定量PCR法和蛋白质印迹法检测转入Drosha-shRNA后对MGC-803和SGC-7901细胞中Drosha mRNA和蛋白表达的影响,Transwell小室迁移和侵袭实验分别检测Drosha下调对MGC-803和SGC-7901细胞迁移和侵袭能力的影响;实时荧光定量PCR检测Drosha下调的MGC-803和SGC-7901细胞中微RNA-195-5P(microRNA-195-5P,miR-195-5P)的表达水平。在Drosha下调的胃癌细胞中采用脂质体法同时转入miR-195-5P-inhibitor,实时荧光定量PCR检测miR-195-5P的下调效果,Transwell小室迁移和侵袭实验分别检测下调miR-195-5P表达对细胞迁移和侵袭能力的影响。采用脂质体法将miR-195-5P-mimics转染MGC-803和SGC-7901细胞,实时荧光定量PCR检测miR-195-5P过表达的效果,Transwell小室迁移和侵袭实验分别检测miR-195-5P过表达对胃癌细胞迁移和侵袭能力的影响。结果 :感染Drosha-shRNA重组慢病毒后,MGC-803和SGC-7901细胞中Drosha mRNA和蛋白表达水平均较对照组明显下调(P值均<0.01),细胞的迁移和侵袭能力均明显被抑制(P值均<0.05),miR-195-5P的表达水平明显上调(P值均<0.001)。成功在Drosha下调的胃癌MGC-803和SGC-7901细胞中下调miR-195-5P的表达水平(P值均<0.05),miR-195-5P表达下调可以恢复Drosha下调对胃癌细胞迁移和侵袭能力的影响(P值均<0.05)。成功将miR-195-5P-mimics瞬时转入MGC-803和SGC-7901细胞,MGC-803和SGC-7901细胞中miRNA-195-5P的表达水平明显上调(P值均<0.01),细胞的迁移和侵袭能力均明显降低(P值均<0.01)。结论 :Drosha可促进胃癌细胞的迁移和侵袭能力。Drosha与miR-195-5P的表达呈负相关性,Drosha通过调控miR-195-5P的表达促进胃癌细胞的迁移和侵袭。
Objective: To investigate the effects of Drosha on the migration and invasion of gastric cancer MGC-803 and SGC-7901 cells, and to explore the possible mechanism.Methods: MGC-803 and SGC-7901 cells were infected with the recombinant lentivirus carrying Drosha-shRNA or the negative control(NC)-shRNA(as the control). The expression levels of Drosha mRNA and protein in MGC-803 and SGC-7901 cells were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The migration and invasion abilities of MGC-803 and SGC-7901 cells with Drosha gene silencing were detected by Transwell assay, and the expression level of microRNA-195-5P(miR-195-5P) was detected by real-time fluorescent quantitative PCR. After Drosha gene silenced cells were transfected with miR-195-5P-inhibitor, the down-regulation of miR-195-5P expression was tested by real-time fluorescent quantitative PCR, then the migration and invasion of gastric cancer cells were detected by Transwell assay. After miR-195-5P-mimics or the control-mimics were transfected into MGC-803 and SGC-7901 cells, the expression level of miR-195-5P was tested by real-time fluorescent quantitative PCR, then the migration and invasion of gastric cancer cells were detected by Transwell assay.Results: The expression levels of Drosha mRNA and protein in MGC-803 and SGC-7901 cells infected with the recombinant lentivirus carrying Drosha-shRNA were down-regulated as compared with the control groups(both P < 0.01). The migration and invasion abilities of MGC-803 and SGC-7901 cells with Drosha gene silencing were inhibited(all P < 0.05), and the expression level of miR-195-5P was up-regulated(both P < 0.001). The miR-195-5P expression level was down-regulated in MGC-803 and SGC-7901 cells with Drosha gene silencing after transfection with miR-195-5P-inhibitor(both P < 0.05), then the migration and invasion abilities of gastric cancer cells were rescued(all P < 0.05). The miR-195-5P expression level was upregulated in MGC-803 and SGC-7901 cells after transfection with miR-195-5P-mimics(both P < 0.05), then the migration and invasion abilities of gastric cancer cells were decreased(all P < 0.05).Conclusion: Drosha can promote the migration and invasion of gastric cancer cells by regulating the expression of miR-195-5P. Drosha is negatively correlated with the expression of miR-195-5P.
Objective: To investigate the effects of Drosha on the migration and invasion of gastric cancer MGC-803 and SGC-7901 cells, and to explore the possible mechanism.Methods: MGC-803 and SGC-7901 cells were infected with the recombinant lentivirus carrying Drosha-shRNA or the negative control(NC)-shRNA(as the control). The expression levels of Drosha mRNA and protein in MGC-803 and SGC-7901 cells were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The migration and invasion abilities of MGC-803 and SGC-7901 cells with Drosha gene silencing were detected by Transwell assay, and the expression level of microRNA-195-5P(miR-195-5P) was detected by real-time fluorescent quantitative PCR. After Drosha gene silenced cells were transfected with miR-195-5P-inhibitor, the down-regulation of miR-195-5P expression was tested by real-time fluorescent quantitative PCR, then the migration and invasion of gastric cancer cells were detected by Transwell assay. After miR-195-5P-mimics or the control-mimics were transfected into MGC-803 and SGC-7901 cells, the expression level of miR-195-5P was tested by real-time fluorescent quantitative PCR, then the migration and invasion of gastric cancer cells were detected by Transwell assay.Results: The expression levels of Drosha mRNA and protein in MGC-803 and SGC-7901 cells infected with the recombinant lentivirus carrying Drosha-shRNA were down-regulated as compared with the control groups(both P < 0.01). The migration and invasion abilities of MGC-803 and SGC-7901 cells with Drosha gene silencing were inhibited(all P < 0.05), and the expression level of miR-195-5P was up-regulated(both P < 0.001). The miR-195-5P expression level was down-regulated in MGC-803 and SGC-7901 cells with Drosha gene silencing after transfection with miR-195-5P-inhibitor(both P < 0.05), then the migration and invasion abilities of gastric cancer cells were rescued(all P < 0.05). The miR-195-5P expression level was upregulated in MGC-803 and SGC-7901 cells after transfection with miR-195-5P-mimics(both P < 0.05), then the migration and invasion abilities of gastric cancer cells were decreased(all P < 0.05).Conclusion: Drosha can promote the migration and invasion of gastric cancer cells by regulating the expression of miR-195-5P. Drosha is negatively correlated with the expression of miR-195-5P.
引文
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[8]XU L,HOU Y,TU G,et al.Nuclear Drosha enhances cell invasion via an EGFR-ERK1/2-MMP7 signaling pathway induced by dysregulated miRNA-622/197 and their targets LAMC2 and CD82 in gastric cancer[J].Cell Death Dis,2017,8(3):e2642.
[9]CAI C,HE H,DUAN X,et al.miR-195 inhibits cell proliferation and angiogenesis in human prostate cancer by downregulating PRR11 expression[J].Oncol Rep,2018,39(4):1658-1670.
[10]YANG C,WU K,WANG S,et al.Long non-coding RNA XIST promotes osteosarcoma progression by targeting YAP via miR-195-5p[J].J Cell Biochem,2018,119(7):5646-5656.
[11]SLATTERY ML,MULLANY LE,SAKODALC,et al.Dysregulated genes and miRNAs in the apoptosis pathway in colorectal cancer patients[J].Apoptosis,2018,23(3/4):237-250.
[12]WANG R,MA Q,JI L,et al.miR-622 suppresses tumor formation by directly targeting VEGFA in papillary thyroid carcinoma[J].Onco Targets Ther,2018,11:1501-1509.
[2]HA M,KIM VN.Regulation of microRNA biogenesis[J].Nat Rev Mol Cell Biol,2014,15(8):509-524.
[3]SHU GS,YAN GZ L,LIU DC.Immunohistochemical study of Dicer and Drosha expression in the benign and malignant lesions of gallbladder and their clinicopathological significances[J].Pathol Res Pract,2012,208(7):392-397.
[4]DíAZ-GARCíA CV,AGUDO-LóPEZA,PéREZ C,et al.DICER1,DROSHA and miRNAs in patients with nonsmall cell lung cancer:Implications for outcomes and histologic classification[J].Carcinogenesis,2013,34(5):1031-1038.
[5]VAN ROOIJ E,OLSON EN.MicroRNAs:powerful new regulators of heart disease and provocative therapeutic targets[J].J Clin Invest,2007,117(9):2369-2376.
[6]ZHAO YF,HAN ML,XIONG YJ,et al.A micRNA-200c/cathepsin L feedback loop determines paclitaxel resistance in human lung cancer A549 cells in vitro through regulating epithelial-mesenchymal transition[J].Acta Pharmacol Sin,2018,39(6):1034-1047.
[7]MIRGHASEMI A,TAHERIAZAM A,KARBASY SH,et al.Down-regulation of mi R-133a and miR-539 are associated with unfavorable prognosis in patients suffering from osteosarcoma[J].Cancer Cell Int,2015,15:86.
[8]XU L,HOU Y,TU G,et al.Nuclear Drosha enhances cell invasion via an EGFR-ERK1/2-MMP7 signaling pathway induced by dysregulated miRNA-622/197 and their targets LAMC2 and CD82 in gastric cancer[J].Cell Death Dis,2017,8(3):e2642.
[9]CAI C,HE H,DUAN X,et al.miR-195 inhibits cell proliferation and angiogenesis in human prostate cancer by downregulating PRR11 expression[J].Oncol Rep,2018,39(4):1658-1670.
[10]YANG C,WU K,WANG S,et al.Long non-coding RNA XIST promotes osteosarcoma progression by targeting YAP via miR-195-5p[J].J Cell Biochem,2018,119(7):5646-5656.
[11]SLATTERY ML,MULLANY LE,SAKODALC,et al.Dysregulated genes and miRNAs in the apoptosis pathway in colorectal cancer patients[J].Apoptosis,2018,23(3/4):237-250.
[12]WANG R,MA Q,JI L,et al.miR-622 suppresses tumor formation by directly targeting VEGFA in papillary thyroid carcinoma[J].Onco Targets Ther,2018,11:1501-1509.