microRNA98靶向N-RAS对鼻咽癌CNE-1细胞增殖与迁移的影响
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摘要
目的探讨miR-98对鼻咽癌CNE-1细胞增殖与迁移的影响及可能机制。方法利用脂质体介导的miR-98模拟物或阴性对照转染鼻咽癌CNE-1细胞,并通过实时定量PCR进行验证。MTT实验检测CNE-1细胞增殖能力的变化,划痕实验检测CNE-1迁移能力变化,采用生物信息学软件预测N-RAS是否为miR-98潜在的靶基因,并以双荧光素酶报告基因实验验证。Western blot检测miR-98过表达后CNE-1细胞N-RAS蛋白的表达变化以及下游ERK、p-ERK、AKT、p-AKT的表达变化。结果 MiR-98过表达能抑制鼻咽癌CNE-1细胞的体外增殖与迁移能力;生物信息学软件分析结果显示N-RAS是miR-98的靶基因之一,双荧光素酶报告基因证实N-RAS为miR-98的下游靶基因。miR-98过表达后,CNE-1细胞中N-RAS表达下调,ERK与Akt蛋白的表达水平不变,但pERK与p-Akt蛋白的表达水平下调。结论 miR-98通过靶向调控N-RAS抑制鼻咽癌CNE-1细胞增殖与迁移。
Objective To investigate the effect and possible mechanism of microRNA-98 on the proliferation and migration of nasopharyngeal carcinoma CNE-1 cells. Methods NPC CNE-1 cells were transfected with liposomemediated mimics of microRNA-98 or negative control, and verified by real-time quantitative PCR. MTT assay was used to detect the proliferation of CNE-1 cells, scratch assay was used to detect the migration of CNE-1 cells, and bioinformatics software was used to predict whether N-RAS was a potential target gene of microRNA-98, which was evaluated by dual luciferase reporter assay. Western blot was used to detect the expression of N-RAS protein and downstream ERK, p-ERK,AKT and p-AKT proteins in CNE-1 cells after over-expression of microRNA-98. Results MiR-98 overexpression inhibited the proliferation and migration of nasopharyngeal carcinoma CNE-1 cells in vitro. Bioinformatics software analysis showed that N-RAS was one of the target genes of microRNA-98, and double luciferase reporter assay confirmed that NRAS was the downstream target gene of microRNA-98. After the overexpression of microRNA-98, the expression level of NRAS was down-regulated in CNE-1 cells, while the expression levels of ERK and Akt proteins remained unchanged, but the expression levels of p-ERK and p-Akt protein were down-regulated. Conclusion Mi-98 inhibited the proliferation and migration of nasopharyngeal carcinoma CNE-1 cells by targeting N-RAS.
Objective To investigate the effect and possible mechanism of microRNA-98 on the proliferation and migration of nasopharyngeal carcinoma CNE-1 cells. Methods NPC CNE-1 cells were transfected with liposomemediated mimics of microRNA-98 or negative control, and verified by real-time quantitative PCR. MTT assay was used to detect the proliferation of CNE-1 cells, scratch assay was used to detect the migration of CNE-1 cells, and bioinformatics software was used to predict whether N-RAS was a potential target gene of microRNA-98, which was evaluated by dual luciferase reporter assay. Western blot was used to detect the expression of N-RAS protein and downstream ERK, p-ERK,AKT and p-AKT proteins in CNE-1 cells after over-expression of microRNA-98. Results MiR-98 overexpression inhibited the proliferation and migration of nasopharyngeal carcinoma CNE-1 cells in vitro. Bioinformatics software analysis showed that N-RAS was one of the target genes of microRNA-98, and double luciferase reporter assay confirmed that NRAS was the downstream target gene of microRNA-98. After the overexpression of microRNA-98, the expression level of NRAS was down-regulated in CNE-1 cells, while the expression levels of ERK and Akt proteins remained unchanged, but the expression levels of p-ERK and p-Akt protein were down-regulated. Conclusion Mi-98 inhibited the proliferation and migration of nasopharyngeal carcinoma CNE-1 cells by targeting N-RAS.
引文
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[3]Chen SS,Peng M,Zhou GZ,et al.Long non-coding RNA HOTAIRregulates the development of non-small cell lung cancer through miR-217/DACH1 signaling pathway[J].Eur Rev Med Pharmacol Sci,2019,23(2):670-678.
[4]Yang L,Zhang X,Li H,et al.The long noncoding RNA HOTAIRactivates autophagy by upregulating ATG3 and ATG7 in hepatocellular carcinoma[J].Mol Biosyst,2016,12(8):2605-2612.
[5]GU Y,Chen T,Li G,et al.LncRNAs:emerging biomarkers in gastric cancer[J].Future Oncol,2015,11(17):2427-2441.
[6]Dong X,He X,Guan A,et al.Long non-coding RNA Hotair promotes gastric cancer progression via miR-217-GPC5 axis[J].Life Sci,2019,217:271-282.
[7]Cheng C,Qin Y,Zhi Q,et al.Knockdown of long non-coding RNAHOTAIR inhibits cisplatin resistance of gastric cancer cells through inhibiting the PI3K/Akt and Wnt/beta-catenin signaling pathways by up-regulating miR-34a[J].Int J Biol Macromol,2018,107:2620-2629.
[8]Towers CG,Thorbutn A.Therapeutic Targeting of Autophagy[J].EBioMedicine,2016,14:15-23.
[9]Li YT,Yi C,Chen CC,et al.A semisynthetic Atg3 reveals that acetylation promotes Atg3 membrane binding and Atg8 lipidation[J].Nat Commun,2017,8:14846-1852.
[10]Fan Q,Yang L,Zhang X,et al.Autophagy promotes metastasis and glycolysis by upregulating MCT1 expression and Wnt/beta-catenin signaling pathway activation in hepatocellular carcinoma cells[J].JExp Clin Cancer Res,2018,37(1):9-15.
[11]Kong F,Jin J,Lv X,et al.Long noncoding RNA RMRPupregulation aggravates myocardial ischemia-reperfusion injury by sponging miR-206 to target ATG3 expression[J].Biomed Pharmacother,2019,109:716-25.