lnc171靶向调控miR-149-5p促进肝癌细胞的迁移

LNC171 targets miR-149-5p promoting the migration of hepatoma cells

目的:探讨长链非编码RNA RP1-171K16.5(lnc171)对肝癌细胞的作用和机制。方法:采用实时荧光定量PCR(qPCR)法检测正常人和肝癌患者血浆外泌体、正常肝细胞和肝癌细胞中lnc171的表达水平;设计siRNA转染肝细胞,分为空白对照组(Blank组)、阴性对照组(siNC组)和干扰组(si171组);采用Transwell实验检测lnc171对细胞迁移的影响;RegRNA、miRDB和LncBase在线靶基因预测网站预测lnc171的靶基因,并采用Venny 2.1对3个数据库预测结果取交集;双荧光素酶报告基因实验验证lnc171和miR-149-5p之间的靶向关系;生物信息学分析miR-149-5p的靶蛋白RAP1并用qPCR验证。结果:肝癌患者血浆外泌体中lnc171的表达水平高于正常对照者(P<0.01),肝癌细胞中lnc171的表达水平高于正常肝细胞(P<0.05);与siNC组比较,转染si171-3后,HL-7702和Hep3B细胞迁移数量明显下降(P<0.05);生物信息学预测lnc171的靶基因为miR-149-5p;与siNC组比较,Hep3B细胞转染si171-3后miR-149-5p的表达量升高(P<0.001);lnc171WT中miR-149-5p mimics组荧光素酶活性较miR-NC组下降(P<0.05);利用TargetScan和MicroT-CDS在线网站预测miR-149-5p的靶基因取得256个交集靶基因,将其放入DAVID数据库中进行KEGG通路富集得到与迁移相关的RAP1信号通路。生存分析发现,Ras-associated protein 1A(RAP1A)高表达的肝癌患者预后差。结论:lnc171在肝癌中高表达,可能通过靶向作用miR-149-5p调控RAP1A表达水平从而促进肝癌细胞迁移。

Objective: To investigate the effects and mechanism of long non-coding RNA RP1-171 k16.5(lnc171) on hepatoma cells. Methods: The expression level of lnc171 in plasma exosomes of healthy subjects and liver cancer patients, hepatocytes and hepatoma cells were detected by RT-qPCR assay;SiRNAs were designed and transfected to cells. This study included blank control group(Blank group), negative control group(siNC group) and interference group(si171 group). Transwell assay was used to detect the effect of lnc171 on cell migration. Target genes of lnc171 were predicted by using the online target gene prediction websites of RegRNA, miRDB and lncBase, and the prediction results of the three databases were intersected by Venny2.1;Dualluciferase reporter gene assay verified the targeted relationship between lnc171 and miR-149-5 p. Target genes of miR-149-5 p were analyzed by bioinformatics and verified by qPCR. Results: The expression level of lnc171 in plasma exosomes of liver cancer patients was higher than that of normal controls(P<0.01), and the expression level of lnc171 in hepatoma cells was higher than that of hepatocytes(P<0.05). Compared with the siNC group,The migration of HL-7702 and Hep3 B cells was significantly decreased after transfected with si171-3(P<0.05).Bioinformatics predicted that the target gene of lnc171 was miR-149-5 p. Compared with the siNC group, the expression level of mi R-149-5 p increased after si171-3 transfection of Hep3 B cells(P<0.001). Luciferase activity in the mi R-149-5 p mimics group decreased compared with the mi R-NC group in lnc171 WT(P<0.05). Using Target Scan and micro T-CDS online sites to predict the target genes of mi R-149-5 p, 256 intersection target genes were obtained, which were put into DAVID online site for KEGG pathway enrichment to obtain the transit-related RAP1 signaling pathway. Survival analysis showed that HCC patients with high Ras-associated protein 1 A(RAP1 A) expression had poor prognosis. Conclusion: lnc171 is highly expressed in HCC, which may promote hepatoma cells migration by targeting mi R-149-5 p to regulate the expression level of RAP1 A.