长链非编码RNA MALAT1促进肝癌细胞系迁移的机制研究

摘要

目的 探索长链非编码RNA(lncRNA)肺腺癌转移相关转录子1(metastasis associated lung adenocarcinoma transcript 1,MALAT1)在肝细胞癌(hepatocellular carcinoma,HCC;简称肝癌)细胞中是否可作为竞争性内源RNA(competing endogenous RNA,ceRNA)发挥促迁移功能.方法 在HepG2细胞中通过瞬时转染MALAT1的小干扰RNA(small interfering RNA,siRNA)敲低MALAT1的表达;利用Transwell实验检测敲低MALAT1后对细胞迁移能力的影响;利用RNA pull-down实验确认MALAT1是否可直接吸附miR-126*;荧光定量逆转录PCR(quantitative reverse transcription PCR,qRT-PCR)和Western印迹检测敲低MALAT1后miR-126*靶基因的mRNA和蛋白水平的变化;利用公共数据集癌症基因组图谱(The Cancer Genome Atlas,TCGA)分析MALAT1和miR-126*在肝癌组织样本中的表达情况和预后价值.结果 与对照组相比,敲低MALAT1可显著抑制肝癌细胞的迁移能力;MALAT1含有3个miR-126*的结合位点,其野生型序列相对于突变体序列可显著吸附miR-126*;敲低MALAT1后,miR-126*的靶基因CXCL12的mRNA和蛋白水平均显著下调;TCGA肝癌数据分析显示MALAT1在癌组织中显著上调表达,且MALAT1高表达组有较低的无病生存率,而miR-126*与其相反.结论 MALAT1可吸附miR-126*,上调CXCL12的表达,进而促进肝癌的转移.%Objective To investigate whether the long non-coding RNA (lncRNA) metastasis associated lung adenocarcinoma transcript 1 (MALAT1) can act as a competitive endogenous RNA (ceRNA) to promote the migration of hepatocellular carcinoma (HCC) cells.Methods Transient transfection of small interfering RNA (siRNA) against MALAT1 was used to knockdown MALAT1 in HepG2 cells.Transwell assays were employed to assess the migration capabilities of HepG2 cells upon MALAT1 knockdown.RNA pull-down assays were performed to validate the direct binding between MALAT1 and miR-126*.Quantitative reverse transcription PCR (qRT-PCR) and Western blotting assays were used to detect the mRNA and protein levels of the miR-126* target genes.The dysregulation and prognostic significance of MALAT1 and miR-126* were analyzed in the public dataset of The Cancer Genome Atlas (TCGA).Results Compared with the control group, MALAT1 knockdown significantly inhibited the migration of HCC cells.MALAT1, with three miR-126* response elements, directly sponged miR-126* in a sequence-specific manner.The mRNA and protein levels of CXCL12, which was the miR-126* target gene, were significantly down-regulated upon MALAT1 knockdown.The TCGA database showed that MALAT1 was significantly up-regulated in HCC and high expression levels of MALAT1 were significantly associated with poor disease-free survival, whereas an opposite pattern of miR-126* was observed.Conclusion This study suggests that MALAT1 directly sponges miR-126* and upregulates the expression of CXCL12, which in turn promotes the migration of HCC cells.