上调lncRNA SNHG12与miR-199a-5p/FZD6轴对肝细胞癌细胞增殖、侵袭和上皮-间质转化的影响

背景与目的：肝细胞癌（HCC）是临床上常见的恶性肿瘤之一，侵袭、转移和术后复发是导致HCC患者死亡的主要原因。目前认为长链非编码RNA（lncRNA）的失调可能与各类癌症的发生和转移有关。有研究显示lncRNA SNHG12在HCC组织表达明显上调，但其具体功能尚不清楚。故本研究探讨lncRNA SNHG12在HCC细胞中的作用及机制。 方法：用qRT-PCR检测SNHG12以及预测到的靶miRNA及靶基因（miR-199a-5p与FZD6）在HCC细胞HepG2细胞与人肝内胆管上皮细胞HIBEC中的基因表达量，观察下调SNHG12对HepG2细胞增殖、侵袭和上皮-间质转化（EMT）相关蛋白表达的影响；采用miRanda和双荧光素酶报告基因实验分析SNHG12和miR-199a-5p之间的关系，分析下调miR-199a-5p对HepG2细胞增殖、侵袭和EMT的影响，以及SNHG12对miR-199a-5p作用的影响；TargetScan和双荧光素酶报告基因实验分析miR-199a-5p和FZD6的关系；检测过表达及下调FZD6对HepG2细胞增殖、侵袭和EMT的影响，以及SNHG12和miR-199a-5p对FZD6表达的影响。 结果：与HIBEC细胞比较，HepG2细胞中SNHG12表达量明显升高、miR-199a-5p表达量明显降低、FZD6表达量明显升高（均P<0.01）。下调SNHG12表达，HepG2细胞增殖、侵袭和EMT被明显抑制，过表达SNHG12作用则相反（均P<0.05）。SNHG12与miR-199a-5p特异性结合，下调miR-199a-5p促进HepG2细胞增殖、侵袭与EMT，过表达miR-199a-5p则起到抑制作用（均P<0.05）。过表达miR-199a-5p对HepG2细胞的作用，能部分被同时过表达SNHG12所逆转（均P<0.05）。miR-199a-5p靶向FZD6，下调FZD6后HepG2细胞增殖、侵袭与EMT明显抑制，过表达FZD6则相反（均P<0.05)。下调SNHG12抑制FZD6的基因和蛋白表达，而下调miR-199a-5p则促进FZD6的表达（均P<0.01）；与单独下调miR-199a-5p比较，同时下调SNHG12和miR-199a-5p，FZD6的表达被抑制（P<0.01）。 结论：HCC细胞中lncRNA SNHG12的上调可能通过影响miR-199a-5p/FZD6轴促进HCC细胞的增殖、侵袭和EMT过程。

Influence of lncRNA SNHG12 up-regulation and miR-199a-5p/FZD6 axis on proliferation, invasion and epithelial-mesenchymal transition of hepatocellular carcinoma cells

Background and Aims: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in clinical practice. Invasion, metastasis and postoperative recurrence are the main causes of death for HCC patients. At present, the dysregulation of long non-coding RNAs (lncRNA) is considered to be related to the occurrence and metastasis of various cancers. Studies have showed that lncRNA SNHG12 expression is increased in HCC tissue, but its specific function is still unclear. Therefore, this study was conducted to investigate the action of lncRNA SNHG12 in HCC cells and the mechanism.  Methods: The gene expressions of SNHG12 as well as the predicted targeted miRNA and gene (miR-199a-5p and FZD6) in HCC cell lines HepG2 and human intrahepatic bile duct cell line HIBEC were detected by qRT-PCR method. The effects of down-regulation of SNHG12 on the proliferation, invasion and the expressions of proteins associated with epithelial-mesenchymal transition (EMT) in HepG2 cells were observed. The association between SNHG12 and miR-199a-5p was analyzed by miRanda and Dual luciferase reporter gene assay, and then, the effects of down-regulation of miR-199a-5p on the proliferation, invasion and EMT in HepG2 cells as well as the influences of SNHG12 exerted by miR-199a-5p were analyzed. The relationship between miR-199a-5p and FZD6 was analyzed by the TargetScan and Dual luciferase reporter gene assay, and then, the effects of up-regulation and down-regulation of FZD6 on the proliferation, invasion and EMT in HepG2 cells as well as the effects of SNHG12 and miR-199a-5p on FZD6 expression were detected. Results: The expression level of SNHG12 was significantly increased, miR-199a-5p was significantly decreased and FZD6 was significantly increased in HepG2 cells compared with those in HIBEC cells (all P<0.01). In HepG2 cells, the proliferation, invasion and EMT were significantly inhibited after down-regulation of SNHG12, while its overexpression resulted in opposite effects (all P<0.05). The 3''UTR of SNHG12 was targeted by miR-199a-5p. The proliferation, invasion and EMT of HepG2 cells were augmented by down-regulation of miR-199a-5p, and were suppressed by its overexpression (all P<0.05). The effects of miR-199a-5p overexpression on HepG2 cells were partially reversed by the simultaneous overexpression of SNHG12 (all P<0.05). The miR-199a-5p were targeted by FZD6. The proliferation, invasion and EMT of HepG2 cells were inhibited by down-regulation of FZD6, while were increased by up-regulation of FZD6 (all P<0.05). Down-regulation of SNHG12 inhibited FZD6 gene and protein expressions, while down-regulation of miR-199a-5p promoted FZD6 expression (all P<0.01). Compared with down-regulation of miR-199a-5p alone, the FZD6 expression was significantly decreased by both SNHG12 and miR-199a-5p down-regulations (P<0.01). Conclusion: Upregulation of lncRNA SNHG12 in HCC cells can promote the proliferation, invasion and EMT process of HCC cells through influencing the miR-199a-5p/FZD6 axis.