SNHG3调控miR-186-5p/ZEB1促进肝癌细胞增殖、迁移、侵袭和上皮-间质转化的机制研究

［摘要］　目的　探究SNHG3调控miR-186-5p/E盒结合锌指蛋白1（ZEB1）促进肝癌细胞增殖、迁移、侵袭和上皮-间质转化（EMT）的机制。方法　在HepG2细胞中转染siRNA干扰片段敲除SNHG3、转染pcDNA3.1(+)/SNHG3使SNHG3过表达，通过实时荧光定量聚合酶链式反应（FQ-PCR）检测是否转染成功。克隆形成实验检测HepG2细胞增殖，细胞划痕实验检测HepG2细胞迁移率，Transwell小室实验检测HepG2细胞侵袭数量，双荧光素酶活性检测验证SNHG3与miR-186-5p、miR-186-5p与ZEB1的靶向关系，FQ-PCR和Western blot法检测SNHG3、ZEB1、E-cadherin、N-cadherin、MMP-2、vimentin、MMP-9、Snail的表达水平。结果　肝癌组织中SNHG3表达量高于正常组织，SNHG3表达量低/中的肝癌患者的生存预后优于SNHG3表达量高的肝癌患者，差异有统计学意义（P<0.05）。克隆形成实验结果显示，与pcDNA3.1(+)组相比，pcDNA3.1(+)/SNHG3组HepG2细胞增殖数量显著增加（P<0.05）。细胞划痕实验结果显示，pcDNA3.1(+)/SNHG3组HepG2细胞迁移率显著高于pcDNA3.1(+)组（P<0.05）。Transwell小室实验结果显示，pcDNA3.1(+)/SNHG3组HepG2细胞侵袭数量显著高于pcDNA3.1(+)组（P<0.05）。双荧光素酶活性检测结果显示，miR-186-5p mimic和pGL6-SNHG3-WT共转染后，荧光素酶活性低于其他组，miR-186-5p mimic和pGL6-ZEB1-WT共转染后，荧光素酶活性低于其他组，差异有统计学意义（P<0.05）。FQ-PCR和Western blot检测结果显示，当SNHG3过表达时，E-cadherin mRNA和蛋白相对表达量显著下调，N-cadherin、MMP-2、vimentin、MMP-9和Snail mRNA和蛋白相对表达量以及ZEB1蛋白相对表达量显著上调；当SNHG3敲除后，E-cadherin mRNA和蛋白相对表达量显著上调，N-cadherin、MMP-2、vimentin、MMP-9和Snail mRNA和蛋白相对表达量以及ZEB1蛋白相对表达量显著下调，差异有统计学意义（P<0.05）。结论　SNHG3调控miR-186-5p/ZEB1可促进肝癌细胞增殖、迁移、侵袭和EMT，表明SNHG3是肝癌潜在的治疗靶点。

A study on the mechanism of SNHG3 promoting proliferation, migration, invasion and epithelial-mesenchymal transition of hepatoma cells by regulating the miR-186-5p/ZEB1 axis

［Abstract］　Objective　To explore the mechanism of SNHG3 promoting proliferation, migration, invasion and epithelial-mesenchymal transition(EMT) of hepatoma cells by regulating the micro ribonucleic acid(miR)-186-5p/zinc finger E-box binding homeobox 1(ZEB1) axis. Methods　Human-derived hepatoma(HepG2) cells were transfected with small interfering RNA(siRNA) to knock out SNHG3 and transfected with pcDNA3.1(+)/SNHG3 to overexpress SNHG3, and real-time fluorescent quantitative polymerase chain reaction(FQ-PCR) was used to detect whether the transfections were successful. HepG2 cell proliferation was detected by colony formation assay. Cell scratch wound healing assay was used to detect the migration rate of HepG2 cells. Transwell assay was used to detect the number of HepG2 cell invasion. Dual-luciferase reporter assay was used to verify the targeting relationship between SNHG3 and miR-186-5p as well as the targeting relationship between miR-186-5p and ZEB1. The expression levels of SNHG3, ZEB1, E-cadherin, N-cadherin, matrix metalloproteinase-2(MMP-2), vimentin, matrix metalloproteinase-9(MMP-9) and Snail were detected by using FQ-PCR and Western blot. Results　The expression of SNHG3 in hepatocellular carcinoma(HCC) tissues was higher than that in normal tissues, and the survival prognosis of the HCC patients with low/moderate SNHG3 expression was better than that of the HCC patients with high SNHG3 expression, and the differences were statistically significant(P<0.05). The results of cloncolony formation assay showed that compared with that in the pcDNA3.1(+) group, the proliferation of HepG2 cells in the pcDNA3.1(+)/SNHG3 group was significantly increased(P<0.05). The results of cell scratch wound healing assay showed that the migration rate of HepG2 cells in the pcDNA3.1(+)/SNHG3 group was significantly higher than that in the pcDNA3.1(+) group(P<0.05). The results of Transwell assay showed that the number of HepG2 cell invasion in the pcDNA3.1(+)/SNHG3 group was significantly higher than that in the pcDNA3.1(+) group(P<0.05). The results of dual-luciferase reporter assay showed that after co-transfection with miR-186-5p mimic and pGL6-SNHG3-WT, the luciferase activity was lower than the other groups, and after co-transfection with miR-186-5p mimic and pGL6-ZEB1-WT, the luciferase activity was lower than the other groups, and the differences were statistically significant(P<0.05). The results of FQ-PCR and Western blot showed that when SNHG3 was overexpressed, the relative expressions of E-cadherin mRNA and protein were significantly downregulated, while the relative expressions of N-cadherin, MMP-2, vimentin, MMP-9, and Snail mRNA and protein, and the relative expressions of ZEB1 protein were significantly upregulated, and after SNHG3 was knocked out, the relative expressions of E-cadherin mRNA and protein were significantly upregulated, while the relative expressions of N-cadherin, MMP-2, vimentin, MMP-9 and Snail mRNA and protein, and the relative expression of ZEB1 protein were significantly downregulated, and the differences were statistically significant(P<0.05). Conclusion　SNHG3 can promote the proliferation, migration, invasion and EMT of HCC cells by regulating the miR-186-5p/ZEB1, indicating that SNHG3 is a potential therapeutic target for HCC.