LncRNA SNHG16 Promotes Hepatocellular Carcinoma Proliferation,migration And Invasion By Regulating MiR-186

Objective:To investigate the expression and role of long non-coding RNA(LncRNA)small nucleolar RNA host gene 16(SNHG16)in hepatocellular carcinoma(HCC)and to confirm whether SNHG16 promotes the proliferation,migration and invasion of HCC by regulating microRNA-186(miR-186).Methods:Real-time quantitative PCR(qRT-PCR)was conducted to evaluate the expression level of SNHG16 in HCC tissues and normal liver tissues.In addition,the expression level of SNHG16 in five HCC cell lines(Hep-3B,Huh7,Sk-hep-1,SMMC-7721 and PLC)and normal hepatocytes(HL-77O2)were detected by qRT-PCR.Hep-3B and Sk-hep-1 cell were infected with lentivirus in order to get HCC cell lines with stably over-expressing and knocking down SNHG16.The effects of SNHG16 on cell proliferation,migration and invasion were detected by CCK-8 assay,transwell migration assay and transwell invasion assay,respectively.Xenograft tumor experiment was used to determine the biological functionof SNHG16 in vivo.Next,the relationship between SNHG16,miR-186 and ROCK1 were analyzed using bioinformatics analysis,qRT-PCR,luciferase reporter assay and western blot.Rescue experiments were conducted to demonstrate that SNHG16 affects proliferation,migration and invasion of HCC cells by regulating miR-186.Results:The results of qRT-PCR showed that compared with normal liver tissues and normal liver cells,the expression of SNHG16 was significantly up-regulated in HCC tissues(P<0.001)and HCC cell lines(P<0.001,P<0.01,P<0.05,P<0.05 and P<0.05).The chi-square test results showed:The expression of SNHG16 in cancer tissues was highly correlated with tumor size(P<0.05),TNM stage(P<0.001),ALT expression level(P<0.05)and HBV DNA level(P<0.01).After lentivirus infection,the expression level of SNHG16 was significantly increased in the SNHG16group(P<0.01);The expression level of SNHG16 was essentially decreased in the sh-SNHG16-2 group(P<0.001).The results of CCK-8 showed that the cell proliferation activity of SNHG16 group was higher than that of NC group,and it was the most obvious at 72 hour(P<0.05).In contrast,the cell proliferation activity of sh-SNHG16-2 group was weaker than that of sh-NC group.Both 48 hour(P<0.05)and 72 hour(P<0.01)were more obvious.The results of Transwell migration and invasion experiments showed that compared with the NC group,the number of cells of migration(P<0.001)and invasion(P<0.01)in the SNHG16 group weresignificantly increased.In contrast,compared with the sh-NC group,the number of cells of migration(P<0.01)and invasion(P<0.01)in the sh-SNHG16-2 group were obviously decreased.The result of Xenograft tumor experiment showed that compared with the NC group,the growth rate of tumors in the SNHG16 group was faster and the tumor volume(P<0.05)as well as weight(P<0.05)were increased.In contrast,compared with the sh-NC group,the growth rate of tumors in the sh-SNHG16-2group was slower and the tumor volume(P<0.05)as well as weight(P<0.01)were reduced.Subsequently,the levels of miR-186 in HCC tissues and cell lines were detected by qRT-PCR.The results of qRT-PCR showed that compared with normal liver tissues and normal liver cells,the expression of miR-186 was significantly down-regulated in HCC tissues(P<0.001)and HCC cell lines(P<0.01,P<0.01,P<0.001,P<0.001 and P<0.001).Correlation analysis showed that the expression of SNHG16 was negatively correlated with the level of miR-186(r=-0.5691).The results of qRT-PCR showed that the expression of miR-186 was lowered in SNHG16 group(P<0.001),and the expression of miR-186 was increased in sh-SNHG16-2 group(P<0.001);The expression of SNHG16 was lowered in miR-186 mimic group(P<0.001),and the expression of SNHG16 was increased in miR-186 inhibitor group(P<0.05).The results of the dual luciferase assay showed that miR-186 mimic reduced the fluorescence activity of cells in the SNHG16-wt group(P<0.01),while miR-186 contorl had no effect on thefluorescence activity of the cells in the SNHG16-wt group.The results of qRT-PCR and Westorn blot showed that miR-186 mimic reversed the stimulative effect of over-expression of SNHG16 on ROCK1,and miR-186 inhibitor reversed the inhibitory effect of knockdown of SNHG16 on ROCK1(P<0.01 and P<0.001).The results of the rescue experiments showed that miR-186 mimic reversed the stimulative effect of over-expression of SNHG16 on proliferation,migration and invasion of Hep3 B cell(P<0.05).MiR-186 inhibitor reversed the inhibitory effect of knockdown of SNHG16 on proliferation,migration and invasion of Sk-hep-1 cell(P<0.05).Conclusion:The expression of SNHG16 is increased in HCC cells and tissues.The expression level of SNHG16 was correlated with tumor size,TNM stage,ALT expression level and HBV DNA level.SNHG16 promotes proliferation,migration and invasion of HCC cell.The expression of SNHG16 was negatively correlated with the level of miR-186 and SNHG16 directly bound to miR-186 to regulate the downstream target gene ROCK1.SNHG16 may be to promote the proliferation,migration and invasion of HCC by regulating the expression of miR-186.SNHG16 may be an important biomarker and therapeutic target for HCC,and it deserves to be studied further.