微小RNA-933靶向调控BDNF表达及对肝癌细胞迁移侵袭的影响

Targeted-regulation of microRNA-933 on BDNF expression and its effect on migration and invasion of hepatoma cells

目的探讨微小RNA-933(miR-933)对脑源性神经营养因子(BDNF)的靶向调控作用及肝癌细胞迁移侵袭的影响。方法收集本院2017年1月至2017年12月经手术切除的30例肝癌组织及对应癌旁组织,采用实时定量PCR(QPCR)检测以上组织和肝癌细胞(Huh7、Hep G2和SK-Hep-1)中的miR-933水平;采用脂质体法向SK-Hep-1细胞分别转染miR-933模拟物(过表达组)和miR-933阴性对照(对照组),采用QPCR法检测转染48 h后各组的miR-933水平以评价转染效率;分别采用Transwell和划痕实验检测各组的侵袭和迁移情况;QPCR和Western blotting分别检测各组的BDNF mRNA和蛋白水平;双荧光素酶报告基因实验验证miR-933与BDNF之间靶向关系和结合位点。结果肝癌组织中的miR-933水平为0.351±0.026,低于癌旁组织的1.124±0.054,差异有统计学意义(P<0.05);Huh7、Hep G2和SK-Hep-1细胞系中miR-933水平分别为0.751±0.062、0.453±0.057和0.017±0.006,均低于健康肝细胞系L02,差异有统计学意义(P<0.05);过表达组转染48h后SK-Hep-1细胞的miR-933水平为3.197±0.354,高于对照组的1.019±0.079,差异有统计学意义(P<0.05)。过表达组的愈合率和穿膜细胞数分别为(24.4±3.9)%和(57.6±6.8)个,均低于对照组的(62.5±4.7)%和(102.6±9.5)个(P<0.05)。过表达组的BDNF mRNA和蛋白水平均低于对照组(P<0.05)。双荧光素酶报告实验结果显示,miR-933显著抑制野生型BDNF-3’UTR质粒转染细胞的荧光素酶活性(P<0.05),但对突变型BDNF-3’UTR荧光素酶活性无影响(P>0.05)。结论 miR-933在肝癌组织和细胞中水平下调,且可抑制肝癌细胞的侵袭迁移,可能是通过靶向调控BDNF来实现,可作为肝癌的潜在治疗靶点。

Objective To investigate the effect of microRNA-933(miR-933)on the regulation of brain-derived neurotrophic factor(BDNF)and the migration and invasion of hepatoma cells.Methods Thirty pairs of hepatoma and paracancerous tissues surgically removed in our hospital were collected from January 2017 to December 2017.Levels of miR-933 in the above tissues and hepatoma cells(Huh7,HepG2 and SK-Hep-1)were detected by real-time quantitative PCR(QPCR).SK-Hep-1 cells were transfected with miR-933 mimic(overexpressing group)and negative control(control group)by liposome method.QPCR was used to detect the miR-933 level of each group at 48 h after transfection to evaluate the transfection efficiency.The invasion and migration of each group were evaluated by Transwell and scratch test,respectively.QPCR and Western blotting were used to detect the mRNA and protein levels of BDNF in each group.The dual luciferase reporter gene assay was applied to confirm the target and binding sites between miR-933 and BDNF.Results The level of miR-933 was 0.351±0.026 in hepatoma tissues,lower than 1.124±0.054 of normal paracancerous tissue(P<0.05).The levels of miR-933 in Huh7,HepG2 and SK-Hep-1 cells were 0.751±0.062,0.453±0.057 and 0.017±0.006,lower than that of the healthy hepatocyte line L02(P<0.05).The miR-933 level of SK-Hep-1 cells in overexpression group at 48 h after transfection was 3.197±0.354,higher than 1.019±0.079 of the control group(P<0.05).The rate of healing and the number of membrane cells in the overexpressed group were(24.4±3.9)%and 57.6±6.8,lower than(62.5±4.7)%and 102.6±9.5 of the control group(P<0.05).The mRNA and protein levels of BDNF in the overexpression group were lower than those in the control group(P<0.05).The results of double fluorescence report showed that miR-933 significantly inhibited the luciferase activity of cells transfected with the wild type BDNF-3’UTR plasmid,but had no significant effect on cells transfected with mutant BDNF-3’UTR.Conclusion The levels of miR-933 in the tissues and cells of liver cancer were reduced,and the invasion and migration of hepatoma cells could be inhibited.It may be realized by targeting BDNF,which can be used as a potential target for the treatment of liver cancer.