微小RNA-451调控COX-2等细胞因子对人肝癌细胞株增殖和侵袭能力的影响

目的 探讨微小RNA-451(miRNA-451)通过调控COX-2等细胞因子对人肝癌细胞株增殖和侵袭能力的影响.方法 采用RT-PCR法检测正常肝细胞株以及人肝细胞癌细胞株中miRNA-451的表达情况;利用慢病毒转染的方法使MHCC-97H细胞过表达miRNA-451,利用RT-PCR法检测MIF 、MAPK 、ERK 、COX-2 、TGF-β 、VEGF和PGE2 mRNA的表达水平;通过平板克隆实验和Transwell小室分别检测miRNA-451过表达后MHCC-97H的增殖和侵袭能力的变化.结果 HCC细胞株HCCLM-3、MHCC-97H、MHCC-97L、Huh-7、SMMC-7721、HepG2中miRNA-451的相对表达量分别为(0.11±0.01)、(0.23±0.01)、(0.44±0.03)、(0.55±0.02)、(0.74±0.03)、(0.70±0.01),与正常肝细胞株L-02细胞比较,差异均有统计学意义(P﹤0.05).转染72 h后,LV-pre-miRNA-451组miRNA-451的相对表达量较空白对照组上调了(12.09 ± 0.15)倍(P﹤0.05).miRNA-451过表达后,MIF 、MAKP 、ERK 、COX-2 、TGF-β 、VEGF 、PGE2 mRNA的相对表达量分别为(0.30 ± 0.13)、(0.68 ± 0.02)、(0.48 ± 0.02)、(0.56±0.01)、(0.51±0.02)、(0.42±0.02)、(0.76±0.03),与空白对照组比较,差异均有统计学意义(P﹤0.05).培养24 h后,LV-pre-miRNA-451组侵袭至Transwell膜背面的细胞数量为(53±5)个,明显低于空白对照组的(79±4)个,差异有统计学意义(t=7.700,P﹤0.01);HCC细胞株MHCC-97H过表达miRNA-451后的平板克隆形成率为(15.7± 0.5)%,明显低于空白对照组细胞的(27.0±0.4)%,差异有统计学意义(t=31.705,P﹤0.01).结论 miRNA-451通过MIF/MAPK/ERK途径可调控肿瘤微环境内的细胞因子,从而影响肿瘤细胞的侵袭能力.

The effect of miRNA-451 on the proliferation and invasion capacity of human hepatocellular carcinoma cell lines via its regulation on COX-2 and other cytokines

Objective To investigate the effect of microRNA-451 (miRNA-451) on the proliferation and invasion ca-pacity of human hepatocellular carcinoma (HCC) by regulating on COX-2 and other cytokines.Method The expres-sions of the miRNA-451 in normal live cell and the HCC cells were detected by RT-PCR.Slow virus transfection was used on MHCC-97H for over-expressing of miRNA-451.The levels of expressions of MIF、MAKP、ERK、COX-2、TGF-β 、VEGF and PGE2 mRNA were detected by RT-PCR.Tablet clone formation test and transwell assay were used to test the migration and invasion abilities of MHCC-97H cell which were over-expressing miRNA-451.Result The relative ex-pressions of miRNA-451 were significantly higher in HCC cell lines,including HCCLM-3 (0.11 ± 0.01),MHCC-97H (0.23±0.01),MHCC-97L (0.44±0.03),Huh-7 (0.55±0.02),SMMC-7721 (0.74±0.03) and HepG2 (0.70±0.01),than that in normal cell line L-02 (P<0.05).After 72 h transfection,compared to the blank control group,the expression of miRNA-451 in a cell line of LV-pre-miRNA-451 group was successfully up-regulated by (12.09±0.15) times (P<0.05).Compared to the blank control group,the expressions of the MIF mRNA (0.30+0.13)、MAKP mRNA (0.68+0.02)、ERK mRNA (0.48+0.02)、COX-2 mRNA (0.56+0.01)、TGF-β mRNA (0.51+0.02)、VEGF mRNA (0.42+0.02)、PGE2 mRNA (0.76+0.03) in the LV-pre-miRNA-451 cell line after over-expressing miRNA-451 were all significantly different (P<0.05).Af-ter 24 h culture,the number of metastatic cell in the LV-pre-miRNA-451 group (53±5)/HP was significantly lower than that in the blank control group (79±4)/HP (t=7.700,P<0.01).Tablet clone formation rate (15.7%±0.5%) of HCC cell line MHCC-97H which over-expressing miRNA-451 was significantly lower than that in the blank control group (27.0 ± 0.4)%;t=31.705,P<0.01].Conclusion miRNA-451 inhibits the expression of cytokines in HCC microenvironment via MIF/MAPK/ERK pathway,thereby impact the invasion ability of tumor cells.