微小RNA-16在肝细胞肝癌生长侵袭中的作用

The role of microRNA一16 in growth and invasion of hepatocellular carcinoma

目的探讨微小RNA(miRNA,miR)-16在肝细胞肝癌生长侵袭中的作用。方法采用实时定量聚合酶链反应(Real-time PCR)检测不同肝癌细胞株及人正常肝细胞(购自中国科学院上海细胞库)中miR-16表达,应用Lipofectamine™3000将miR-16 mimics、miR-16 NC转染到肝癌细胞,分别记为miR-16 mimics组和miR-16 NC组,并测定miR-16表达量,细胞活力、细胞凋亡、细胞周期、细胞侵袭能力及B细胞淋巴瘤/白血病-2(bcl-2)、髓细胞白血病-1(Mcl-1)、细胞周期素D1(CCND1)、基质金属蛋白酶(MMP)-2、MMP-9、Wnt3a及β-连环蛋白(β-catenin)表达。计量资料以均值±标准差(Mean±SD)表示,组间比较采用t检验。结果SMMC-7721(0.65±0.03),HepG2(0.44±0.04),Bel-7402(0.28±0.03),Huh 7(0.87±0.06)和MHCC97(0.23±0.02)肝癌细胞中miR-16表达量显著低于L02正常肝细胞(2.16±0.22)(t=11.291,P<0.05),差异有统计学意义。miR-16 mimics组(2.03±0.20)miR-16表达量较miR-16 NC组(0.54±0.05)提高(t=7.311,P<0.01)。miR-16 mimics组(0.36±0.04)细胞活力较miR-16 NC组(0.59±0.06)降低(t=9.436,P<0.05),差异有统计学意义。miR-16 mimics组细胞早期凋亡率(18.54±1.85)%及晚期凋亡率(20.37±2.03)%较miR-16 NC组[(2.35±0.20)%,(3.24±0.32)%]提高(t=6.624,P<0.05),差异有统计学意义,miR-16 mimics组细胞周期G1期(64.17±6.42)%较miR-16 NC组(48.45±4.85)%延长(t=7.612,P<0.05),差异有统计学意义。miR-16 mimics组(42.53±1.36)细胞侵袭能力较miR-16 NC组(134.54±13.45)降低(t=7.311,P<0.05),差异有统计学意义。Western blot结果表明miR-16 mimics组中bcl-2、Mcl-1、CCND1、MMP-2、MMP-9、Wnt3a及β-catenin表达量较miR-16 NC组下调(t=9.154,P<0.05),差异有统计学意义。结论miR-16过表达可能通过阻断Wnt/β-catenin信号通路抑制HepG2肝癌细胞的生长与侵袭。

Objective To explore the role of microRNA(miRNA,miR)-16 in the growth and invasion of hepatocellular carcinoma(HCC).Methods The expression of miR-16 in different HCC cell lines and human normal hepatocytes was detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR).Which were recorded as miR-16 mimics group and miR-16 NC group respectively.After miR-16 mimics and miR-16 NC were transfected into HepG2 cells by liposome Lipofectamine™3000,the expression of miR-16,cell viability,cell invasion ability,cell apoptotic rate,cell cycle,cell invasion,the expression of B cell lymphoma/lewkmia-2(bcl-2),myeloid cell leukemia-1(Mcl-1),CCND1,matrix metalloproteinase(MMP)-2,MMP-9,Wnt3a andβ-catenin were detected.Results The expression of miR-16 in SMMC-7721(0.65±0.03),HepG2(0.44±0.04),Bel-7402(0.28±0.03),Huh 7(0.87±0.06)and MHCC97(0.23±0.02)hepatoma cells were significantly lower than L02 normal hepatocytes(2.16±0.22)(t=11.291,P<0.05).The expression of miR-16 in miR-16 mimics group(2.03±0.20)was higher than that in miR-16 NC(0.54±0.05)(t=7.311,P<0.05).Cell viability in miR-16 mimics group(0.36±0.04)was lower than that in miR-16 NC group(0.59±0.06)(t=9.436,P<0.05).The early apoptosis rate(18.54±1.85)%and late apoptosis rate(20.37±2.03)%was higher than that in miR-16 NC group[(2.35±0.20)%,(3.24±0.32)%](t=6.624,P<0.05).The cell cycle in miR-16 mimics group(64.17±6.42)%was longer than miR-16 NC group(48.45±4.85)%(t=7.612,P<0.05).Cell invasion number in miR-16 mimics group(42.53±1.36)was lower than that in miR-16 NC group(134.54±13.45)(t=7.311,P<0.05).The results of western blot was showed the expression of bcl-2,Mcl-1,CCND1,MMP-2,MMP-9,Wnt3a andβ-catenin in miR-16 mimics group was lower than that in miR-16 NC group(t=9.154,P<0.05).Conclusion Over-expression of miR-16 might inhibit the growth and invasion of HepG2 hepatoma cells by blocking the Wnt/β-catenin signaling pathway.