摘要目的 探讨miR-181b对靶基因的金属蛋白酶组织抑制因子-3(tissue inhibitor of met-allo proteinases 3,TIMP-3)的调控作用及其对肝癌细胞体外侵袭迁移能力的影响.方法 采用qRT-PCR检测肝细胞癌(HCC)组织和不同人HCC细胞系中miR-181b的表达.Western-blot检测TIMP-3在HCC中蛋白的表达.选择miR-181b高表达HCC细胞系SKHep-1,通过报告基因检测试验,观察miR-181b对TIMP-3的调控作用.分别通过Transwell小室和细胞划痕试验,观察阻断SKHep-1细胞miR-181b表达对其侵袭、迁移能力的影响.结果 miR-181b在HCC组织中mRNA的表达明显高于癌旁组织和正常肝组织(P＜0.01),其高表达与HCC高侵袭转移密切相关(P＜0.01).TIMP-3在HCC中蛋白表达明显低于癌旁组织和正常肝组织(P＜0.05).miR 181b在人HCC细胞Hep3B、HepG2、Huh-7、SKHep-1、SNU182、SNU449和肝细胞中均有表达,其中在SKHep-1中表达最高(P＜0.01).高表达miR-181b抑制带有TIMP-3' UTR的荧光素酶的表达(P＜0.05),抑制miR-181b不仅能降低SK-hep1的HCC细胞迁移能力(P＜0.05),还可降低其侵袭能力(P＜0.01).结论 miR-181b可能通过下调TIMP-3基因表达促进HCC侵袭和迁移能力.

abstractsObjective To explore the impact of TIMP 3 regulated by miR-181b as a target gene on invasion and migration of hepatocellular carcinoma (HCC) in vitro.Methods The expressions of miR-181b were detected using SYBR Green real-time fluorescence quantitative polymerase chain reaction on liver cancer specimens and on HCC cell lines.The protein expression of TIMP 3 in HCC was detected using westen blot,and SKHep-1 as a cell line expressing high miR-181b was chosen through reporter gene experiment.TIMP-3 as a target gene regulated by miR-181b and its effect on invasion and migration treated by anti-miR-181 b were studied using transwell and cell scarification test,respectively.Results The expression of miR-181b in HCC was higher than cancer-adjacent tissues and normal liver tissues.The differences among them were significant.There was a correlation between the high expression of miR-181b and invasiveness and metastasis in HCC.The protein expression of TIMP-3 in HCC was significantly lower than normal liver tissues and cancer-adjacent tissues.Expression of miR-181b mRNA was detected in various HCC cell lines such as Hep3B,HepG2,Huh 7,SKHep-1,SNU182,SNU449 and hepatocyte,with the expression of miR-181b in SKHep-1 being the highest (P＜0.01).TIMP3-3UTR was low when the expression of miR-181b was high (P＜0.05).The invasion and migration abilities of SKHep-1 were significantly inhibited by anti-miR-181b (P＜0.05).Conclusion The data suggested that miR-181b promoted invasion and migration of SKHep-1 by down-regulating TIMP-3 in HCC.