miR-590-5p和miR-590-3p参与肝细胞肝癌发展的机制

Roles of miR-590-5p and miR-590-3p in the development of hepatocellular carcinoma

目的研究miR-590的两个臂—miR-590-5p和miR-590-3p对肝癌细胞增殖能力的影响,以及参与肿瘤发生发展的机制。方法利用miRNA芯片、QPCR对肝癌标本和各细胞株miR-590的表达谱系进行分析和验证。通过脂质体将miR-590 mimic或inhibitor转染正常肝细胞或肝癌细胞,MTT生长曲线分析和软琼脂克隆形成实验检测转染后细胞增殖及存活能力的变化。Targetscan预测miR-590-5p和miR-590-3p的靶基因,并通过萤光素酶报告系统以及western blot进行验证。结果 miRNA芯片结果显示3个肝细胞肝癌(hepatocellular carcinoma,HCC)组织相对于癌旁正常组织,miR-590-5p和miR-590-3p表达上调。通过QPCR验证了10例临床HCC肿瘤标本发现80%的HCC组织相对于癌旁组织miR-590-5p和miR-590-3p均同步显著上调。QPCR结果进一步发现,HepG2、Hep3B、Huh7三株HCC细胞株相对于正常肝细胞株L-O2 miR-590-5p/3p同样表达上调。MTT和软琼脂克隆形成结果显示,L-O2分别过表达miR-590-5p和miR-590-3p,细胞的增殖能力都显著增加(P<0.05);而HepG2、Hep3B、Huh7分别下调miR-590-5p和miR-590-3p的表达后,细胞的增殖能力也都显著降低(P<0.05)。Targetscan预测,PDCD4和PTEN分别作为miR-590-5p和miR-590-3p的潜在靶基因。萤光素酶报告系统以及western blot结果显示miR-590-5p和miR-590-3p可以分别直接下调靶基因PDCD4和PTEN的表达(P<0.05)。进一步我们发现miR-590-3p通过下调PTEN激活PI3K-AKT信号通路,促进AKT1-S473的磷酸化水平。结论在HCC发展过程中miR-590扮演着重要的致癌因子角色,我们结果发现miR-590的两个臂都具有促进肿瘤发生的生物学功能,它们分别通过靶向调节抑癌基因PDCD4和PTEN的表达来促进HCC细胞的增殖和存活,并激活核心的PI3K-AKT肿瘤信号通路。由此可见miR-590在HCC发生发展过程中具有重要的意义,也可作为临床诊治潜在的新靶标。

Objective To study the effect of the two arms of miR-590, miR-590-Sp and miR-590-3p, on hepatoma cell proliferation and their roles in tumor development. Methods We analyzed and verified the expression pattern of miR-590 in liver cancer specimens and cell lines by miRNA microarrays and QPCR. MiR-590 mimic or inhibitor was transfected into normal liver cells or liver cancer cells via liposome, and the changes in cell proliferation and survival were determined by MTT assay and soft agar colony formation assay. The target genes of miR-590-5p and miR-590-3p were predicated with Targetscan and validated by luciferase reporter system and Western blotting. Results The expressions of miR-590-5p and miR-590-3ps were up-regulated in 3 hepatocellular carcinoma （HCC） tissues and their synchronization was significantly up-regulated in 8 out of 10 HCC tissues as compared with the adjacent tissues. QPCR further showed that miR-590-5p/3p was up-regulated in 3 HCC cell lines （HepG2, Hep3B, and Huh7） in comparison with the normal liver cell line L-O2. L-O2 cells over-expressing miR-590-5p and miR-590-3p exhibited significantly increased proliferation （P〈0.05）, while down-regulation of miR-590-5p and miR-590-3p caused significantly suppressed proliferation in HepG2, Hep3B, and Huh7 cells. Targetscan predicted PDCD4 and PTEN as the potential target genes of miR-590-5p and miR-590-3p, which was verified by luciferase reporter system and Western blotting, miR-590-3p was found to activate PI3K-AKT signaling pathway by down-regulating PTEN to promote AKT1-S473 phosphorylation. Conclusion MiR-590 is an important tumorigenic factor for HCC, and its two arms can both promote tumorigenesis by regulating the expression of their target tumor suppressor gene, PDCD4 and PTEN, to promote HCC cell proliferation and survival and activate the core tumor signal pathway PI3K-AKT.