Institute of Microbiology & Immunology, National Yang-Ming University, Taipei, Taiwan.
J Biomed Sci. 2010 May 6;17(1):35. doi: 10.1186/1423-0127-17-35.
Protein Kinase C (PKC) is a serine/threonine kinase that involved in controlling of many cellular processes such as cell proliferation and differentiation. We have observed previously that TPA (12-O-tetradecanoylphorbol 13-acetate) induces cell cycle arrest in G0/G1 phase in human hepatoma HepG2 cells. However, is there any miRNA involved in PKCalpha mediated cell growth arrest is still unknown.
We first surveyed 270 miRNA expression profiles in 20 pairs of human hepatoma tissues. We identified 11 up-regulated and 23 down-regulated miRNAs (FDR < = 0.01; fold-change > = 2) in human hepatoma tissue after Student's T-test and Mann-Whitney rank test. We then examined miRNAs expression profile in TPA treated HepG2 cells. Two miRNAs, miR-101, and miR-29c, were shown to be significantly down regulated in human hepatoma tissues and induced over 4-fold in HepG2 cells under TPA treatment.
In this study, we examined TPA regulated miRNA expression profile in human hepatoma HepG2 cells. We identified two miRNAs, 101 and 29c, were induced by TPA and down regulated in human hepatoma tissues suggest that they might play as tumor suppressor gene and in tumor formation of HCC. Since induction kinetics of miR-101 by TPA was much faster than miR-29c suggests that the induction of miR-101 may be the primary response of TPA treatment. We then further investigated how miR-101 was regulated by TPA. MiR-101 targets two subunits of PRC2 complex, enhancer of zeste homolog 2 (EZH2) and EED, and was shown to play as a tumor suppressor gene in human prostate, breast and liver cancers. The target sequence of miR-101 located in the 3' UTR of both EZH2 and EED's mRNA was identified by bioinformatic analysis and was validated by reporter luciferase activity assay. Then we showed that TPA not only up regulated miR-101 expression, but also reduced protein level of EZH2, EED and H3K27me3 in HepG2 cells. Using lenti-virus-mediated shRNA to knockdown endogenous PKCalpha expression, we observed that TPA induced growth arrest, elevation of miR-101 and reduction of EZH2, EED and H3K27me3 proteins were all PKCalpha dependent. Specific inhibitor of ERK completely blocked TPA induced miR-101 expression.
Therefore, this is the first time to show that PKCalpha and ERK pathway play important role to activate miR-101 expression, reduce PRC2 complex and H3K27me3 level. This epigenetic regulatory pathway may represent a novel mechanism of carcinogenesis and deserve further investigation.
蛋白激酶 C（PKC）是一种丝氨酸/苏氨酸激酶，参与控制许多细胞过程，如细胞增殖和分化。我们之前观察到，TPA（12-O-十四烷酰佛波醇 13-乙酸酯）可诱导人肝癌 HepG2 细胞的细胞周期停滞在 G0/G1 期。然而，PKCalpha 介导的细胞生长抑制是否涉及任何 miRNA 仍不清楚。
我们首先调查了 20 对人肝癌组织中的 270 种 miRNA 表达谱。通过学生 t 检验和曼惠特尼秩检验，我们在人肝癌组织中鉴定出 11 种上调和 23 种下调的 miRNA（FDR <= 0.01；倍数变化>= 2）。然后，我们检查了 TPA 处理的 HepG2 细胞中的 miRNA 表达谱。miR-101 和 miR-29c 在人肝癌组织中显著下调，在 TPA 处理的 HepG2 细胞中诱导超过 4 倍。
在这项研究中，我们检查了 TPA 调节的人肝癌 HepG2 细胞中的 miRNA 表达谱。我们鉴定出两种 miRNA，101 和 29c，被 TPA 诱导，并在人肝癌组织中下调，表明它们可能作为肿瘤抑制基因，并在 HCC 的肿瘤形成中发挥作用。由于 miR-101 被 TPA 诱导的动力学比 miR-29c 快得多，这表明 miR-101 的诱导可能是 TPA 治疗的主要反应。然后，我们进一步研究了 miR-101 是如何被 TPA 调节的。miR-101 靶向 PRC2 复合物的两个亚基，增强子 of zeste 同源物 2（EZH2）和 EED，并被证明在人类前列腺癌、乳腺癌和肝癌中作为肿瘤抑制基因发挥作用。生物信息学分析鉴定出 miR-101 的靶序列位于 EZH2 和 EED mRNA 的 3'UTR 中，并通过报告荧光素酶活性测定得到验证。然后，我们发现 TPA 不仅上调了 miR-101 的表达，还降低了 HepG2 细胞中 EZH2、EED 和 H3K27me3 的蛋白水平。利用慢病毒介导的 shRNA 敲低内源性 PKCalpha 表达，我们观察到 TPA 诱导的生长停滞、miR-101 的升高和 EZH2、EED 和 H3K27me3 蛋白的降低均依赖于 PKCalpha。ERK 的特异性抑制剂完全阻断了 TPA 诱导的 miR-101 表达。
因此，这是首次表明 PKCalpha 和 ERK 通路在激活 miR-101 表达、降低 PRC2 复合物和 H3K27me3 水平方面发挥重要作用。这种表观遗传调控途径可能代表一种新的致癌机制，值得进一步研究。
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