探討人類肝細胞癌內蛋白激酵素Cα的異常功能與基因調節

Investigation of the abnormal function and gene regulation of protein kinase Cα in human hepatocellular carcinoma

謝逸憲(Yi-Hsien Hsieh)

指導教授 ： 劉哲育

中山醫學大學/醫學院/生化暨生物科技研究所/博士(2006年)

https://doi.org/10.6834/CSMU.2006.00042

摘要

蛋白激酵素C（Protein Kinase C﹔PKC）是一種鈣和磷酸酯依賴的Ser/Thr 蛋白激酵素。至今已有10種PKC異構物被發現，這些異構物在不同的組織或細胞中有不同的分佈及功能，且也被認為與腫瘤生長、移動和侵襲有關。但是PKC在人類肝細胞癌形成過程的生物功能和基因調節機制，仍然未知。最近我們的研究顯示PKCα在人類肝癌組織表現量比正常組織高，並且與腫瘤大小、癌化期數和存活率有正相關。為了了解PKCα在人類肝細胞癌內的生物功能和訊息傳遞路徑，我們進行細胞實驗，結果發現低度分化人類肝癌細胞株（HA22T/VGH、SK-Hep-1）內PKCα 的表現都較高度分化人類肝癌細胞株（PLC/PRF/5、Hep3B 和HepG2）顯著增加，且反譯股寡核甘酸PKCα (antisense oligonucleotide PKCα)實驗證明PKCα參與細胞增殖、移動和侵襲的角色。另外，利用PKCα siRNA處理發現PKCα參與細胞移動和侵襲是透過活化p38MAPK，並且影響下游MMP-1和u-PA基因表現。為了了解PKCα mRNA過度表現的機制，我們利用EMSA和ChIP方式證明PKCα mRNA是藉由MZF-1和Elk-1轉錄因子所調控，而且抑制MZF-1和Elk-1基因表現也會影響細胞增殖、移動、侵襲能力和腫瘤形成。接下來我們想要了解MZF-1和Elk-1是否共同結合作用來調控PKCα的表現，我們採用luciferase assay、ChIP和Re-ChIP方式證明MZF-1和Elk-1會形成複合物來共同調控PKCα promoter。當這兩個轉錄因子缺乏DNA結合位置時會減少與PKCα promoter結合能力，此時也會抑制PKCα的表現、減少細胞增殖、移動、侵襲能力和腫瘤形成。以上結果將有助於我們了解PKCα在人類肝癌末期細胞侵入轉移的分子機制，同時也將提供轉錄因子的基因治療策略。

關鍵字

人類肝細胞癌 ； 蛋白激酵素Cα

並列摘要

Protein kinase C (PKC), a Ca2+/phospholipid dependent Ser/Thr kinase. To date, ten isozymes of PKC with distinct enzymological characteristics and intracellular localization have been identified, and it is also believed to be correlated with the tumor proliferation, migration and invasion. However, the biological function and gene regulation of PKC in the progression of human HCC remains unclear. In this study, we found that the level of PKCα in the HCC tissues were significantly higher than that in the nontumor tissues, and it positive correlation with tumor size, tumor stage and survival rate. Furthermore, we investigated the biological function and signal pathways of PKCα in HCC cells. The data showed that the level of PKCα in the low differentiated human hepatoma cell lines (HA22T/VGH and SK-Hep-1) were significantly higher than that in the high differentiated human hepatoma cell lines (PLC/PRF/5, Hep3B and HepG2). By antisense oligonucleotide PKCα assay, PKCα was considered to involve in cell proliferation, migration and invasion. In addition, the reduction of PKCα in SK-Hep-1 cells by treatment with PKCα-siRNA was associated with the decreases in cell migration and invasion, and the reduce in the downstream of MMP-1 and u-PA gene expression. Moreover, to understanding the mechanism of PKCα overexpression, EMSA and ChIP assays were performed. We found that PKCα may be regulated by Elk-1 and MZF-1 transcription factor, and the MZF-1 and Elk-1 gene expression were also associated with the potentials of cell proliferation, migration, invasion and tumorigenicity. To determine whether the cooperative interaction of MZF-1 and Elk-1 enchances the transcriptional binding activity in PKCα promoter. Luciferase, ChIP and Re-ChIP assays were performed. The data suggested that the transcriptional binding activity of PKCα promoter was regulated by the Elk-1 and MZF-1 complex formation. When the truncated-MZF-1 or -Elk-1 with lack of DNA binding domain was added to cells, it decrease the binding activity in PKCα promoter, they also inhibit PKCα expression, cell proliferation, migration, invasion and tumorigenicity. These findings will help us understand the molecular mechanism in the late stage of HCC cell invasion and metastasis, and will provide new strategy for transcription factors gene therapy.

並列關鍵字

HCC ； PKCα

參考文獻

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