Amiloride對於人類肝癌細胞株Huh7 mRNA選擇性剪接表現之影響

Effects of Alternative mRNA Splicing by Amiloride in Human Hepatocellular Carcinoma Huh-7 cells

林惠華(Hui-Hua Lin)

指導教授 ： 張建國

高雄醫學大學/醫學院/醫學研究所/碩士(2009年)

摘要

選擇性剪接是真核生物控制基因表達的一種重要機制，生物體透過這種機制使有限的基因得以表達大量複雜的蛋白質，在形成生物複雜性和多樣性上具有極其重要的作用。這項生物機制不僅影響細胞的分化及個體的發育，同時與疾病的發生和未來治療趨勢更息息相關。在本篇實驗中，我們發現amiloride可以改變人類肝癌細胞株 Huh-7 cells Bcl-x、HIPK3、Ron 基因的選擇性剪接。Amiloride會使splicing factor SF2/ASF磷酸化下降，SRp20表現量下降。且amiloride也會使AKT, ERK1/2 and PP1α磷酸化下降，p38 and JNK磷酸化增加。投與PI3K-AKT signal pathways的抑制劑，雖然影響AKT phosphorylation卻沒有改變SF2/ASF的活性及Bcl-x and HIPK3 mRNA alternative splicing。當我們投與PP1 phosphatase的抑制劑，則可以有效抑制amiloride造成的SF2/ASF dephosphorylation和Bcl-x、HIPK3基因選擇性剪接改變。綜合上述的實驗結果，amiloride透過PP1 phosphatase影響alternative splicing factors來調控人類肝癌細胞株 Huh-7 cells的選擇性剪接。

關鍵字

amiloride ； 選擇性剪接

並列摘要

Alternative splicing（AS） is a regulatory mechanism that allows genes to encode for multiple protein isoforms that often play different biological roles. Altered AS of proto-oncogenes and tumor suppressor genes often result in neoplastic phenotypes of cancer. Screening various drugs and small organic molecules for AS modulating activity, we have found that amiloride, a potassium-sparing diuretic drug with Na+/H+ exchanger inhibitory activity, can affect the oncogenic alternative splicing of Bcl-x, HIPK3 and Ron transcripts in human hepatocellular carcinoma Huh-7 cells. Our proteomic analyses detected the splicing factor SF2/ASF mainly in unphosphorylated and hypo-phosphorylated forms and also decreased expression of SRp20 in the amiloride-treated cells. Amiloride treatment also decreased the phosphorylation of AKT, ERK1/2 and PP1α, but increased the phosphorylation of p38 and JNK. Inhibitors of PI3K-AKT signal pathways, although affecting the AKT phosphorylation, did not cause significant change in SF2/ASF or alternate splicing of HIPK3 and Bcl-x. The amiloride-induced alterations of SF2/ASF phosphorylation and the alternative splicing are inhibited by okadaic acid. Take together, our results indicate that amiloride affects the alternative mRNA splicing by perturbation of PP1 phosphatase.

並列關鍵字

amiloride ； RNA alternative splicing

參考文獻

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