组蛋白H3Ser10磷酸化对人鼻咽癌细胞增殖和转化的调控作用

发布时间：2018-04-24 07:16

本文选题：鼻咽肿瘤 + 组蛋白类　；参考：《肿瘤》2014年10期

【摘要】：目的 :探讨组蛋白H3第10位丝氨酸(serine 10,Ser10)磷酸化在调控鼻咽癌CNE1细胞增殖和恶性转化中的作用及可能的作用机制。方法 :采用蛋白质印迹法检测表皮生长因子(epidermal growth factor,EGF)作用下鼻咽癌CNE1细胞中组蛋白H3Ser10磷酸化水平的改变;构建组蛋白H3野生型(H3WT)和突变型(H3S10A)真核表达质粒,转染CNE1细胞,采用杀稻瘟菌素筛选获得稳定过表达野生型或突变型组蛋白H3细胞株;蛋白质印迹法检测转染组蛋白H3野生型和突变型重组质粒后对CNE1细胞中组蛋白H3Ser10磷酸化水平的影响;采用CCK-8(cell counting kit-8)法和软琼脂集落形成实验检测过表达野生型和突变型组蛋白H3对CNE1细胞增殖和克隆形成能力的影响;分别采用蛋白质印迹法和双荧光素酶报告基因系统检测过表达野生型和突变型组蛋白H3对CNE1细胞中c-Jun、c-Fos和Bcl-2蛋白表达以及激活蛋白-1(activator protein-1,AP-1)转录活性的影响。结果 :EGF可诱导CNE1细胞中组蛋白H3Ser10磷酸化水平的上调;成功构建了稳定过表达野生型和突变型组蛋白H3的CNE1细胞株,组蛋白H3Ser10位点的突变可明显抑制EGF作用下组蛋白H3的磷酸化水平;与转染空质粒细胞相比,过表达野生型组蛋白H3可促进EGF作用下CNE1细胞增殖和软琼脂克隆形成能力,并且上调c-Jun和c-Fos蛋白的表达以及提高AP-1的转录活性,但过表达突变型组蛋白H3则没有引起上述明显的改变。结论 :组蛋白H3,尤其是组蛋白H3Ser10磷酸化,在调控EGF诱导的CNE1细胞增殖和恶性转化中具有重要作用,其机制与促进c-Jun和c-Fos基因的转录,进而增强AP-1的活性有关。
[Abstract]:Aim: to investigate the role and possible mechanism of phosphorylation of histone H3 in regulating proliferation and malignant transformation of nasopharyngeal carcinoma (CNE1) cells. Methods: the phosphorylation of histone H3Ser10 in nasopharyngeal carcinoma (NPC) CNE1 cells was detected by Western blotting, and the eukaryotic expression plasmids of histone H3 wild-type H3WTand mutant H3S10A were constructed and transfected into CNE1 cells. Overexpression wild type or mutant histone H3 cell lines were obtained by screening rice blast fungicide, and the effect of recombinant plasmid transfected with wild type of histone H3 and mutant plasmid on the phosphorylation of histone H3Ser10 in CNE1 cells was detected by Western blotting. The effects of overexpression of wild type and mutant histone H3 on the proliferation and clone formation of CNE1 cells were detected by CCK-8(cell counting kit-8 and soft Agar colony forming assay. The effects of overexpression of wild type histone H3 and mutant histone H3 on the expression of c-Junc-Fos and Bcl-2 protein and the transcriptional activity of activator protein-1AP-1 in CNE1 cells were detected by Western blotting and double luciferase reporter gene system, respectively. Results CNE1 cells were induced to up-regulate the level of histone H3Ser10 phosphorylation, and stable overexpression of wild type and mutant histone H3 was successfully constructed into CNE1 cell lines. The mutation of histone H3Ser10 site could significantly inhibit the phosphorylation of histone H3 under the action of EGF, and overexpression of wild histone H3 could promote the proliferation of CNE1 cells and the ability of soft Agar clone formation compared with empty plasmid cells transfected with EGF. The expression of c-Jun and c-Fos proteins and the transcriptional activity of AP-1 were up-regulated, but overexpression of mutant histone H3 did not cause these changes. Conclusion: histone H3, especially histone H3Ser10 phosphorylation, plays an important role in regulating the proliferation and malignant transformation of CNE1 cells induced by EGF, and its mechanism is related to promoting the transcription of c-Jun and c-Fos genes and thus enhancing the activity of AP-1.
【作者单位】：广东医学院病理生理学教研室;广东医学院广东省医学分子诊断重点实验室;
【基金】：国家自然科学基金资助项目(编号:81071638) 湛江市科技攻关计划项目(编号:2013B01074)
【分类号】：R739.63

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