KDM3A全长及截短质粒构建及其在细胞中的定位探究  

Construction and Subcellular Distribution of KDM3A Full-length and Its Truncated Mutants in Cancer Cells

作　　者：周宝生 王春玉[1] 王安琦 赵越[1] Zhou Baosheng;Wang Chunyu;Wang Anqi;Zhao Yue(Chromatin Biology Laboratory,Key Laboratory of the Department of Medical Cell Biology Education,Key Laboratory of Cell Biology,Ministryof Health,School of Life Sciences,China Medical University,Shenyang,110122;Northestem Yucai Bilingual School,High School,Shenyang,110164)

机构地区：[1]中国医科大学生命科学学院,卫生部细胞生物学重点实验室,医学细胞生物学教育部重点实验室,染色质生物学研究室,沈阳110122 [2]东北育才双语学校,高中部,沈阳110164

出　　处：《基因组学与应用生物学》2019年第7期3362-3367,共6页Genomics and Applied Biology

基　　金：国家自然科学基金项目(31871286;81872015)资助

摘　　要：赖氨酸去甲基化酶3A(lysine demethylase 3A,KDM3A)是组蛋白去甲基化酶家族成员之一,通过特异性对组蛋白H3K9甲基化进行去甲基化的作用调控基因转录。目前研究证实KDM3A在肝癌、雌激素受体(estrogen receptorα,ERα)阳性乳腺癌及前列腺癌等肿瘤中发挥促癌作用,但其具体作用机制仍有待更深层次的研究。为深入解析KDM3A在各种肿瘤中的作用及其分子机制,本研究利用分子生物学方法构建了KDM3A基因的全长和截短基因表达质粒,酶切鉴定及测序证实KDM3A全长及截短基因的表达质粒构建成功。在此基础上,再将构建成功的上述多种表达质粒分别转染到HEK293细胞和MCF7细胞中,进行Western blotting和免疫荧光共聚焦实验,进一步对各重组表达质粒在培养的哺乳动物细胞中的蛋白表达及蛋白细胞定位进行初步探究。Western blotting结果显示KDM3A全长基因表达的蛋白约147 kD,其他KDM3A截短基因表达的蛋白均对应其特异性的蛋白条带;免疫荧光共聚焦实验结果显示KDM3A全长质粒以及pN1、pN2、p N3、pΔ截短质粒表达的蛋白分布在细胞核,pC1、pC2截短质粒表达的蛋白分布在细胞质,pC3截短质粒表达的蛋白分别分布于细胞核与细胞质。本实验为后续深入研究KDM3A在肿瘤中的作用及分子机制提供了实验基础。Lysine demethylase 3A(KDM3A),a member of histone demethylase family is involved in modulation of gene transcription via specific demethylation of histone H3 lysine 9 methylation.KDM3A participates in promoting the progression of various tumors,including hepatocellular carcinoma,estrogen receptorα(ERα)positive breast cancer and prostate cancer.However,the specific mechanism of action of KDM3A remains to be further studied.In order to further analyze molecular mechanism and the role of KDM3A in tumors,the expression plasmids of KDM3A full-length and its truncated mutants were constructed.The restriction endonuclease digestion and DNA sequence analysis have been performed to confirm that KDM3A full-length expression plasmid and its truncated mutants were successfully generated.Moreover,a series of KDM3A expression plasmids were transfected into HEK293 cells and MCF7 cells,respectively.Western blotting and immunofluorescence confocal experiments were further performed to assess the protein expression and subcellular distribution of each recombinant plasmid in cultured mammalian cells.Western blotting data have shown that the protein encoded KDM3A full length is about 147 kD,the proteins encoded KDM3A truncated mutants are corresponded with the different protein sizes as indicated.The results from confocal microscopy have demonstrated that the expressed proteins carrying KDM3A full length,pN1,pN2,pN3,pΔtruncated mutants are distributed in the nucleus,the expressed proteins carrying pC1,pC2 are distributed in the cytoplasm,the expressed protein encoded pC3 is located in both nucleus and cytoplasm.Our study provides an experimental basis for further investigate the role and molecular mechanism of KDM3A in tumors.

关 键 词：KDM3A 重组质粒构建 蛋白表达 细胞定位 

分 类 号：Q78[生物学—分子生物学]