去甲基化酶JMJD6影响BRAF突变型黑色素瘤耐药性形成的机制研究  

Study on the mechanism of demethylase JMJD6 affecting the formation of drug resistance in BRAF mutant melanoma

作　　者：唐永吉 朱智鑫 尚艳 樊丹 赵海龙 Tang Yongji;Zhu Zhixin;Shang Yan;Fan Dan;Zhao Hailong(Nanshan Class,Class of 2020,Zunyi Medical University,Zunyi Guizhou 563006,China;Department of Pathophysiology,Zunyi Medical University,Zunyi Guizhou 563006,China)

机构地区：[1]遵义医科大学,贵州遵义563006 [2]遵义医科大学病理生理学教研室,贵州遵义563006

出　　处：《遵义医科大学学报》2024年第8期749-758,共10页Journal of Zunyi Medical University

基　　金：国家自然科学基金资助项目(NO:82060503);贵州省科技计划项目[NO:黔科合基础-ZK(2022)一般622];遵义医科大学大学生创新创业训练计划项目(N0:S202310661178)。

摘　　要：目的探究去甲基化酶JMJD6影响BRAF突变型黑色素瘤耐药性形成的可能机制。方法利用荷瘤小鼠腹腔给药的方式建立针对BRAF抑制剂(达拉非尼)和MEK抑制剂(曲美替尼)具有稳定耐药表型的BRAF突变型黑色素瘤体内模型,使用TCGA、Oncomine、OSskcm等数据库分析、Western blot和免疫组化研究JMJD6在黑色素瘤中的表达情况及预后价值,同时在下调其表达水平后通过TUNEL凋亡试剂盒与体内成瘤实验检测耐药表型在体内外的逆转情况,最后通过生物信息学分析、shRNA干扰、Western blot和免疫荧光实验验证转录因子E2F2是否为JMJD6潜在靶点。结果在依次建立3代耐药体内模型后,成功获得具有稳定耐药表型的BRAF突变型黑色素瘤体内模型A375-R和SK-Mel-28-R,TCGA、Oncomine、OSskcm等数据库均显示JMJD6在黑色素瘤中的表达较正常健康者明显升高且具有更差的预后(P<0.05)。Western blot和免疫组化结果显示,JMJD6在耐药型BRAF突变型黑色素瘤中出现高表达;利用shRNA下调JMJD6表达水平后,A375-R和SK-Mel-28-R在药物压力下凋亡水平和成瘤能力显著降低(P<0.05),呈现体内外耐药表型逆转现象;通过GeneMANIA、GEPIA数据库分析发现JMJD6与E2F2存在表达相关性(P<0.05),同时Western blot和免疫荧光验证下调JMJD6可以抑制E2F2表达以及RNA聚合酶Ⅱ的结合,并在shRNA下调E2F2表达水平的基础上验证其位于JMJD6分子机制下游。结论JMJD6通过调控E2F2表达进而影响BRAF突变型黑色素瘤对BRAF和MEK抑制剂耐药性的形成。Objective To explore the possible mechanism of demethylase JMJD6 affecting the formation of drug resistance in BRAF mutant melanoma.Methods In vivo model of BRAF mutant melanoma with stable drug-resistant phenotype against BRAF inhibitor(Dabrafenib)and MEK inhibitor(Trametinib)was established by intraperitoneal administration in tumor-bearing mice.TCGA,Oncomine and OSskcm database analysis,Western blot and immunohistochemistry were used to study the expression and prognostic value of JMJD6 in melanoma.At the same time,after down-regulating its expression level,TUNEL apoptosis kit and in vivo tumorigenesis experiment were used to detect the reversal of drug-resistant phenotype in vitro and in vivo.Finally,bioinformatics analysis,shRNA interference,Western blot and immunofluorescence experiment were used to verify whether transcription factor E2F2 was a potential target of JMJD6.Results After establishing three generations of drug-resistant models in vivo,the models A375-R and SK-Mel-28-R of BRAF mutant melanoma with stable drug-resistant phenotypes were successfully obtained.The databases such as TCGA,Oncomine and OSskcm all showed that the expression of JMJD6 in melanoma was significantly higher than that in normal healthy people,with a worse prognosis(P<0.05).At the same time,Western blot and immunohistochemical results showed that JMJD6 was expressed higher in drug-resistant BRAF mutant melanoma.After the expression level of JMJD6 was down-regulated by shRNA,the apoptosis level and tumorigenicity of A375-R and SK-Mel-28-R were significantly decreased under drug pressure(P<0.05),showing the phenomenon of drug resistance phenotype reversal in vitro and in vivo.Through the analysis of Genemania and GEPIA databases,it was found that there was a correlation between the expression of JMJD6 and E2F2(P<0.05).At the same time,Western blot and immunofluorescence proved that down-regulation of JMJD6 could inhibit E2F2 expression and RNA polymeraseⅡbinding,and down-regulation of E2F2 expression by shRNA verified that it

关 键 词：BRAF突变型黑色素瘤 耐药性 Jumonji结构域蛋白6 E2F家族转录因子2 

分 类 号：R739.5[医药卫生—肿瘤]