EHMT2 (G9a) activation in mantle cell lymphoma and its associated DNA methylation and gene expression

Objective:The function of euchromatic histone-lysine N-methyltransferase 2(EHMT2)has been studied in several cancers;however,little is known about its role in mantle cell lymphoma(MCL).Thus,this study aimed to characterize the significance and function of EHMT2 in MCL.Methods:EHMT2 expression in MCL and reactive hyperplasia(RH)were investigated by immunohistochemistry.Genome-wide analysis of DNA methylation was performed on EHMT2+MCL samples.The function of EHMT2 was determined by CCK&flow cytometry,and western blot assays.Gene expression profile analysis was performed before and after EHMT2 knockdown to search for EHMT2-regulated genes.Co-immunoprecipitation(Co-IP)experiments were conducted to identify the proteins interacting with EHMT2.Results:EHMT2 was expressed in 68.57%(24/35)of MCLs but not in any RHs.Genome-wide analysis of DNA methylation on EHMT2+MCLs revealed that multiple members of the HOX,FOX,PAX,SOX,and CDX families were hypermethylated or hypomethylated in EHMT2+MCLs.BIX0I294,a EHMT2 inhibitor,inhibited MCL cell growth and stalled cells in the G1 phase.Additionally,BIX01294 downregulated the expressions of cell cycle proteins,cyclin DI,CDK4,and P21,but upregulated the expressions of apoptosis-related proteins,Bax and caspase-3.Co-IP experiments revealed that EHMT2 interacted with UHRF1,HDAC1,and HDAC2 but not with HDCA3.After EHMT2 knockdown,multiple genes were regulated,including CD5 and CCND1,mostly enriched in the Tec kinase signaling pathway.In addition,several genes(e.g.,MARCH 1,CCDC50,HIP1,and WNT3)were aberrantly methylated in EHMT2+MCLs.Conclusions:For the first time,we determined the significance of EHMT2 in MCL and identified potential EHMT2-regulated genes.

EHMT2 promotes tumorigenesis in GNAQ/11-mutant uveal melanoma via ARHGAP29-mediated RhoA pathway

Constitutive activation of GNAQ/11 is the initiative oncogenic event in uveal melanoma(UM).Direct targeting GNAQ/11 has yet to be proven feasible as they are vital for a plethora of cellular functions.In search of genetic vulnerability for UM,we found that inhibition of euchromatic histone lysine methyltransferase 2(EHMT2)expression or activity significantly reduced the proliferation and migration capacity of cancer cells.Notably,elevated expression of EHMT2 had been validated in UM samples.Furthermore,Kaplan-Meier survival analysis indicated high EHMT2 protein level was related to poor recurrence-free survival and a more advanced T stage.Chromatin immunoprecipitation sequencing analysis and the following mechanistic investigation showed that ARHGAP29 was a downstream target of EHMT2.Its transcription was suppressed by EHMT2 in a methyltransferasedependent pattern in GNAQ/11-mutant UM cells,leading to elevated RhoA activity.Rescuing constitutively active RhoA in UM cells lacking EHMT2 restored oncogenic phenotypes.Simultaneously blocking EHMT2 and GNAQ/11 signaling in vitro and in vivo showed a synergistic effect on UM growth,suggesting the driver role of these two key molecules.In summary,our study shows evidence for an epigenetic program of EHMT2 regulation that influences UM progression and indicates inhibiting EHMT2 and MEK/ERK simultaneously as a therapeutic strategy in GNAQ/11-mutant UM.

纳布啡改善瑞芬太尼诱导的大鼠痛觉过敏的机制

目的探讨纳布啡对瑞芬太尼诱导的大鼠痛觉过敏的改善作用及对脊髓背角常染色质组蛋白赖氨酸N-甲基转移酶2(G9a)和钠激活钾通道(Slack)表达的影响。方法55只大鼠随机抽取30只作为对照组、瑞芬太尼组和切口痛组,每组10只,其余25只建造瑞芬太尼诱发大鼠术后痛觉过敏反应模型,20只建模成功大鼠随机分为模型组、纳布啡组,每组10只。瑞芬太尼组、模型组和纳布啡组静脉输注瑞芬太尼,对照组、切口痛组输注等体积生理盐水,输注5 min后,切口痛组、模型组、纳布啡组接受左后足底切口术,对照组、瑞芬太尼组仅仰卧固定到手术台相同时间。纳布啡组泵注瑞芬太尼前3 min静注纳布啡,其余各组静注等体积的氯化钠注射液。大鼠造模后2~48 h进行痛觉行为学测定;用酶联免疫吸附法测定脊髓背角细胞炎症因子;用免疫荧光法检验脊髓背角的星形胶质细胞激活状态;用蛋白免疫印迹法测定造模后48 h脊髓背角G9a、Slack蛋白的表达水平。结果随造模时间延长,瑞芬太尼组、切口痛组、模型组大鼠的机械缩足反应阈(paw withdrawal mechanical threshold,PWMT)、热缩足反应潜伏期(paw withdrawal thermal latency,PWTL)值持续减小,而纳布啡组持续增大(P<0.05),对照组无明显变化;与对照组比较,瑞芬太尼组、切口痛组、模型组、纳布啡组大鼠的PWMT和PWTL值均减小(P<0.05);与瑞芬太尼组比较,模型组大鼠的PWMT和PWTL值减小、纳布啡组PWMT和PWTL值增大(P<0.05),切口痛组与之比较,差异无统计学意义;与模型组比较,纳布啡组PWMT和PWTL值增大(P<0.05)。与对照组比较,造模组肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)水平升高(P<0.05);与瑞芬太尼组比较,模型组TNF-α、IL-1β水平较高,纳布啡组TNF-α、IL-1β水平降低(P<0.05),切口痛组与之比较差异无统计学意义;与模型组比较,纳布啡组TNF-α、IL-1β水平降低(P<0.05)。与对照组比较,瑞芬太尼组、切口痛组的星形胶质细胞明显激活,模型组激活星形胶质细胞密度最大,纳布啡组与之相比差异无统计学意义。与对照组比较,模型组G9a蛋白相对表达量升高,Slack总蛋白与膜蛋白的相对表达量降低(P<0.05);与瑞芬太尼组比较,模型组G9a蛋白的相对表达量升高,Slack总蛋白与膜蛋白的相对表达量降低(P<0.05),纳布啡组G9a蛋白的相对表达量降低,Slack总蛋白与膜蛋白的相对表达量升高(P<0.05),切口痛组与之比较差异无统计学意义;与模型组比较,纳布啡组G9a蛋白的相对表达量降低,Slack总蛋白与膜蛋白的相对表达量升高(P<0.05)。结论对瑞芬太尼诱导的术后痛觉过敏反应大鼠模型进行纳布啡预处理,可有效改善大鼠的痛觉过敏,可能是通过下调G9a、上调Slack的表达,达到镇痛的效果。

膀胱癌组织MAGE-A3表达变化机制研究

目的膀胱癌是最常见的恶性肿瘤之一,严重危害人类健康。本研究检测膀胱癌组织中黑色素瘤抗原3(melanoma-associated antigen 3,MAGE-A3)的表达变化并探讨其内在机制,为膀胱癌的防治提供理论基础。方法收集2013-06-01-2016-05-31武汉科技大学附属武昌医院泌尿外科膀胱癌患者120例,取术中切取的膀胱癌组织和正常膀胱组织(距离肿瘤边缘>3cm)。实时荧光定量PCR(Real-time fluorescence quantitative PCR,RT-qPCR)和蛋白质印迹法检测膀胱癌组织和正常膀胱组织中MAGE-A3,DNA甲基转移酶1(DNA methyltransferase 1,DNMT1)及常染色体组蛋白赖氨酸甲基转移酶2(euchromatic histone-lysine N-methyltransferase 2,EHMT2)mRNA和蛋白表达变化;巢式降落式甲基化特异性PCR(nested methylated specific PCR,nMS-PCR)检测膀胱癌组织和正常膀胱组织中MAGE-A3启动子区DNA甲基化水平变化;染色质免疫共沉淀(chromatin immunoprecipitation,CHIP)检测膀胱癌组织和正常膀胱组织中MAGE-A3启动子区组蛋白H3K9二甲基化(H3K9me2)水平。结果膀胱癌组织中MAGE-A3mRNA相对表达量为5.41±0.85,蛋白相对表达量为0.76±0.08;正常膀胱组织中MAGE-A3mRNA相对表达量为0.92±0.15,蛋白相对表达量为0.20±0.07,两组比较差异有统计学意义,t值分别为15.56和15.60,均P=0.001。膀胱癌组织中DNMT1mRNA相对表达量为0.37±0.10,蛋白相对表达量为0.36±0.10;EHMT2mRNA相对表达量为0.81±0.09,蛋白相对表达量为0.27±0.08。正常膀胱组织中DNMT1mRNA相对表达量为1.02±0.25,蛋白相对表达量为0.91±0.15,RNA相对表达量为4.32±0.82,蛋白的相对表达量为1.09±0.17,两组比较差异有统计学意义,t值分别为5.95,9.16,11.58,11.08,均P<0.001。膀胱癌组织中MAGE-A3启动子区DNA甲基化水平为0.27±0.07,H3K9me2水平为0.25±0.13;正常膀胱组织MAGE-A3启动子区DNA甲基化水平为0.73±0.08,H3K9me2水平为0.61±0.24,两组比较差异有统计学意义,t值分别为7.189和3.860,均P<0.001。结论膀胱癌组织中MAGE-A3启动子区DNA甲基化及H3K9me2水平降低使MAGE-A3表达量升高,此机制可能参与膀胱癌的发生发展。