H3K36me3及其表观遗传修饰蛋白质SETD2和KDM4B在结直肠癌中的临床病理意义

Clinicopathological significance of H3K36me3 and its epigenetic modification proteins SETD2 and KDM4B in colorectal cancer

目的:组蛋白修饰是表观遗传调控的一种重要形式。其中,组蛋白甲基化是染色质状态的关键决定因素。组蛋白H3第36位赖氨酸三甲基化产物(histone H3 trimethylation at lysine 36,H3K36me3)可以介导多种转录相关事件,有助于DNA损伤修复。组蛋白赖氨酸三甲基转移酶SET结构域2(SET domain containing 2,SETD2)、赖氨酸特异性去甲基化酶4B(lysine-specific demethylase 4B,KDM4B)分别是H3K36me3甲基转移酶及去甲基酶,SETD2失活及KDM4B活化均可下调H3K36me3水平从而促进肿瘤的发生和发展。本研究主要探讨H3K36me3、SETD2、KDM4B在结直肠癌中表达的临床病理意义。方法:收集2018年10月至2020年10月就诊于福建中医药大学附属人民医院的80例结直肠癌患者术后石蜡包埋的组织样本。采用免疫组织化学染色法检测H3K36me3、KDM4B、SETD2及4种错配修复(mismatch repair,MMR)蛋白质(MSH2、MSH6、MLH1、PMS2)在结直肠癌及正常肠黏膜中的表达水平。采用多重荧光PCR-毛细管电泳检测结直肠癌及正常肠黏膜的微卫星状态,分为微卫星高度不稳定性(microsatellite instability-high,MSI-H)组与微卫星稳定(microsatellite stable,MSS)组,检测2组结直肠癌中H3K36me3及SETD2、KDM4B、MMR蛋白质表达情况并分析其临床病理意义。结果:STED2、H3K36me3表达与结直肠癌发生呈负相关(分别为r=-0.745,P<0.001;r=-0.160,P<0.05),KDM4B表达与结直肠癌发生呈正相关(r=0.660,P<0.001)。不同的患者性别、年龄,不同的肿瘤发生部位、大体类型、肿瘤最大径、浸润深度、淋巴结转移的临床病理参数之间SETD2及H3K36me的表达差异均无统计学意义(均P>0.05),除在不同的肿瘤浸润深度KDM4B的表达差异有统计学意义(χ^(2)=0.349,P<0.05)外,在其他临床病理参数上KDM4B的表达差异均无统计学意义(均P>0.05)。结直肠癌样本中检测出14例为MSI-H结直肠癌、66例为MSS结直肠癌;SETD2及H3K36me3的表达均与结直肠癌微卫星状态相关(分别为χ^(2)=3.916,P<0.05及χ^(2)=41.608,P<0.001);KDM4B的表达与结直肠癌微卫星状态无明显相关性(χ^(2)=0.067,P>0.05)。SETD2及H3K36me3表达与MSI-H结直肠癌发生呈负相关(分别为r=-0.221,P<0.05及r=-0.721,P<0.001)。KDM4B表达与MSS结直肠癌发生呈正相关(r=0.200,P<0.05)。免疫组织化学染色结果显示13例(16.25%)为dMMR,67例(83.75%)为pMMR;PCR-毛细管电泳验证结果显示14例为MSI-H结直肠癌,其中13例为dMMR,1例为pMMR;66例为MSS结直肠癌。免疫组织化学染色与多重荧光PCR-毛细管电泳检测结果具有强一致率(Kappa=0.955)。结论:H3K36me3及其表观遗传修饰蛋白质SETD2和KDM4B蛋白质的不同表达程度与结直肠癌发生、发展具有相关性。

Objective:Histone modification is a crucial form of epigenetic regulation.Among them,histone methylation plays a pivotal role in determining chromatin status.Histone H3 trimethylation at lysine 36(H3K36me3)serves as a mediator in various transcription-related events and contributes to DNA damage repair.SET domain containing 2(SETD2),a histone lysine trimethyltransferase,and lysine-specific demethylase 4B(KDM4B),a histone demethylase,are responsible for H3K36me3 methylation and demethylation,respectively.Inactivation of SETD2 and activation of KDM4B can both down-regulate the level of H3K36me3,thus promoting tumorigenesis and progression.This study aims to investigate the clinicopathological significance of H3K36me3 and its epigenetic modification proteins SETD2 and KDM4B in colorectal cancer.Methods:The paraffin-embedded tissue samples from 80 postoperative colorectal cancer patients,who were admitted to the People’s Hospital of Fujian University of Traditional Chinese Medicine from October 2018 to October 2020 were collected.Immunohistochemical staining was performed to detect the expression levels of H3K36me3,KDM4B,SETD2,and 4 mismatch repair(MMR)proteins(MSH2,MSH6,MLH1,PMS2)in both colorectal cancer and normal intestinal mucosa.Multiplex fluorescence PCR-capillary electrophoresis was used to detect microsatellite status of colorectal cancer and normal intestinal mucosa.The samples were divided into a microsatellite instability-high(MSI-H)group and a microsatellite stable(MSS)group.The expressions of H3K36me3,SETD2,KDM4B,and MMR proteins in the 2 groups of colorectal cancer were detected and their clinicopathological significance was analyzed.Results:Expression of STED2 and H3K36me3 showed a negative correlation with the occurrence of colorectal cancer(r=−0.745,P<0.001;r=−0.160,P<0.05,respectively),while the expression of KDM4B exhibited a positive correlation with the occurrence of colorectal cancer(r=0.660,P<0.001).There were no statistically significant differences in the expression of SETD2 and H3K36me among clinicopathological parameters such as patient sex,age,tumor location,macroscopic type,tumor maximum diameter,depth of infiltration,and lymph node metastasis(all P>0.05).Except for the expression of KDM4B showing statistically significant differences in different depths of tumor infiltration(χ^(2)=0.349,P<0.05),there were no statistically significant differences in the expression of KDM4B in relation to other clinicopathological parameters(P>0.05).Among the colorectal cancer samples,14 cases were identified as MSI-H colorectal cancer,and 66 cases were MSS colorectal cancer.Expression of SETD2 and H3K36me3 correlated with microsatellite status of colorectal cancer(χ^(2)=3.916,P<0.05 andχ^(2)=41.608,P<0.001,respectively).However,there was no significant correlation between the expression of KDM4B and the microsatellite status of colorectal cancer(χ^(2)=0.067,P>0.05).The expression of SETD2 and H3K36me3 was negatively correlated with the occurrence of MSI-H colorectal cancer(r=−0.221,P<0.05 and r=−0.721,P<0.001,respectively),while the expression of KDM4B was positively correlated with the occurrence of MSS colorectal cancer(r=0.200,P<0.05).Immunohistochemical staining results revealed that 13(16.25%)cases were dMMR,and 67(83.75%)cases were pMMR.PCR-capillary electrophoresis results demonstrated that 14 cases were MSI-H colorectal cancer,including 13 cases of dMMR and 1 case of pMMR.Sixty-six cases were MSS colorectal cancer.The results of immunohistochemical staining were strongly consistent with those of multiplex fluorescence PCR-capillary electrophoresis(Kappa=0.955).Conclusion:The different levels of expression of H3K36me3 and its epigenetic modification proteins SETD2 and KDM4B are correlated with the occurrence and progression of colorectal cancer.