组蛋白去甲基化酶5B在喉癌中的表达及体外对Hep-2细胞增殖的影响  

Expression of KDM5B in laryngeal carcinoma and its effect on Hep-2 cell proliferation in vitro

作　　者：孙红村[1] 胡建道[1] 邱小雯[1] 江文博[1] 方来福 SUN Hongcun;HU Jiandao;QIU Xiaowen;JIANG Wenbo;FANG Laifu(Department of Otolaryngology Head and Neck Surgery,the Affiliated People's Hospital of Ningbo University,Ningbo,Zhejiang,315040,China;Department of Pathology,the Affiliated People's Hospital of Ningbo University,Ningbo,Zhejiang,315040,China)

机构地区：[1]宁波大学附属人民医院耳鼻咽喉头颈外科,浙江宁波315040 [2]宁波大学附属人民医院病理科,浙江宁波315040

出　　处：《中国耳鼻咽喉头颈外科》2023年第6期344-349,共6页Chinese Archives of Otolaryngology-Head and Neck Surgery

基　　金：宁波市鄞州区科技局基金资助项目(2021AS0017)。

摘　　要：目的明确组蛋白去甲基化酶5B(lysine-specific demethylase 5B,KDM5B)在喉癌中的表达情况,探讨其对喉癌Hep-2细胞增殖的影响及相关机制。方法收集121例喉癌患者手术切除组织标本和60例癌旁组织标本,对KDM5B表达量进行免疫组化分析,了解KDM5B表达与各临床因素间的关系。设计重组KDM5B-shRNA干扰表达质粒,并转染入Hep-2细胞内。研究分为空白组、NC-shRNA组和KDM5B-shRNA组。qRT-PCR检测KDM5B mRNA表达水平,CCK-8法观察细胞增殖能力的变化,平板克隆形成实验观察单克隆集落的形成,流式细胞术检测细胞周期分布和凋亡情况,Western-blot检测KDM5B、H3K4me3、p21、p27、CyclinD1、CDK4等蛋白的表达情况。结果KDM5B在喉癌组织中的表达明显高于癌旁组织(P=0.000),与病理类型(P=0.007)、临床分期(P=0.002)和淋巴结转移(P=0.000)均有关。KDM5B-shRNA组细胞内KDM5B mRNA水平明显低于空白组和NC-shRNA组(P<0.01)。随着转染时间的持续延长,KDM5B-shRNA组细胞的吸光度值与空白组和NC-shRNA组相比均有较大差异(P=0.000),且KDM5B-shRNA组细胞的单克隆集落数明显减少(P=0.000)。KDM5B蛋白表达被抑制后,CyclinD1和CDK4蛋白表达随转染时间延长均逐渐减少,而H3K4me3、p21和p27蛋白表达量则均逐渐升高。与空白组和NC-shRNA组相比,KDM5B-shRNA组细胞的G1期分布明显升高,而S期和G2期分布比例均明显降低,细胞凋亡率也明显升高(P均=0.000)。结论KDM5B参与喉癌增殖的调控,可作为一个潜在治疗喉癌的靶点。OBJECTIVE To study the expression of KDM5B in laryngeal cancer and explore the mechanism of KDM5B on the proliferation of laryngeal carcinoma Hep-2 cells.METHODS Expression of KDM5B was examined with immunohistochemical staining method in 121 laryngeal carcinoma tissue samples and 60 adjacent normal tissue samples of laryngeal cancer patients.The relationship between expression of KDM5B and clinicopathological factors in 121 patients was analyzed.The recombinant KDM5B-shRNA plasmid was designed and transfected into Hep-2 cells.The study was divided into three groups:blank group,NC-shRNA group and KDM5B-shRNA group.The mRNA level of KDM5B was detected by qRT-PCR.CCK-8 assay was used to observe proliferation of Hep-2 cells.The number of colonies were observed in a colony formation assay.Flow cytometry was used to detect the cell cycle distribution and cell apoptosis.Western blot analysis was used to detect the expression of KDM5B,H3K4me3,p21,p27,CyclinD1 and CDK4.RESULTS The expression of KDM5B was higher in laryngeal cancer tissue than in adjacent normal tissues(P=0.000).The expression of KDM5B was associated with histological type,clinical stage and lymph node metastasis(P=0.007,0.002 and 0.000,respectively).The expression of KDM5B mRNA in KDM5B-shRNA group was significantly lower than that in blank and NC-shRNA group(P=0.000).Optical density at a wave length of 450 nm in KDM5B-shRNA group was lower than that in blank and NC-shRNA group at 24h and 48h after transfection(P=0.000).The number of colonies were significant differences among three groups(P=0.000).The expression of KDM5B,CyclinD1 and CDK4 protein in KDM5B-shRNA group was gradually decreased while the expression of H3K4me3,p21 and p27 protein was gradually increased.There was the highest proportion of Hep-2 cells in G1 phase and the lowest proportion in S and G2 phase in KDM5B-shRNA group,compared with that in blank and NC-shRNA group(P=0.000).Meanwhile,the number of cells in the apoptotic stage increased much more apparently in KDM5B-shRNA group than in

关 键 词：喉肿瘤 细胞增殖 组蛋白去甲基化酶5B 

分 类 号：R739.65[医药卫生—肿瘤]