摘要
背景与目的:食管癌是中国癌症死亡的主要原因之一,其发病率和死亡率居高不下。表观遗传学上的乙酰化修饰参与并调控多种肿瘤细胞的增殖、侵袭和转移,乙酰基转移酶参与的肿瘤蛋白的乙酰化修饰可能是食管癌发生的重要机制之一。本研究探讨食管癌中乙酰基转移酶3b(acetyltransferase 3b,KAT3b)的表达及ATP结合盒转运子E1(ATP-combined box transporter E1,ABCE1)蛋白乙酰化水平,分析两者之间的相关性,在食管癌发病过程中的作用及其机制。方法:选取2020年1月—2021年5月承德医学院附属医院胸外科手术切除食管癌标本及癌旁组织各55例。采用免疫组织化学SP法及蛋白质印迹法(Western blot)检测ABCE1和KAT3b蛋白表达,采用免疫共沉淀(coimmunoprecipitation,Co-IP)检测KAT3b蛋白表达和ABCE1蛋白乙酰化水平,采用生物信息学预测KAT3b以ABCE1为底物发生乙酰化的情况,采用荧光免疫组织化学(fluorescence immunohistochemistry,IF)验证ABCE1和KAT3b在癌组织中的定位及表达,分析ABCE1乙酰化与KAT3b的相关性及两者与临床病理学特征的相关性。构建下调KAT3b si-RNA,采用脂质体介导法转染食管癌EC109细胞。采用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)和Western blot检测KAT3b mRNA表达和蛋白水平,采用Co-IP实验检测细胞内KAT3b表达和ABCE1蛋白乙酰化水平。结果:食管癌组织中ABCE1和KAT3b蛋白表达率显著高于正常食管黏膜(P<0.05);ABCE1蛋白在食管癌组织中发生了乙酰化,且ABCE1蛋白乙酰化率显著高于正常黏膜组织(P<0.05);生物信息学预测乙酰基转移酶KAT3b可以催化ABCE1为底物发生乙酰化,且IF证实ABCE1和KAT3b蛋白在癌组织中同时呈现高表达状态;食管癌组织中ABCE1乙酰化和KAT3b表达具有相关性,两者呈正相关;ABCE1乙酰化及KAT3b与食管癌分期、组织分化及淋巴结转移密切相关,而与性别及年龄无关。体外实验显示,在食管癌细胞内RNA干扰KAT3b后,可抑制KAT3b mRNA和蛋白的表达,同时ABCE1蛋白乙酰化水平也显著下降。结论:在食管癌病变过程中KAT3b与ABCE1乙酰化具有相关性,KAT3b高表达可能是催化ABCE1发生乙酰化的重要上游分子,在食管癌诊治中具有重要的潜在价值。
Background and purpose:Esophageal cancer is one of the main causes of cancer death in China,and the morbidity and death rates have remained high.Epigenetic acetylation modifications are involved in and regulate the proliferation,invasion and metastasis of a wide variety of tumor cells,and the acetylation modification of tumor proteins mediated by acetyltransferases may be one of the important mechanisms for esophageal carcinogenesis.This study investigated the expression of acetyltransferase 3b(KAT3b)and the ATP-combined box transporter E1(ABCE1)acetylation level in esophageal cancer and analyzed the correlation and molecular mechanism in the pathogenesis of esophageal cancer.Methods:Fifty-five esophageal cancer tissue samples and 55 para-cancerous mucosal tissues were collected in Affiliated Hospital of Chengde Medical College from January 2020 to May 2021.The protein expressions of ABCE1 and KAT3b were measured by immunohistochemistry and Western blot.The acetylation level of ABCE1and KAT3b protein expression were determined by coimmunoprecipitation(Co-IP).Bioinformatics predicts the acetylation transferase KAT3b undergoing acetylation using ABCE1 as a substrate,and fluorescence immunohistochemistry(IF)verified the location and expressions of ABCE1 and KAT3b in cancerous tissues.The correlation between ABCE1 acetylation and KAT3b and their relationship with the clinical pathology characteristics were analyzed by statistics.RNA interference sequence of KAT3b for downregulation was constructed,and then transfected into esophageal cancer EC109 cells using liposome-mediated methods.The KAT3b mRNA expression and protein levels were determined by real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)and Western blot.ABCE1 protein acetylation levels were detected using Co-IP experiments.Results:Protein expressions of ABCE1 and KAT3b were significantly higher compared with normal esophageal mucosa(P<0.05).The ABCE1 protein was acetylated in esophageal cancer tissue,and the level of ABCE1 protein acetylation was significantly higher in cancer tissues than in normal mucosal tissues(P<0.05).Bioinformatics predicts that the acetyltransferase KAT3b could catalyze the acetylation of ABCE1 as a substrate,and IF confirmed that ABCE1 and KAT3b proteins exhibited a high expression state in cancerous tissues.The ABCE1 acetylation levels and the KAT3b expression were correlated in esophageal cancer tissues,and they were correlated positively.ABCE1 acetylation and KAT3b were associated with esophageal cancer stage,tissue differentiation and lymph node metastasis,and were independent of gender and age.The in vitro experiments showed that the expressions of KAT3b mRNA and protein were inhibited after RNA interference of KAT3b in esophageal cancer cells,and the ABCE1 protein acetylation levels were also decreased significantly.Conclusion:There is correlation between KAT3b and ABCE1 protein acetylation in pathogenesis of esophageal cancer.Highly expressed KAT3b may be an important upstream molecule that catalyze acetylation of ABCE1 and have important potential value in the diagnosis and treatment of esophageal cancer.
作者
梁宗英
杨阳
孙光蕊
赵宝山
辛国华
张乐
侯继申
LIANG Zongying;YANG Yang;SUN Guangrui;ZHAO Baoshan;XIN Guohua;ZHANG Le;HOU Jishen(Thoracic Surgery Department,Affiliated Hospital of Chengde Medical College,Chengde 067000,Hebei Province,China;The Chest Surgery Department of Hebei Provincial Chest Hospital,Shijiazhuang 050000,Hebei Province,China)
出处
《中国癌症杂志》
CAS
CSCD
北大核心
2022年第4期316-323,共8页
China Oncology
基金
河北省2018年医学科学研究重点课题计划(20181159)。
