乙酰基转移酶KAT3b与ABCE1乙酰化在食管癌中的相关性研究

doi:10.19401/j.cnki.1007-3639.2022.04.004

摘要/Abstract

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。

关键词: 乙酰基转移酶3b, ATP结合盒转运子E1, 乙酰化, 食管癌

Abstract:

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.

Key words: Acetyltransferase 3b, ATP combined box transporter E1, Acetylation, Esophageal cancer

中图分类号:

R735.1

梁宗英, 杨阳, 孙光蕊, 赵宝山, 辛国华, 张乐, 侯继申. 乙酰基转移酶KAT3b与ABCE1乙酰化在食管癌中的相关性研究[J]. 中国癌症杂志, 2022, 32(4): 316-323.

LIANG Zongying, YANG Yang, SUN Guangrui, ZHAO Baoshan, XIN Guohua, ZHANG Le, HOU Jishen. A study on correlation of acetyltransferase KAT3b and ABCE1 acetylation in esophageal cancer[J]. China Oncology, 2022, 32(4): 316-323.

图/表 11

图1

图2

图3

表1

图4

表2

表3

图5

图6

图7

图8

参考文献 13

[1]	BRAY F,, FERLAY J,, SOERJOMATARAM I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2018, 68(6): 394-424. doi: 10.3322/caac.21492
[2]	DUDASH M J,, SLIPAK S,, DOVE J, et al. Lymph node harvest as a measure of quality and effect on overall survival in esophageal cancer: a national cancer database assessment[J]. Am Surg, 2019, 85(2): 201-205.
[3]	李强,, 王九江,, 罗水发, 等. ABCE1基因沉默对人膀胱癌T24细胞增殖和迁移能力的影响[J]. 中国病理生理杂志, 2015, 31(5): 929-933.
	LI Q,, WANG J J,, LUO S F, et al. Effect of ABCE1 gene silencing on proliferation and migration of human bladder cancer cell line T24[J]. Chin J Pathophysiol, 2015, 31(5): 929-933.
[4]	李小瑞,, 岳军艳,, 张清琴, 等. 沉默ABCE1基因对人食管癌EC109细胞凋亡、增殖、侵袭及迁移的影响[J]. 中国肿瘤生物治疗杂志, 2013, 20(5): 569-574.
	LI X R,, YUE J Y,, ZHANG Q Q, et al. Effect of ABCE1 gene silencing on apoptosis, proliferation, migration and invasion of human esophageal carcinoma EC-109 cells[J]. Chin J Cancer Biotherapy, 2013, 20(5): 569-574.
[5]	NARITA T,, WEINERT B T,, CHOUDHARY C. Functions and mechanisms of non-histone protein acetylation[J]. Nat Rev Mol Cell Biol, 2019, 20(3): 156-174. doi: 10.1038/s41580-018-0081-3
[6]	WANG L K,, DU Y P,, LU M, et al. ASEB: a web server for KAT-specific acetylation site prediction[J]. Nucleic Acids Res, 2012, 40(W1): W376-W379.
[7]	SOCINSKI M A,, OBASAJU C,, GANDARA D, et al. Current and emergent therapy options for advanced squamous cell lung cancer[J]. J Thorac Oncol, 2018, 13(2): 165-183. doi: 10.1016/j.jtho.2017.11.111
[8]	MARTÍNEZ-REYES I,, CHANDEL N S. Acetyl-CoA-directed gene transcription in cancer cells[J]. Genes Dev, 2018, 32(7/8): 463-465. doi: 10.1101/gad.315168.118
[9]	LOVINSKY-DESIR S,, MILLER R L. Epigenetics, asthma, and allergic diseases: a review of the latest advancements[J]. Curr Allergy Asthma Rep, 2012, 12(3): 211-220. doi: 10.1007/s11882-012-0257-4
[10]	GARAGNANI P,, PIRAZZINI C,, FRANCESCHI C. Colorectal cancer microenvironment: among nutrition, gut microbiota, inflammation and epigenetics[J]. Curr Pharm Des, 2013, 19(4): 765-778. doi: 10.2174/1381612811306040765
[11]	LIANG Z Y,, YU Q,, JI H T, et al. Tip60-siRNA regulates ABCE1 acetylation to suppress lung cancer growth via activation of the apoptotic signaling pathway[J]. Exp Ther Med, 2019, 17(4): 3195-3202.
[12]	刘佳楠,, 唐芳兰,, 宁宁, 等. 组蛋白去乙酰化酶抑制剂抗卵巢癌的机制研究进展[J]. 医学综述, 2018, 24(24): 4842-4846.
	LIU J N,, TANG F L,, NING N, et al. Research advances in mechanism of histone deacetylase inhibitor for treatment of ovarian cancer[J]. Med Recapitul, 2018, 24(24): 4842-4846.
[13]	申艳佳,, 杨冉,, 陈渺, 等. HDAC抑制剂调控免疫细胞功能及在自身免疫病中的应用进展[J]. 中国药理学通报, 2020, 36(11): 1481-1486.
	SHEN Y J,, YANG R,, CHEN M, et al. Research progress in applications of histone deacetylase inhibitors in modulating function of immune cells and autoimmune disease[J]. Chin Pharmacol Bull, 2020, 36(11): 1481-1486.

KAT	Site	Sequence	P value
CBP/p300	397	IRMLAGRLKPDEGGEVP	0.030 5
CBP/p300	19	VNHDKCKPKKCRQECKK	0.041 6
CBP/p300	415	LNVSYKPQKISPKSTGS	0.108 4
CBP/p300	15	RIAIVNHDKCKPKKCRQ	0.153 8

KAT3b	ABCE1 acetylation		χ²	P value	r value
KAT3b	Positive	Negative	χ²	P value	r value
Positive	35	8	7.466	0.003	0.414
Negative	5	7

Clinicopathological features	Case n	ABCE1 acetylation rate	P value	KAT3b positive	P value
Gender			0.348		0.274
Male	44	62.28±3.54		34
Female	11	58.97±2.92		9
Age/year			0.432		0.263
≥60	42	58.76±1.42		33
<60	13	55.24±2.16		10
TNM stage			0.004		0.017
Ⅰ	7	26.93±1.25		2
Ⅱ	32	45.54±2.42		24
Ⅲ	16	62.14±2.86		15
Histodifferentiation			0.017		0.033
Well	7	24.53±2.45		4
Moderately	40	56.46±3.41		31
Poorly	8	68.71±2.35		8
Lymphatic metastasis			0.008		0.014
No	41	42.31±1.34		31
Yes	14	68.15±2.52		12

[1]	唐波, 赵夏, 刘红兵, 张清峰, 刘奎. 端端分层吻合加胃底套入包埋在微创食管癌切除术中的应用[J]. 中国癌症杂志, 2022, 32(3): 251-257.
[2]	陈劭赓, 何荣琦, 张万飞, 林贤钻, 陈河山, 许荣誉. miR-122-5p通过靶向CREB1抑制食管癌细胞及移植瘤的生长[J]. 中国癌症杂志, 2021, 31(1): 35-44.
[3]	张晓慧 , 罗建民 , 索晓慧 , 孙国锋 , 刘洪峰 , 李　静 , 牛广旭 . SOCS1的表观遗传学修饰与急性髓系白血病的关系[J]. 中国癌症杂志, 2020, 30(8): 577-585.
[4]	唐曦平, 刘爱群, 刘立义, 黄月丽. 胃镜检查在下咽癌共病食管癌中的重要性评价及其临床特点分析[J]. 中国癌症杂志, 2020, 30(8): 626-631.
[5]	颜　芳, 应明真, 陈龙佩, 傅　强. 白蛋白结合型紫杉醇联合奈达铂一线治疗晚期食管癌患者的临床观察[J]. 中国癌症杂志, 2020, 30(8): 632-635.
[6]	雷海科, 廖忠莉, 李小升, 周　琦, 赵玉兰, 何　美, 陈伟庆, 周　宏, 张　维, 吴永忠 . 重庆市食管癌患者生存随访调查及预后影响因素研究[J]. 中国癌症杂志, 2020, 30(2): 98-105.
[7]	晋瑞,金迎迎,王敏聪,惠文涛,李毅,李芳,贾辉,潘继元,马红兵 . 抑制USP9x表达增强食管癌Ec9706-R细胞的放射敏感性[J]. 中国癌症杂志, 2019, 29(7): 486-493.
[8]	胡文斌,张　婷,秦　威,等. 江苏省昆山市1981—2015年去食管癌死因期望寿命和潜在减寿年分析[J]. 中国癌症杂志, 2018, 28(4): 263-269.
[9]	徐　瞳,胡丽娜,郭显智,等. 5-氮杂胞苷可通过抑制Notch1通路诱导食管癌细胞凋亡[J]. 中国癌症杂志, 2018, 28(10): 726-732.
[10]	曹燕飞,任　睿,杨　晔,等. LRRC3B对食管癌细胞Eca109迁移、侵袭及PI3K/Akt信号通路的影响[J]. 中国癌症杂志, 2017, 27(5): 345-352.
[11]	程凌霄,刘敏,靳雨辰,等. MAPK抑制剂与组蛋白去乙酰化酶抑制剂对甲状腺癌细胞的联合再分化作用[J]. 中国癌症杂志, 2017, 27(11): 841-846.
[12]	刘志坤,张魏丽,祝淑钗,等. 慢病毒介导沉默食管癌细胞53BP1基因表达对裸鼠移植瘤放射敏感性的研究[J]. 中国癌症杂志, 2016, 26(7): 574-580.
[13]	温仁祝,陈铭伍,冼　磊,等. 食管癌术后吻合口狭窄预防方法的探讨[J]. 中国癌症杂志, 2016, 26(6): 552-555.
[14]	刘　琪,余　雯,蔡旭伟,等. PET-CT用于评价食管鳞癌放疗中¹⁸F-FDG高摄取区域的空间动态变化的前瞻性研究[J]. 中国癌症杂志, 2016, 26(2): 161-167.
[15]	陈　赟,艾沓杉,夏　怡,等. 紫杉醇联合氟尿嘧啶同期放疗治疗初治局部晚期食管鳞癌患者的Ⅱ期临床研究[J]. 中国癌症杂志, 2016, 26(11): 926-931.