敲除<i>Usp13</i>促进棕榈酸诱导的小鼠肝实质细胞凋亡<sup>*</sup>

doi:10.13523/j.cb.2112036

中国生物工程杂志

2022, Vol. 42

Issue (4): 9-16 DOI: 10.13523/j.cb.2112036

研究报告

敲除Usp13促进棕榈酸诱导的小鼠肝实质细胞凋亡^*

王婷¹,刘凯²,李柯颖³,陈旭²,任广明^2,^**(

),杨晓明^1,^2,^3,^**(

)

1 安徽医科大学基础医学院合肥 230032
2 军事科学院军事医学研究院生命组学研究所北京 100850
3 青岛大学青岛 266071

Knockdown of Usp13 Promotes Palmitic Acid-induced Apoptosis in Mouse Hepatocytes

WANG Ting¹,LIU Kai²,LI Ke-ying³,CHEN Xu²,REN Guang-ming^2,^**(

),YANG Xiao-ming^1,^2,^3,^**(

)

1 School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China
2 Beijing Institute of Lifeomics, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 100850, China
3 Qingdao University, Qingdao 266071, China

全文: PDF(3275 KB) HTML

摘要：

目的: 研究去泛素化酶Usp13对棕榈酸诱导的肝实质细胞凋亡的影响,并对其机制进行初步探究。方法: 两步胶原酶灌注法分离小鼠原代肝实质细胞并采用棕榈酸处理;甘油三酯酶法及油红O染色检测肝实质细胞中脂质的累积;MTS和LDH试剂盒检测细胞活力;流式细胞术检测细胞凋亡;qPCR检测凋亡相关基因的表达以及免疫印迹法检测Caspase-3的活化。结果: 敲除Usp13基因并不影响棕榈酸诱导下肝实质细胞的甘油三酯累积,但导致细胞损伤加重,凋亡增加。机制研究显示,敲除Usp13促使肝实质细胞中抗凋亡基因Bcl-2、Bcl-xl表达下调,促凋亡基因Bid、p53表达上调,以及Caspase-3活化增强。结论: 初步揭示了Usp13调控棕榈酸诱导下肝实质细胞凋亡的机制,为深入研究Usp13调控非酒精性脂肪性肝炎的发生、发展奠定了基础。

关键词： 非酒精性脂肪性肝炎; Usp13; 细胞凋亡; 棕榈酸; 小鼠原代肝实质细胞

Abstract:

Objective: To study the effect of ubiquitin-specific protease 13 (Usp13) on the apoptosis of mouse hepatocytes stimulated by palmitic acid,and to explore the underlying molecular mechanism. Methods: Mouse primary hepatocytes were isolated by two-step collagenase perfusion technique,and then stimulated with palmitic acid after cultured in vitro.Lipid accumulation was detected by triglyceride quantitative method and oil red O staining. Cell viability was measured by MTS and LDH assay. The apoptosis of hepatocytes was tested by Annexin V-FITC/7-AAD assay. Finally the expression levels of anti-apoptotic genes and pro-apoptotic genes were detected by qPCR and the activation of Caspase-3 was detected by Western blotting. Results: Knockdown of Usp13 did not affect the accumulation of triglyceride in palmitic acid-induced hepatocytes,but the cell viability was decreased and apoptosis was increased.Studies show that knockdown of Usp13 resulted in the down-regulated expression of anti-apoptotic genes Bcl-2 and Bcl-xl, and the up-regulated expression of pro-apoptotic genes Bid and p53.The Caspase-3 activity in Usp13 knockdown hepatocytes was enhanced after being exposed to palmitic acid. Conclusion: This study revealed the mechanism of Usp13 regulating the apoptosis of hepatocytes induced by palmitic acid, which laid a foundation for further study of Usp13 regulating the occurrence and development of non-alcoholic steatohepatitis.

Key words: Non-alcoholic steatohepatitis Usp13 Apoptosis Palmitic acid Mouse primary hepatocytes

收稿日期: 2021-12-08 出版日期: 2022-05-05

ZTFLH:

Q819

基金资助: * 国家自然科学基金青年科学基金(82001666)

通讯作者: 任广明,杨晓明 E-mail: max19920503@163.com;xiaomingyang@sina.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	王婷
	刘凯
	李柯颖
	陈旭
	任广明
	杨晓明

引用本文:

王婷,刘凯,李柯颖,陈旭,任广明,杨晓明. 敲除Usp13促进棕榈酸诱导的小鼠肝实质细胞凋亡^*[J]. 中国生物工程杂志, 2022, 42(4): 9-16.

WANG Ting,LIU Kai,LI Ke-ying,CHEN Xu,REN Guang-ming,YANG Xiao-ming. Knockdown of Usp13 Promotes Palmitic Acid-induced Apoptosis in Mouse Hepatocytes. China Biotechnology, 2022, 42(4): 9-16.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2112036 或 https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I4/9

表1 qPCR引物序列

图1 Usp13-/-小鼠基因型鉴定

图2 敲除Usp13不影响肝实质细胞脂质累积

图3 敲除Usp13降低棕榈酸诱导下肝实质的细胞活力

图4 敲除Usp13促进棕榈酸诱导下肝实质细胞凋亡

图5 敲除Usp13使棕榈酸诱导下肝实质细胞的凋亡通路活化增强

[1]	Eslam M, Valenti L, Romeo S. Genetics and epigenetics of NAFLD and NASH: clinical impact. Journal of Hepatology, 2018, 68(2): 268-279. doi: 10.1016/j.jhep.2017.09.003
[2]	Oseini A M, Sanyal A J. Therapies in non-alcoholic steatohepatitis (NASH). Liver International, 2017, 37(Suppl 1): 97-103. doi: 10.1111/liv.13302
[3]	Cobbina E, Akhlaghi F. Non-alcoholic fatty liver disease (NAFLD) - pathogenesis, classification, and effect on drug metabolizing enzymes and transporters. Drug Metabolism Reviews, 2017, 49(2): 197-211. doi: 10.1080/03602532.2017.1293683 pmid: 28303724
[4]	Williams C D, Stengel J, Asike M I, et al. Prevalence of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis among a largely middle-aged population utilizing ultrasound and liver biopsy: a prospective study. Gastroenterology, 2011, 140(1): 124-131. doi: 10.1053/j.gastro.2010.09.038
[5]	Sumida Y, Yoneda M. Current and future pharmacological therapies for NAFLD/NASH. Journal of Gastroenterology, 2018, 53(3): 362-376. doi: 10.1007/s00535-017-1415-1
[6]	Sheka A C, Adeyi O, Thompson J, et al. Nonalcoholic steatohepatitis. JAMA, 2020, 323(12): 1175. doi: 10.1001/jama.2020.2298 pmid: 32207804
[7]	Guo J, Zhang J L, Liang L, et al. Potent USP10/ 13 antagonist spautin-1 suppresses melanoma growth via ROS-mediated DNA damage and exhibits synergy with cisplatin. Journal of Cellular and Molecular Medicine, 2020, 24(7): 4324-4340. doi: 10.1111/jcmm.15093
[8]	Huang J M, Ye Z Y, Wang J, et al. USP 13 mediates PTEN to ameliorate osteoarthritis by restraining oxidative stress, apoptosis and inflammation via AKT-dependent manner. Biomedicine & Pharmacotherapy, 2021, 133: 111089. doi: 10.1016/j.biopha.2020.111089
[9]	Chu Y Y, Dong X Q, Kang Y J, et al. The chaperone BAG 6 regulates cellular homeostasis between autophagy and apoptosis by holding LC3B. iScience, 2020, 23(11): 101708. doi: 10.1016/j.isci.2020.101708
[10]	Xie W H, Jin S H, Wu Y X, et al. Auto-ubiquitination of NEDD4- 1 recruits USP13 to facilitate autophagy through deubiquitinating VPS34. Cell Reports, 2020, 30(8): 2807-2819.e4. doi: 10.1016/j.celrep.2020.01.088
[11]	Xie W H, Jin S H, Cui J. The NEDD4-USP13 axis facilitates autophagy via deubiquitinating PIK3C3. Autophagy, 2020, 16(6): 1150-1151. doi: 10.1080/15548627.2020.1743071
[12]	Zhang J, Zhang P, Wei Y, et al. Deubiquitylation and stabilization of PTEN by USP13. Nature Cell Biology, 2013, 15(12):1486-1494. doi: 10.1038/ncb2874
[13]	Man X J, Piao C Y, Lin X Y, et al. USP 13 functions as a tumor suppressor by blocking the NF-κB-mediated PTEN downregulation in human bladder cancer. Journal of Experimental & Clinical Cancer Research: CR, 2019, 38(1): 259.
[14]	Zhang S, Zhang M, Jing Y, et al. Deubiquitinase USP13 dictates MCL1 stability and sensitivity to BH3 mimetic inhibitors. Nature Communications, 2018, 9(1):215. doi: 10.1038/s41467-017-02693-9
[15]	Huang J, Gu Z L, Chen W, et al. Knockdown of ubiquitin-specific peptidase 13 inhibits cell growth of hepatocellular carcinoma by reducing c-Myc expression. The Kaohsiung Journal of Medical Sciences, 2020, 36(8): 615-621. doi: 10.1002/kjm2.12209
[16]	Marra F, Svegliati-Baroni G. Lipotoxicity and the gut-liver axis in NASH pathogenesis. Journal of Hepatology, 2018, 68(2): 280-295. doi: 10.1016/j.jhep.2017.11.014
[17]	Zhou F, Zhou J H, Wang W X, et al. Unexpected rapid increase in the burden of NAFLD in China from 2008 to 2018: a systematic review and meta-analysis. Hepatology (Baltimore, Md), 2019, 70(4): 1119-1133. doi: 10.1002/hep.30702
[18]	Hu S S, Bai S C, Dai Y Y, et al. Deubiquitination of MITF-M regulates melanocytes proliferation and apoptosis. Frontiers in Molecular Biosciences, 2021, 8: 692724. doi: 10.3389/fmolb.2021.692724
[19]	朱海珍, 黄湘, 王静静, 等. USP13抑制溶瘤病毒诱导的肝癌细胞凋亡机制. 湖南大学学报(自然科学版), 2019, 46(12): 107-113.
	Zhu H Z, Huang X, Wang J J, et al. USP13 restrains apoptosis of liver cancer cells induced by NDV. Journal of Hunan University (Natural Sciences), 2019, 46(12): 107-113.
[20]	Fang X G, Zhou W C, Wu Q L, et al. Deubiquitinase USP13 maintains glioblastoma stem cells by antagonizing FBXL14-mediated Myc ubiquitination. The Journal of Experimental Medicine, 2017, 214(1): 245-267. doi: 10.1084/jem.20151673
[21]	陶守松, 任广明, 尹荣华, 等. 敲低去泛素化酶USP13抑制K562细胞的增殖. 中国生物工程杂志, 2021, 41(5): 1-7.
	Tao S S, Ren G M, Yin R H, et al. Knockdown of deubiquitinase USP13 inhibits the proliferation of K562 cells. China Biotechnology, 2021, 41(5): 1-7.
[22]	屈直. USP13在口腔鳞癌中的作用及其分子机制. 沈阳: 中国医科大学, 2020.
	Qu Z. The role of USP13 in oral squamous cell carcinoma and its molecular mechanism. Shenyang: China Medical University, 2020.
[23]	Wang K W. Autophagy and apoptosis in liver injury. Cell Cycle (Georgetown, Tex), 2015, 14(11): 1631-1642. doi: 10.1080/15384101.2015.1038685

[1]	陶守松,任广明,尹荣华,杨晓明,马文兵,葛志强. 敲低去泛素化酶USP13抑制K562细胞的增殖^*[J]. 中国生物工程杂志, 2021, 41(5): 1-7.
[2]	朱永朝,陶金,任萌萌,熊燃,何亚琴,周瑜,卢震辉,杜勇,杨芝红. 自噬抑制肿瘤坏死因子α诱导人胎盘胎儿来源间充质干细胞发生凋亡 ^*[J]. 中国生物工程杂志, 2019, 39(9): 62-67.
[3]	刘叶,潘玥,郑魏,胡晶. miR-186-5p在酒精诱导的心肌细胞中高表达并通过靶基因XIAP调控细胞凋亡水平 ^*[J]. 中国生物工程杂志, 2019, 39(5): 53-62.
[4]	代立婷, 吴忠南, 黄翔, 杨杰, 曾慧兰, 王国才, 蒋建伟. 卤地菊乙醇提取物W40单体诱导GLC-82细胞凋亡的分子机制研究[J]. 中国生物工程杂志, 2017, 37(8): 1-7.
[5]	徐安健, 李艳萌, 李斯文, 乌姗娜, 张蓓, 黄坚. PHP14沉默对肺癌细胞凋亡的影响及其机制[J]. 中国生物工程杂志, 2017, 37(7): 12-17.
[6]	白欣艳, 温丽敏, 王玉晶, 王海龙, 解军, 郭睿. ANKRD49通过上调Bcl-xL的表达抑制UV诱导GC-1细胞的凋亡[J]. 中国生物工程杂志, 2017, 37(4): 40-47.
[7]	万春红, 张志, 李圣纳, 彭以元, 许亮国. TRAF7的研究进展[J]. 中国生物工程杂志, 2016, 36(3): 93-101.
[8]	陈娜子, 姜潮, 李校堃. 内质网应激与疾病[J]. 中国生物工程杂志, 2016, 36(1): 76-85.
[9]	邱华丽, 穰杰, 丁学知, 胡胜标, 张友明, 朱道奇, 夏立秋. 苦瓜MAP30蛋白的原核表达及其生物活性研究[J]. 中国生物工程杂志, 2014, 34(06): 40-46.
[10]	韩笑, 李娜, 杜培革. 抗肿瘤多肽研究进展[J]. 中国生物工程杂志, 2013, 33(6): 93-98.
[11]	张曦, 刘北忠, 高艳军, 黎亮, 高远梅, 胡秀秀, 马鹏鹏, 钟梁. *干扰 GINS2* 表达对HL60细胞增殖和凋亡的影响**[J]. 中国生物工程杂志, 2013, 33(3): 41-46.
[12]	魏东, 邹浩, 王琳, 王文举, 骆志玲, 张小文. 靶向miRNA干扰Bmi-1诱导胆囊癌细胞凋亡及上调Caspase-3表达的研究[J]. 中国生物工程杂志, 2013, 33(12): 1-8.
[13]	林颖, 李璞, 单敬轩, 陈晓静, 施慧莉, 霍克克. RIOK3促进了caspase-10对PAK2的酶解激活[J]. 中国生物工程杂志, 2012, 32(08): 1-8.
[14]	秦晓林, 徐勇, 范晓卿, 李武县, 匡文斌, 成凤, 涂植光. TFPI-2对人肝癌细胞生长增殖、凋亡及AFP合成的影响[J]. 中国生物工程杂志, 2011, 31(12): 33-38.
[15]	唐敏, 曾文彬, 曹亚. 分子探针在细胞凋亡检测中的研究进展[J]. 中国生物工程杂志, 2011, 31(06): 124-128.

Viewed

Full text

Abstract

Cited

Shared

Discussed