ZNF384调控GCLM对肝细胞癌转移的机制研究

张明; 于成龙; 曹锦鹏

doi:10.12259/j.issn.2095-610X.S20231108

ZNF384调控GCLM对肝细胞癌转移的机制研究

doi: 10.12259/j.issn.2095-610X.S20231108

1.
青岛市市南区人民医院检验科
2.
青岛市第三人民医院健康管理中心，山东青岛　266000

基金项目: 青岛市输血协会科技计划项目（2020-qdsx11）

详细信息

作者简介:
张明（1971～），男，山东青岛人，医学学士，副主任技师，主要从事临床基础检验工作

通讯作者:
曹锦鹏，E-mail：epeng12345@sina.com

中图分类号: R735.7
计量
- 文章访问数: 1003
- HTML全文浏览量: 678
- PDF下载量: 15
- 被引次数: 0
出版历程
- 收稿日期: 2023-09-05
- 网络出版日期: 2023-11-04
- 刊出日期: 2023-11-30

ZNF384 Regulates Metastasis of Hepatocellular Carcinoma via GCLM

1.
Dept. of Clinical Laboratory，Shinan District People’s Hospital of Qingdao
2.
Health Management Center Affiliated Qingdao Third People’s Hospital，Qingdao University，Shandong Qingdao 266000，China

摘要

摘要: 目的探讨ZNF384通过调控GCLM对肝细胞癌（hepatocellular carcinoma，HCC）增殖和转移的影响及具体调控机制。方法分别在HCC细胞系HepG2和HuH7中转染si-ZNF384和pcDNA-GCLM。Western blot检测ZNF384和GCLM蛋白表达以及上皮间质转化（epithelial-mesenchymal transition，EMT）标志物N-cadherin、Vimentin、E-cadherin的表达，CCK-8检测细胞增殖活力，Transwell检测细胞迁移。双荧光素酶报告基因实验验证ZNF384和GCLM结合关系。荧光原位杂交（fluorescence in situ hybridization，FISH）技术检测ZNF384和GCLM在HepG2细胞中的定位。结果 ZNF384和GCLM在HepG2和HuH7细胞中表达升高（P < 0.05），敲降ZNF384抑制HepG2和HuH7细胞增殖（P < 0.001）、迁移（P < 0.001）以及间质标志物N-cadherin（P < 0.01）和Vimentin（P < 0.01）的表达，促进上皮标志物E-cadherin的表达（P < 0.01）。数据库以及双荧光素酶实验证实ZNF384与GCLM启动子结合。过表达GCLM逆转敲降ZNF384对HepG2和HuH7细胞增殖、迁移以及EMT的抑制作用（P < 0.05）。结论敲降ZNF384通过抑制GCLM，抑制HCC细胞增殖和转移。
- 肝细胞癌 /
- ZNF384 /
- GCLM /
- 增殖 /
- 转移
Abstract: Objective To investigate the effect of ZNF384 on the proliferation and metastasis of HCC by regulating GCLM and demonstrate the specific mechanism. Methods si-ZNF384 and pcDNA-GCLM were transfected into HepG2 and HuH7 cell lines of HCC, respectively. Western blot was carried out to detect the expressions of ZNF384 and GCLM proteins as well as the expressions of EMT-markers N-cadherin, Vimentin and E-cadherin. CCK-8 assessed cell proliferative activity. Transwell assay was conducted to measure cell migration. Dual-luciferase experiment verified the binding relationship between ZNF384 and GCLM. The localization of ZNF384 and GCLM in HepG2 cells was detected by FISH assay. Results The expressions of ZNF384 and GCLM in HepG2 and HuH7 cells were increased（P < 0.05）. Knockdown of ZNF384 inhibited the proliferation（P < 0.001） and migration（P < 0.001） of HepG2 and HuH7 cells, suppressed the expression of interstitial markers N-cadherin（P < 0.01） and Vimentin（P < 0.01）, and promoted the expression of epithelial marker E-cadherin（P < 0.01）. Database and dual-luciferase experiments confirmed that ZNF384 is bound to the promoter of GCLM. Overexpression of GCLM reversed the inhibitory effect of knocking down ZNF384 on the proliferation, migration and EMT of HepG2 and HuH7 cells（P < 0.05）. Conclusion Knocking down ZNF384 can repress the proliferation and metastasis of HCC cells by inhibiting GCLM.
- Hepatocellular carcinoma /
- ZNF384 /
- GCLM /
- Proliferation /
- Metastasis

HTML全文

图 1 敲降ZNF384抑制HCC细胞增殖和转移

A～B：Western blot检测HCC细胞系中ZNF384蛋白表达，与LO2组相比，^*P < 0.05，^***P < 0.001。C～E：Western blot检测敲降ZNF384对ZNF384蛋白表达的作用；F：CCK-8检测细胞增殖活力；G～H：Transwell检测细胞迁移能力；I-K：Western blot检测EMT相关标志物的表达。与si-NC组相比，^*P < 0.05，^**P < 0.01，^***P < 0.001。

Figure 1. Knockdown of ZNF384 inhibited the proliferation and metastasis of HCC cells

下载: 全尺寸图片幻灯片

图 2 ZNF384作为转录因子调控GCLM的表达

A：AnimalTFD3.0数据库预测ZNF384与GCLM的启动子结合位点;B：双荧光素酶报告基因实验验证ZNF384与GCLM的结合关系，与pcDNA-NC组相比，^***P < 0.001；C：FISH检测ZNF384和GCLM在HepG2细胞中的定位；D：Western blot检测HCC细胞中GCLM的蛋白表达，与LO2组相比，^**P < 0.01，^***P < 0.001；E～F：Western blot检测敲降ZNF384对GCLM蛋白表达的作用。与si-NC组相比，^**P < 0.01，^***P < 0.001。

Figure 2. ZNF384 as a transcription factor regulated the expression of GCLM

下载: 全尺寸图片幻灯片

图 3 ZNF384调控GCLM抑制肝细胞癌细胞的增殖和转移

A～C：Western blot检测ZNF384和GCLM在HepG2和HuH7细胞中的表达；D：CCK-8检测细胞增殖活力；E～G：Transwell检测细胞迁移能力；H～J：Western blot检测EMT相关标志物N-cadherin、Vimentin和E-cadherin的蛋白表达。^*P < 0.05，^**P < 0.01，^***P < 0.001。

Figure 3. ZNF384 suppressed the proliferation and metastasis of HCC cells by regulating GCLM

下载: 全尺寸图片幻灯片

参考文献(24)

[1]	Brown Z J,Tsilimigras D I,Ruff S M,et al. Management of hepatocellular carcinoma: A review[J]. JAMA Surg,2023,158(4):410-420. doi: 10.1001/jamasurg.2022.7989
[2]	Xie D,Shi J,Zhou J,et al. Clinical practice guidelines and real-life practice in hepatocellular carcinoma: A Chinese perspective[J]. Clin Mol Hepatol,2023,29(2):206-216. doi: 10.3350/cmh.2022.0402
[3]	Liu S,Liu X,Lin X,et al. Zinc finger proteins in the war on gastric cancer: Molecular mechanism and clinical potential[J]. Cells,2023,12(9):1314. doi: 10.3390/cells12091314
[4]	Singh J K,Smith R,Rother M B,et al. Zinc finger protein ZNF384 is an adaptor of Ku to DNA during classical non-homologous end-joining[J]. Nat Commun,2021,12(1):6560. doi: 10.1038/s41467-021-26691-0
[5]	Meng Q X,Wang K N,Li J H,et al. ZNF384-ZEB1 feedback loop regulates breast cancer metastasis[J]. Mol Med,2022,28(1):111. doi: 10.1186/s10020-022-00541-1
[6]	Yan Z,Zhou Y,Yang Y,et al. Zinc finger protein 384 enhances colorectal cancer metastasis by upregulating MMP2[J]. Oncol Rep,2022,47(3):49. doi: 10.3892/or.2022.8260
[7]	He L,Fan X,Li Y,et al. Overexpression of zinc finger protein 384 (ZNF 384),a poor prognostic predictor,promotes cell growth by upregulating the expression of Cyclin D1 in Hepatocellular carcinoma[J]. Cell Death Dis,2019,10(6):444. doi: 10.1038/s41419-019-1681-3
[8]	Yang H, Wang J, Huang Z Z, et al. Cloning and characterization of the 5'-flanking region of the rat glutamate-cysteine ligase catalytic subunit[J]. Biochem J, 2001, 357（Pt 2）: 447-455.
[9]	Yang H,Li G,Qiu G. Bioinformatics analysis using ATAC-seq and RNA-seq for the identification of 15 gene signatures associated with the prediction of prognosis in hepatocellular carcinoma[J]. Front Oncol,2021,11:726551.
[10]	Tsai M C,Yen Y H,Chang K C,et al. Elevated levels of serum urokinase plasminogen activator predict poor prognosis in hepatocellular carcinoma after resection[J]. BMC Cancer,2019,19(1):1169. doi: 10.1186/s12885-019-6397-3
[11]	Luo Y,Teng F,Fu H,et al. Immunotherapy in liver transplantation for hepatocellular carcinoma: Pros and cons[J]. World J Gastrointest Oncol,2022,14(1):163-180. doi: 10.4251/wjgo.v14.i1.163
[12]	Gauthier A,Ho M. Role of sorafenib in the treatment of advanced hepatocellular carcinoma: An update[J]. Hepatol Res,2013,43(2):147-154. doi: 10.1111/j.1872-034X.2012.01113.x
[13]	Fang Y,Zhan Y,Xie Y,et al. Integration of glucose and cardiolipin anabolism confers radiation resistance of HCC[J]. Hepatology,2022,75(6):1386-1401. doi: 10.1002/hep.32177
[14]	Zhu L,Bai W,Cheng Q,et al. ZNF384-related fusion genes in acute lymphoblastic leukemia[J]. Cancer Control,2023,30:10732748231182787.
[15]	Grammatico S,Vitale A,La Starza R,et al. Lineage switch from pro-B acute lymphoid leukemia to acute myeloid leukemia in a case with t(12;17)(p13;q11)/TAF15-ZNF384 rearrangement[J]. Leuk Lymphoma,2013,54(8):1802-1805. doi: 10.3109/10428194.2012.753450
[16]	Sim J,Park J,Kim S,et al. Association of Tim-3/Gal-9 Axis with NLRC4 inflammasome in glioma malignancy: Tim-3/Gal-9 induce the NLRC4 inflammasome[J]. Int J Mol Sci,2022,23(4):2028. doi: 10.3390/ijms23042028
[17]	Mori S,Takeuchi T,Ishii Y,et al. Identification of APOBEC3B promoter elements responsible for activation by human papillomavirus type 16 E6[J]. Biochem Biophys Res Commun,2015,460(3):555-560. doi: 10.1016/j.bbrc.2015.03.068
[18]	Xiao Y,Yang K,Liu P,et al. Deoxyribonuclease 1-like 3 inhibits hepatocellular carcinoma progression by inducing apoptosis and reprogramming glucose metabolism[J]. Int J Biol Sci,2022,18(1):82-95. doi: 10.7150/ijbs.57919
[19]	Kendig E L,Chen Y,Krishan M,et al. Lipid metabolism and body composition in Gclm(-/-) mice[J]. Toxicol Appl Pharmacol,2011,257(3):338-348. doi: 10.1016/j.taap.2011.09.017
[20]	Wang T,Li C,Han B,et al. Neuroprotective effects of danshensu on rotenone-induced parkinson's disease models in vitro and in vivo[J]. BMC Complement Med Ther,2020,20(1):20. doi: 10.1186/s12906-019-2738-7
[21]	Wang S, Wang H, Zhu S, et al. Systematical analysis of ferroptosis regulators and identification of GCLM as a tumor promotor and immunological biomarker in bladder cancer[J]. Front Oncol, 2022, 24（12）: 1040892.
[22]	Inoue Y,Tomisawa M,Yamazaki H,et al. The modifier subunit of glutamate cysteine ligase (GCLM) is a molecular target for amelioration of cisplatin resistance in lung cancer[J]. Int J Oncol,2003,23(5):1333-1339.
[23]	Wu N,Zhu D,Li J,et al. CircOMA1 modulates cabergoline resistance by downregulating ferroptosis in prolactinoma[J]. J Endocrinol Invest,2023,46(8):1573-1587. doi: 10.1007/s40618-023-02010-w
[24]	Cheng M L,Lu Y F,Chen H,et al. Liver expression of Nrf2-related genes in different liver diseases[J]. Hepatobiliary Pancreat Dis Int,2015,14(5):485-491. doi: 10.1016/S1499-3872(15)60425-8

相关文章(20)

[1]	王玲, 秦祥川, 李金秋, 阿仙姑·哈斯木. CD147通过AIM2炎症小体介导宫颈癌细胞焦亡和增殖, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20240103
[2]	张晔琳, 马丽娅, 彭旭晖, 杨禾丰, 佘睿. hsa-let-7a-5p调控牙周膜干细胞增殖及凋亡, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20231028
[3]	张梁, 王保全, 雷喜锋, 王旭, 柯阳, 张玮. miR-29c-3p/IGF1分子轴对肝星状细胞活化，增殖和凋亡的作用机制, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20230926
[4]	吴华杰, 屠德桢, 李磊, 乔涵, 李国萍. 白藜芦醇对鼻咽癌转移能力的影响及其分子机制, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20230220
[5]	冯毅, 王小峰, 白西民, 姚胜, 党俊涛, 赵云洁, 蔡冰. miR-149-5p通过MSH5/Wnt信号通路调控胶质瘤细胞恶性生物学行为, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20230823
[6]	张玮, 王保全, 雷喜锋, 王旭, 张梁. miR-125b-5p调控HK2抑制胆囊癌细胞增殖和糖酵解, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20221206
[7]	廖周俊, 杨少华, 刘立鑫, 胡晟, 陈轶晖, 康强, 张小文. AK4对肝内胆管癌细胞HUCCT1增殖、迁移的影响, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20220611
[8]	王保全, 张伟, 田园, 雷喜锋, 王旭. miR-142-5p通过CCND1调控胆囊癌细胞的增殖和转移, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20220223
[9]	杨兰, 贾霄, 姜奕彤, 崔琪, 刘光赐, 何颖红. UBE2C基因沉默表达对人胃癌AGS细胞增殖和迁移的影响, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20210504
[10]	雷喜锋, 侯峰强, 杨少华, 张伟. miR-373在人肝细胞癌中的表达及其作用, 昆明医科大学学报.
[11]	雷喜锋, 侯峰强, 刘韬, 张伟. FOXP2抑制人肝细胞癌增殖及其机制, 昆明医科大学学报.
[12]	贾楠楠. Gnaq对SH-SY5Y细胞增殖的作用及机制, 昆明医科大学学报.
[13]	魏东. 脆性位点基因WWOX调控人胆囊癌细胞的体外增殖效应, 昆明医科大学学报.
[14]	邱建武. P38MAPK特异性抑制剂SB203580对肝癌细胞增殖的影响, 昆明医科大学学报.
[15]	熊伟. 奥沙利铂化疗对荧光HepG2肝癌细胞裸鼠原位移植瘤侵袭转移潜能的影响, 昆明医科大学学报.
[16]	王海峰. 上调microRNA-101沉默EZH2基因表达对人膀胱癌T24细胞系增殖和凋亡的影响, 昆明医科大学学报.
[17]	周喆焱. E-cadherin对非小细胞肺癌的转移和靶向治疗的影响, 昆明医科大学学报.
[18]	武斌. 大豆苷元对人乳牙牙髓干细胞增殖和成骨分化的影响, 昆明医科大学学报.
[19]	夏英杰. SD大鼠髁状突颈部骨折对大鼠髁状突软骨细胞增殖与凋亡的影响, 昆明医科大学学报.
[20]	李雄. 氧自由基在血管紧张素Ⅱ诱导ECV304细胞增殖中的作用, 昆明医科大学学报.