MCM2通过抑制p53信号通路促进胆管癌细胞的增殖、迁移和侵袭
收稿日期: 2021-12-20
修回日期: 2022-01-18
网络出版日期: 2022-01-29
基金资助
国家自然科学基金项目资助(31771397)
MCM2 promotes the proliferation, migration and invasion of cholangiocarcinoma cells by reducing the p53 signaling pathway
Received date: 2021-12-20
Revised date: 2022-01-18
Online published: 2022-01-29
Supported by
Supported by the National Natural Science Foundation of China(31771397)
微小染色体维持蛋白2 (minichromosome maintenance protein 2, MCM2)的异常表达与多种恶性肿瘤的发生发展密切相关。为探索MCM2基因对胆管癌细胞肿瘤生物学行为的影响及其潜在的作用机制,本研究首先采用cell counting kit-8 (CCK-8)、平板细胞克隆形成、Transwell和Invasion实验证实,MCM2可促进胆管癌细胞的增殖、迁移和侵袭能力;流式细胞术检测结果显示,MCM2可加快细胞周期进程,抑制细胞凋亡。进一步通过对胆管癌组织RNA测序数据集的分析发现,MCM2基因与p53信号通路的激活显著负相关。采用实时定量PCR (quantitative real time polymerase chain reaction, qRT-PCR)和蛋白质免疫印迹(Western blotting, WB)实验发现,在胆管癌细胞中MCM2可显著下调p53和BAX的mRNA和蛋白表达水平,显著上调BCL2和CCND1的mRNA和蛋白表达水平。采用流式细胞术、qRT-PCR和WB实验进一步证实MCM2基因依赖于p53通路发挥影响胆管癌细胞周期和凋亡的功能。最后,通过分析MCM2的表达水平与胆管癌患者临床预后的相关性发现,MCM2在胆管癌组织中的表达显著高于癌旁组织,且其高表达与患者的不良预后显著相关。综上所述,本研究初步揭示MCM2可能通过抑制p53信号通路促进胆管癌的发生发展,并提示MCM2的高表达与胆管癌的不良预后显著相关,为未来胆管癌新的临床诊治措施的研发提供了理论依据。
王翠玲, 刘信燚, 王亚会, 张争, 王治东, 周钢桥 . MCM2通过抑制p53信号通路促进胆管癌细胞的增殖、迁移和侵袭[J]. 遗传, 2022 , 44(3) : 230 -244 . DOI: 10.16288/j.yczz.21-426
The abnormal expressions of minichromosome maintenance protein 2 (MCM2) are closely related to the development of various kinds of cancers. We aimed to explore the functions and potential molecular mechanisms of MCM2 gene in cholangiocarcinoma (CCA) cell lines (Huh28 and RBE). First, the cell counting kit-8 (CCK-8), plate clone formation, transwell and invasion assays showed that MCM2 promotes the proliferation, migration and invasion of CCA cells. Flow cytometry assays showed that MCM2 significantly promotes the cell cycle, and inhibits the apoptosis of CCA cells. Further, by analyzing the RNA sequencing data of cholangiocarcinoma, we found that MCM2 gene is significantly negatively correlated with p53 signaling pathway. Quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting (WB) assays confirmed that MCM2 in CCA cells significantly down-regulated the mRNA and protein expression levels of p53 and BAX, and up-regulated the mRNA and protein expression levels of BCL2 and CCND1. Flow cytometry, qRT-PCR and WB assays confirmed that MCM2 promotes CCA through p53 pathway. Finally, we found that MCM2 is up-regulated in CCA tissues compared to the matched non-tumor cholangiocarcinoma tissues, and the high expressions of MCM2 are significantly associated with the poor clinical outcomes of CCA patients. In conclusion, this study revealed that MCM2 promotes the development of CCA by reducing the p53 pathway, and its high expression levels predict poor prognosis in CCA patients. These results provide a theoretical basis for the development of new clinical diagnosis and treatment of cholangiocarcinoma in the future.
Key words： cholangiocarcinoma; MCM2; proliferation; migration; invasion; cell cycle; apoptosis; p53
| [1] | Razumilava N, Gores GJ. Cholangiocarcinoma. Lancet, 2014,383(9935):2168-2179. |
| [2] | Khan SA, Genus T, Morement H, Murphy A, Rous B, Tataru D. Global trends in mortality from intrahepatic and extrahepatic cholangiocarcinoma. J Hepatol, 2019,71(6):1261-1262. |
| [3] | Khan SA, Taylor-Robinson SD, Toledano MB, Beck A, Elliott P, Thomas HC. Changing international trends in mortality rates for liver, biliary and pancreatic tumours. J Hepatol, 2002,37(6):806-813. |
| [4] | Guglielmi A, Ruzzenente A, Valdegamberi A, Bagante F, Conci S, Pinna AD, Ercolani G, Giuliante F, Capussotti L, Aldrighetti L, Iacono C. Hepatolithiasis-associated cholangiocarcinoma: results from a multi-institutional national database on a case series of 23 patients. Eur J Surg Oncol, 2014,40(5):567-575. |
| [5] | Zhang H, Yang T, Wu MC, Shen F. Intrahepatic cholangiocarcinoma: epidemiology, risk factors, diagnosis and surgical management. Cancer Lett, 2016,379(2):198-205. |
| [6] | Banales JM, Marin JJG, Lamarca A, Rodrigues PM, Khan SA, Roberts LR, Cardinale V, Carpino G, Andersen JB, Braconi C, Calvisi DF, Perugorria MJ, Fabris L, Boulter L, Macias RIR, Gaudio E, Alvaro D, Gradilone SA, Strazzabosco M, Marzioni M, Coulouarn C, Fouassier L, Raggi C, Invernizzi P, Mertens JC, Moncsek A, Rizvi S, Heimbach J, Koerkamp BG, Bruix J, Forner A, Bridgewater J, Valle JW, Gores GJ. Cholangiocarcinoma 2020: the next horizon in mechanisms and management. Nat Rev Gastroenterol Hepatol, 2020,17(9):557-588. |
| [7] | Weber SM, Ribero D, O'reilly EM, Kokudo N, Miyazaki M, Pawlik TM. Intrahepatic cholangiocarcinoma: expert consensus statement. HPB (Oxford), 2015,17(8):669-680. |
| [8] | Parker MW, Botchan MR, Berger JM. Mechanisms and regulation of DNA replication initiation in eukaryotes. Crit Rev Biochem Mol Biol, 2017,52(2):107-144. |
| [9] | Dudderidge TJ, Stoeber K, Loddo M, Atkinson G, Fanshawe T, Griffiths DF, Williams GH. Mcm2, Geminin, and KI67 define proliferative state and are prognostic markers in renal cell carcinoma. Clin Cancer Res, 2005,11(7):2510-2517. |
| [10] | Guzinska-Ustymowicz K, Pryczynicz A, Kemona A, Czyzewska J. Correlation between proliferation markers: PCNA, KI-67, MCM-2 and antiapoptotic protein Bcl-2 in colorectal cancer. Anticancer Res, 2009,29(8):3049-3052. |
| [11] | Deng MJ, Sun JJ, Xie SH, Zhen H, Wang YC, Zhong AL, Zhang HQ, Lu RQ, Guo L. Inhibition of MCM2 enhances the sensitivity of ovarian cancer cell to carboplatin. Mol Med Rep, 2019,20(3):2258-2266. |
| [12] | Seo J, Seong D, Lee SR, Oh DB, Song J. Post-translational regulation of ARF: perspective in cancer. Biomolecules, 2020,10(8):1143. |
| [13] | Mercadante A A, Kasi A. Genetics, cancer cell cycle phases. In: Statpearls, Treasure Island (FL): StatPearls Publishing, 2021. |
| [14] | Lee EW, Seong D, Song J. Cytoplasmic myc is an anti- necroptotic protein. Mol Cell Oncol, 2020,7(6):1817697. |
| [15] | Yang WX, Pan YY, You CG. CDK1, CCNB1, CDC20, BUB1, MAD2L1, MCM3, BUB1B, MCM2, and RFC4 may be potential therapeutic targets for hepatocellular carcinoma using integrated bioinformatic analysis. Biomed Res Int, 2019,2019:1245072. |
| [16] | Zhou GY, Soufan O, Ewald J, Hancock REW, Basu N, Xia JG. Networkanalyst 3.0: a visual analytics platform for comprehensive gene expression profiling and meta-analysis. Nucleic Acids Res, 2019,47(W1):W234-W241. |
| [17] | Kuleshov MV, Jones MR, Rouillard AD, Fernandez NF, Duan QN, Wang ZC, Koplev S, Jenkins SL, Jagodnik KM, Lachmann A, Mcdermott MG, Monteiro CD, Gundersen GW, Ma'ayan A. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res, 2016,44(W1):W90-W97. |
| [18] | Joerger AC, Fersht AR. The p53 pathway: origins, inactivation in cancer, and emerging therapeutic approaches. Annu Rev Biochem, 2016,85:375-404. |
| [19] | El-Diwany R, Pawlik TM, Ejaz A. Intrahepatic cholangiocarcinoma. Surg Oncol Clin N Am, 2019,28(4):587-599. |
| [20] | Stoeber K, Tlsty TD, Happerfield L, Thomas GA, Romanov S, Bobrow L, Williams ED, Williams GH. DNA replication licensing and human cell proliferation. J Cell Sci, 2001,114(Pt 11):2027-2041. |
| [21] | Kristensen TP, Maria Cherian R, Gray FC, Macneill SA. The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines. Front Microbiol, 2014,5:123. |
| [22] | Semple JW, Duncker BP. ORC-associated replication factors as biomarkers for cancer. Biotechnol Adv, 2004,22(8):621-631. |
| [23] | Cheung CHY, Hsu CL, Chen KP, Chong ST, Wu CH, Huang HC, Juan HF. MCM2-regulated functional networks in lung cancer by multi-dimensional proteomic approach. Sci Rep, 2017,7(1):13302. |
| [24] | Yang J, Xie Q, Zhou H, Chang L, Wei W, Wang Y, Li H, Deng ZX, Xiao YL, Wu JZ, Xu P, Hong XC. Proteomic analysis and NIR-II imaging of MCM2 protein in hepatocellular carcinoma. J Proteome Res, 2018,17(7):2428-2439. |
| [25] | Forsburg SL. Eukaryotic MCM proteins: beyond replication initiation. Microbiol Mol Biol Rev, 2004,68(1):109-131. |
| [26] | Snyder M, Huang XY, Zhang JJ. The minichromosome maintenance proteins 2-7 (mcm2-7) are necessary for RNA polymerase II (pol II)-mediated transcription. J Biol Chem, 2009,284(20):13466-13472. |
| [27] | Holland L, Gauthier L, Bell-Rogers P, Yankulov K. Distinct parts of minichromosome maintenance protein 2 associate with histone H3/H4 and RNA polymerase II holoenzyme. Eur J Biochem, 2002,269(21):5192-5202. |
| [28] | Yankulov K, Todorov I, Romanowski P, Licatalosi D, Cilli K, Mccracken S, Laskey R, Bentley DL. MCM proteins are associated with RNA polymerase II holoenzyme. Mol Cell Biol, 1999,19(9):6154-6163. |
| [29] | Hubbi ME, Luo WB, Baek JH, Semenza GL. MCM proteins are negative regulators of hypoxia-inducible factor 1. Mol Cell, 2011,42(5):700-712. |
| [30] | Liu LM, Zhang P, Bai M, He LJ, Zhang L, Liu T, Yang Z, Duan ML, Liu MN, Liu BJ, Du R, Qian Q, Sun SR. p53 upregulated by HIF-1α promotes hypoxia-induced G2/M arrest and renal fibrosis in vitro and in vivo. J Mol Cell Biol, 2019,11(5):371-382. |
/
| 〈 |
|
〉 |
www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:
