NUDT9 promotes the proliferation of malignant tumors by stabilizing MCM3 protein expression

doi:10.3969/j.issn.1009-976X.2023.06.010

Abstract

Abstract: Objective To explore the molecular mechanism of ADP-ribose pyrophosphatase NUDT9 inpromoting tumor proliferation. Methods The expression level of NUDT9 in various tumors was determined by analyzing GEPIA2 database. The protein expression was detected by Western blot. The proliferation rate capability was measured by colony forming assay. The cell cycle was detected by flow cytometry analysis. The proportion of cells in S phasewas detected by EdUstaining assay. Ectopic expression of SFB-NUDT9 constructs combined with coimmunoprecipitation （Co-IP） assayand quantitative mass spectrometry were used to identify the interacting proteins of NUDT9. Two sgRNA and siRNA were designed to silence NUDT9 gene expression in tumor cells, and Western blot was used to detect NUDT9 protein expression level and its effect on the stability of proliferation-related proteins. Results Analysis of GEPIA2 database showed that NUDT9 was highly expressed in various tumor cells. The expression of NUDT9was silenced efficiently by two individual sgRNA and siRNA respectively. Clonal formation assay, flow cytometry analysisand EdU proliferation experiments indicated that NUDT9 knockdownsignificantly inhibited the cell proliferation and S phase progression of cancer cells. Co-IP assay further demonstrated that NUDT9 interacted with cell proliferation related MCM2-7 complex, Moreover, NUDT9 knockdown obviously decreased the protein levels of MCM3, while no significant changes were observed in other components of MCM complex. Both clonal formation and EdU proliferation experiments showed that MCM3 knockdown greatly inhibited the proliferation ability as well as the proportion of cells in S-phase. Conclusion NUDT9 accelerated S phase progressions by regulating the protein expression of MCM3,suggesting that it plays an important role in modulating the proliferation capability of tumor cells.

Key words: NUDT9, tumor proliferation, DNA replication, cell cycle, MCM3

摘要： 目的探讨ADP-核糖焦磷酸酶NUDT9促进肿瘤增殖的分子机制。方法分析GEPIA2数据库中NUDT9在多种肿瘤中的表达水平;利用Western Blot免疫印迹法检测蛋白表达水平;通过克隆形成数量检测细胞增殖速度;使用流式细胞术检测细胞周期变化;EdU染色实验检测S期细胞比例;转染外源质粒SFB-NUDT9联合免疫共沉淀法和定量质谱技术鉴定NUDT9的相互作用蛋白;分别设计两条sgRNA和siRNA在肿瘤细胞中沉默NUDT9基因的表达,结合免疫印迹法检测NUDT9蛋白表达水平及其对细胞增殖相关蛋白表达水平的影响。结果 GEPIA2数据库显示NUDT9在多种肿瘤细胞中高表达;利用CRISPR和小干扰RNA技术均有效沉默NUDT9基因的表达;克隆形成、流式细胞术及EdU增殖实验均表明敲低NUDT9后抑制癌细胞的增殖及G1/S期转换;转染外源质粒SFB-NUDT9结合免疫沉淀法和定量质谱技术鉴定出与细胞增殖相关的NUDT9互作蛋白MCM2-7;通过转染siRNA沉默NUDT9再结合免疫印迹法,发现NUDT9的敲低影响MCM复合物中MCM3的蛋白表达水平;最后转染siRNA沉默MCM3基因的表达,克隆形成和EdU增殖实验均显示MCM3的敲低影响肿瘤细胞的增殖,S期细胞比例减少。结论 NUDT9通过调控MCM3的蛋白表达,促进S期细胞周期进程,对肿瘤细胞的增殖过程起到非常重要的作用。

关键词: NUDT9, 肿瘤增殖, DNA复制, 细胞周期, MCM3

CLC Number:

R73-37

ZHUANG Shu-ting, HU Kai-shun. NUDT9 promotes the proliferation of malignant tumors by stabilizing MCM3 protein expression[J]. Lingnan Modern Clinics In Surgery, 2023, 23(6): 504-513.

庄淑婷, 胡开顺. NUDT9通过稳定MCM3的蛋白表达促进恶性肿瘤增殖[J]. 岭南现代临床外科, 2023, 23(6): 504-513.

References

[1] Roy PS, Saikia BJ.Cancer and cure: A critical analysis[J]. Indian J Cancer, 2016, 53(3): 441-442.
[2] 郑荣寿, 张思维, 孙可欣, 等. 2016年中国恶性肿瘤流行情况分析[J]. 中华肿瘤杂志, 2023, 212-220.
[3] Maomao C, He L, Dianqin S, et al.Current cancer burden in China: epidemiology, etiology, and prevention[J]. Cancer Biol Med, 2022, 19(8): 1121-1138.
[4] Siegel RL, Miller KD, Wagle NS, Jemal A.Cancer statistics, 2023[J]. CA Cancer J Clin, 2023, 73(1): 17-48.
[5] Cheng Z, Li M, Dey R, Chen Y.Nanomaterials for cancer therapy: current progress and perspectives[J]. J Hematol Oncol, 2021, 14(1): 85.
[6] Wang S, Osgood AO, Chatterjee A.Uncovering post-translational modification-associated protein-protein interactions[J]. Curr Opin Struct Biol, 2022, 74: 102352.
[7] Chen L, Liu S, Tao Y.Regulating tumor suppressor genes: post-translational modifications[J]. Signal Transduct Target Ther, 2020, 5(1): 90.
[8] Tan S, Zhao J, Wang P.DYRK1A-mediated PLK2 phosphorylation regulates the proliferation and invasion of glioblastoma cells[J]. Int J Oncol, 2023, 63(2): 94.
[9] He W, Li Q, Li X.Acetyl-CoA regulates lipid metabolism and histone acetylation modification in cancer[J]. Biochim Biophys Acta Rev Cancer, 2023, 1878(1): 188837.
[10] Alemasova EE, Lavrik OI.Poly(ADP-ribosyl)ation by PARP1: reaction mechanism and regulatory proteins[J]. Nucleic Acids Res, 2019, 47(8): 3811-3827.
[11] O′Sullivan J, Tedim Ferreira M, Gagné JP, et al. Emerging roles of eraser enzymes in the dynamic control of protein ADP-ribosylation[J]. Nat Commun, 2019, 10(1): 1182.
[12] Benjamin RC, Gill DM.ADP-ribosylation in mammalian cell ghosts. Dependence of poly(ADP-ribose) synthesis on strand breakage in DNA[J]. J Biol Chem, 1980, 255(21): 10493-10501.
[13] Harrision D, Gravells P, Thompson R, Bryant HE.Poly(ADP-Ribose) Glycohydrolase (PARG) vs. Poly(ADP-Ribose) Polymerase (PARP) - Function in Genome Maintenance and Relevance of Inhibitors for Anti-cancer Therapy[J]. Front Mol Biosci, 2020, 7: 191.
[14] Fu H, Liu R, Jia Z, et al.Poly(ADP-ribosylation) of P-TEFb by PARP1 disrupts phase separation to inhibit global transcription after DNA damage[J]. Nat Cell Biol, 2022, 24(4): 513-525.
[15] Menzel S, Koudelka T, Rissiek B, et al.ADP-Ribosylation Regulates the Signaling Function of IFN-γ[J]. Front Immunol, 2021, 12: 642545.
[16] Rosado MM, Pioli C.ADP-ribosylation in evasion, promotion and exacerbation of immune responses[J]. Immunology, 2021, 164(1): 15-30.
[17] Li P, Lei Y, Qi J, et al.Functional roles of ADP-ribosylation writers, readers and erasers[J]. Front Cell Dev Biol, 2022, 10: 941356.
[18] Daniels CM, Thirawatananond P, Ong SE, et al.Nudix hydrolases degrade protein-conjugated ADP-ribose[J]. Sci Rep, 2015, 5: 18271.
[19] Palazzo L, Thomas B, Jemth AS, et al.Processing of protein ADP-ribosylation by Nudix hydrolases[J]. Biochem J, 2015, 468(2): 293-301.
[20] Chen LY, Chen TH, Wen PY, et al.Differential expression of NUDT9 at different phases of the menstrual cycle and in different components of normal and neoplastic human endometrium[J]. Taiwan J Obstet Gynecol, 2009, 48(2): 96-107.
[21] Kennedy CH, Cueto R, Belinsky SA, et al.Overexpression of hMTH1 mRNA: a molecular marker of oxidative stress in lung cancer cells[J]. FEBS Lett, 1998, 429(1): 17-20.
[22] Yoon B, Yang EG, Kim SY.The ADP-ribose reactive NUDIX hydrolase isoforms can modulate HIF-1α in cancer cells[J]. Biochem Biophys Res Commun, 2018, 504(1): 321-327.
[23] Wang Y, Chen H, Zhang J, et al.MCM family in gastrointestinal cancer and other malignancies: From functional characterization to clinical implication[J]. Biochim Biophys Acta Rev Cancer, 2020, 1874(2): 188415.
[24] Nowińska K, Dzięgiel P.[The role of MCM proteins in cell proliferation and tumorigenesis][J]. Postepy Hig Med Dosw (Online), 2010, 64: 627-635.
[25] Hyrien O.How MCM loading and spreading specify eukaryotic DNA replication initiation sites[J]. F1000Res, 2016, 5.
[26] Najar MA, Aravind A, Dagamajalu S, et al.Hyper activation of MEK/ERK pathway by Ca(2+)/calmodulin-dependent protein kinase kinase 2 promotes cellular proliferation by activating cyclin-dependent kinases and minichromosome maintenance protein in gastric cancer cells[J]. Mol Carcinog, 2021, 60(11): 769-783.
[27] Baxley RM, Bielinsky AK.Mcm10: A Dynamic Scaffold at Eukaryotic Replication Forks[J]. Genes (Basel), 2017, 8(2): 73.
[28] Alver RC, Zhang T, Josephrajan A, et al.The N-terminus of Mcm10 is important for interaction with the 9-1-1 clamp and in resistance to DNA damage[J]. Nucleic Acids Res, 2014, 42(13): 8389-8404.
[29] Lutzmann M, Bernex F, da Costa de Jesus C, et al. MCM8- and MCM9 Deficiencies Cause Lifelong Increased Hematopoietic DNA Damage Driving p53-Dependent Myeloid Tumors [J]. Cell Rep, 2019, 28(11): 2851-2865.e2854.