The effects of integrator complex subunits 10(INTS10)on cell cycle,apoptosis,growth and migration of hepatocellular carcinoma(HCC)cells and its potential molecular mechanism were explored. The HCC cell lines with stable overexpression or knockdown of INTS10 were obtained by lentivirus infection. The mRNA and protein expression levels of INTS10 were detected by qRT-PCR and Western blotting. The cell growth was detected by CCK-8, clonal formation and BrdU assay. The cell migration ability was detected by Transwell assay. Flow cytometry was used to detect the number of cell cycles and apoptosis. The results showed that overexpression of INTS10 could significantly inhibit the apoptosis, growth- DOI:10.3969/j.issn.1000-1565.2024.03.008INTS10对HCC细胞周期、凋亡、生长和迁移能力的影响王雪婷1,2,齐欣3,魏小军4,杨爱清2,周钢桥1,2,3(1.河北大学 化学与材料科学学院,河北 保定 071002;2.军事医学研究院 辐射医学研究所,北京 100850;3.贵州大学 医学院,贵州 贵阳 550025;4.航天中心医院 肝胆外科,北京 100049)摘 要:为了探究整合因子复合物亚基10(integrator complex subunits 10,INTS10 )对人肝细胞癌(hepatocellular carcinoma, HCC)细胞周期、凋亡、生长和迁移能力的影响及其潜在的分子作用机制,利用慢病毒感染法获得稳定过表达或敲低INTS10的HCC细胞系,采用qRT-PCR和Western blotting检测INTS10 mRNA和蛋白表达水平,接着采用 CCK-8法、克隆形成和BrdU实验检测细胞生长情况,采用 Transwell小室实验检测细胞迁移能力,采用流式分析术检测细胞的周期和凋亡.结果显示:过表达INTS10可显著抑制HCC细胞的凋亡、生长和迁移能力,促进G1期细胞数量的增加,而敲低INTS10则呈现相反的表型.通过通路富集分析发现,周期相关通路被显著富集,过表达INTS10后,CDC25A和CDK4的mRNA和蛋白质水平显著减少,而CDKN1A的水平显著增加,敲低INTS10则呈现相反趋势.综上,本研究初步揭示了INTS10在HCC细胞中可能通过影响G1/S期相关蛋白质的表达而发挥抑癌基因的功能,为下一步更为深入的功能和机制研究提供了基础.关键词:肝细胞癌(HCC);整合因子复合物亚基10(INTS10);CDC25A;CDKN1A;CDK4中图分类号:Q279 文献标志码:A 文章编号:1000-1565(2024)03-0290-11Effect of INTS10 on cell cycle, apoptosis, growth and migration capacity of HCC WANG Xueting1,2, QI Xin3, WEI Xiaojun4, YANG Aiqing2, ZHOU Gangqiao1,2,3(1. College of Chemistry and Materials Science, Hebei University, Baoding 071002, China; 2. Institute of Radiation Medicine,Academy of Military Medical Sciences, Beijing 100850, China; 3. Medical College, Guizhou University, Guiyang 550025, China; 4. Department of Hepatobiliary Surgery,Space Center Hospital, Beijing 100049,China)Abstract: The effects of integrator complex subunits 10(INTS10)on cell cycle,apoptosis,growth and migration of hepatocellular carcinoma(HCC)cells and its potential molecular mechanism were explored. The HCC cell lines with stable overexpression or knockdown of INTS10 were obtained by lentivirus infection. The mRNA and protein expression levels of INTS10 were detected by qRT-PCR and Western blotting. The cell growth was detected by CCK-8, clonal formation and BrdU assay. The cell migration ability was detected by Transwell assay. Flow cytometry was used to detect the number of cell cycles and apoptosis. The results showed that overexpression of INTS10 could significantly inhibit the apoptosis, growth- 收稿日期:2023-05-11;修回日期:2023-06-15 基金项目:国家自然科学基金青年基金资助项目( 8190061543) 第一作者:王雪婷(1998—),女,河北大学硕士研究生,主要从事遗传学和整合组学研究.E-mail:1379402926@qq.com 通信作者:周钢桥(1972—),男,军事医学研究院研究员,主要从事遗传学和整合组学研究.E-mail:zhougq114@126.com第3期王雪婷等:INTS10对HCC细胞周期、凋亡、生长和迁移能力的影响and migration of HCC, and promote the increase of the number of G1 phase cells, while knockdown of INTS10 showed the opposite effects. Through pathway enrichment analysis, it was found that the cycle-dependent pathway was significantly enriched. Overexpression of INTS10 could significantly reduce the mRNA and protein levels of CDC25A and CDK4, while the level of CDKN1A was significantly increased, and INTS10 knockdown showed the opposite trend. In conclusion, it is preliminarily revealed that INTS10 may play a role as a tumor suppressor gene in HCC by affecting the expression of G1/S phase related proteins, which provides a foundation for further functional and mechanism studies.
[1] HOLVOET T, RAEVENS S, VANDEWYNCKEL Y P, et al. Systematic review of guidelines for management of intermediate hepatocellular carcinoma using the appraisal of guidelines research and evaluation Ⅱ instrument[J]. Dig Liver Dis, 2015, 47(10): 877-883. DOI: 10.1016/j.dld.2015.07.005.
[2] GILLES H, GARBUTT T, LANDRUM J. Hepatocellular carcinoma[J]. Crit Care Nurs Clin N Am, 2022, 34(3): 289-301. DOI: 10.1016/j.cnc.2022.04.004.
[3] LUO P, WU S Y, YU Y L, et al. Current status and perspective biomarkers in AFP negative HCC: towards screening for and diagnosing hepatocellular carcinoma at an earlier sage[J]. Pathol Oncol Res, 2020, 26(2): 599-603. DOI: 10.1007/s12253-019-00585-5.
[4] WANG W Y, WEI C. Advances in the early diagnosis of hepatocellular carcinoma[J]. Genes Dis, 2020, 7(3): 308-319. DOI: 10.1016/j.gendis.2020.01.014.
[5] LEE J S, LIM T S, LEE H W, et al. Suboptimal performance of hepatocellular carcinoma prediction models in patients with hepatitis B virus-related cirrhosis[J]. Diagnostics, 2022, 13(1): 3. DOI: 10.3390/diagnostics13010003.
[6] MARENGO A, ROSSO C, BUGIANESI E. Liver cancer: connections with obesity, fatty liver, and cirrhosis[J]. Annu Rev Med, 2016, 67: 103-117. DOI: 10.1146/annurev-med-090514-013832.
[7] GONG C W, AI J Y, FAN Y, et al. NCAPG promotes the proliferation of hepatocellular carcinoma through PI3K/AKT signaling[J]. Onco Targets Ther, 2019, 12: 8537-8552.. DOI: 10.2147/OTT.S217916
[8] 思兰兰. INTS10蛋白抑制乙型肝炎病毒复制的分子机制的初步研究[D].南宁:广西医科大学, 2015. DOI: 10.7666/d.Y2876795
[9] LUI K Y, ZHAO H, QIU C H, et al. Integrator complex subunit 6(INTS6)inhibits hepatocellular carcinoma growth by Wnt pathway and serve as a prognostic marker[J]. BMC Cancer, 2017, 17(1): 644. DOI: 10.1186/s12885-017-3628-3.
[10] LI Z B, ZHU P W, WANG M L, et al. Correlation between oncogene integrator complex subunit 7 and a poor prognosis in lung adenocarcinoma[J]. J Thorac Dis, 2022, 14(12): 4815-4827. DOI: 10.21037/jtd-22-1533.
[11] ELENA S, MOHINI L, ANNAMIL A T, et al. NACK and INTEGRATOR act coordinately to activate Notch-mediated transcription in tumorigenesis[J]. Cell Commun Signal, 2021, 19(1): 96. DOI:10.1186/s12964-021-00776-1.
[12] INAGAKI Y, YASUI K, ENDO M, et al. CREB3L4, INTS3, and SNAPAP are targets for the 1q21 amplicon frequently detected in hepatocellular carcinoma[J]. Cancer Genet Cytogenet, 2008, 180(1): 30-36. DOI: 10.1016/j.cancergencyto.2007.09.013.
[13] JUNG H M, CHOI S J, KIM J K. Expression profiles of SV40-immortalization-associated genes upregulated in various human cancers[J]. J Cell Biochem, 2009, 106(4): 703-713. DOI: 10.1002/jcb.22063.
[14] SIMPSON H M, KHAN R Z, SONG C, et al. Concurrent Mutations in ATM and genes associated with common γ chain signaling in peripheral T cell lymphoma[J]. PLoS One, 2015, 10(11): e0141906. DOI: 10.1371/journal.pone.0141906.
[15] LI Y F, SI L L, ZHAI Y, et al. Genome-wide association study identifies 8p21.3 associated with persistent hepatitis B virus infection among Chinese[J].Nat Commun, 2016, 7: 11664. DOI: 10.1038/ncomms11664.
[16] 张静,程敏,金倩,等.多聚嘧啶区结合蛋白1通过调控基因的可变剪接促进胆管癌细胞的生长、迁移及侵袭能力[J].中国生物化学与分子生物学报, 2022, 38(7): 899-910. DOI:10.13865/j.cnki.cjbmb.2022.06.1058.
[17] 霍励耘,魏英城,谭文亮,等.SMC基因家族在肝癌中的表达及其预测肝癌预后的临床意义[J].岭南现代临床外科, 2021, 21(1): 24-28. DOI: 10.3969/j.issn.1009-976X.2021.01.005.
[18] 马孟杰,彭小忠,舒鹏程.COMPASS核心成员Ash2l通过调控细胞周期影响神经祖细胞增殖[J].中国生物化学与分子生物学报, 2022, 38(6): 742-748. DOI: 10.13865/j.cnki.cjbmb.2022.05.1090.
[19] KREIS N N, LOUWEN F, YUAN J P. The multifaceted p21(Cip1/Waf1/CDKN1A)in cell differentiation, migration and cancer therapy[J]. Cancers, 2019, 11(9): 1220. DOI: 10.3390/cancers11091220.
[20] CHANDRASEKARAN A P, SURESH B, SARODAYA N, et al. Ubiquitin specific protease 29 functions as an oncogene promoting tumorigenesis in colorectal carcinoma[J]. Cancers, 2021, 13(11): 2706. DOI: 10.3390/cancers13112706.
[21] SHEN T, HUANG S L. The role of Cdc25A in the regulation of cell proliferation and apoptosis[J]. Anticancer Agents Med Chem, 2012, 12(6): 631-639. DOI: 10.2174/187152012800617678.
[22] WANG C, ZENG J, LI L J, et al. Cdc25A inhibits autophagy-mediated ferroptosis by upregulating ErbB2 through PKM2 dephosphorylation in cervical cancer cells[J]. Cell Death Dis, 2021, 12(11): 1055. DOI: 10.1038/s41419-021-04342-y.
[23] KAYA Z, KARAN B M, ALMAL1 N. CDKN1A(p21 gene)polymorphisms correlates with age in esophageal cancer[J]. Mol Biol Rep, 2022, 49(1): 249-258. DOI: 10.1007/s11033-021-06865-1.
[24] HAN J, ZHANG Z X. Screening of pyroptosis-related genes influencing the therapeutic effect of dehydroabietic acid in liver cancer and construction of a survival nomogram[J]. Biochem Biophys Res Commun, 2021, 585: 103-110. DOI: 10.1016/j.bbrc.2021.11.027.
[25] TANG Z P, ZHAO P, ZHANG W X, et al. SALL4 activates PI3K/AKT signaling pathway through targeting PTEN, thus facilitating migration, invasion and proliferation of hepatocellular carcinoma cells[J]. Aging, 2022, 14(24): 10081-10092. DOI: 10.18632/aging.204446.
[26] SUN J F, HU J Y, WANG G J, et al. RETRACTED ARTICLE: LncRNA TUG1 promoted KIAA1199 expression via miR-600 to accelerate cell metastasis and epithelial-mesenchymal transition in colorectal cancer[J]. J Exp Clin Cancer Res, 2018, 37(1): 1-11. DOI: 10.1186/s13046-018-0771-x.
[27] PAWLOTSKY J M, NEGRO F, AGHEMO A, et al. EASL recommendations on treatment of hepatitis C: Final update of the series[J]. J Hepatol, 2020, 73(5): 1170-1218. DOI: 10.1016/j.jhep.2020.08.018.
[28] DOS SANTOS W, DOS REIS M B, PORTO J, et al. Somatic targeted mutation profiling of colorectal cancer precursor lesions[J]. BMC Med Genom, 2022, 15(1): 143. DOI: 10.1186/s12920-022-01294-w.
[29] FEDERICO A, RIENZO M, ABBONDANZA C, et al. Pan-cancer mutational and transcriptional analysis of the integrator complex[J]. Int J Mol Sci, 2017, 18(5): 936. DOI: 10.3390/ijms18050936.
[30] FILLEUR S, HIRSCH J, WILLE A, et al. INTS6/DICE1 inhibits growth of human androgen-independent prostate cancer cells by altering the cell cycle profile and Wnt signaling[J]. Cancer Cell Int, 2009, 9(1): 1-9. DOI: 10.1186/1475-2867-9-28.
[31] PFLEIDERER M M, GALEJ W P. Emerging insights into the function and structure of the Integrator complex[J]. Transcription, 2021, 12(5): 251-265. DOI: 10.1080/21541264.2022.2047583.
[32] WILLIAMS L H, FROMM G, GOKEY N G, et al. Pausing of RNA polymerase Ⅱ regulates mammalian developmental potential through control of signaling networks[J]. Mol Cell, 2015, 58(2): 311-322. DOI: 10.1016/j.molcel.2015.02.003.
[33] SABATH K, STÄUBLI M L, MARTI S, et al. INTS10-INTS13-INTS14 form a functional module of Integrator that binds nucleic acids and the cleavage module[J]. Nat Commun, 2020, 11: 3422. DOI: 10.1038/s41467-020-17232-2. (
