为了探索CCCTC结合因子(CCCTC-binding factor, CTCF)对人胆管癌细胞的生长、迁移和侵袭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CTCF的胆管癌细胞系,采用蛋白质免疫印迹法(Western blotting, Wb)检测CTCF蛋白表达水平,采用Cell Counting Kit-8(CCK-8)法和克隆形成实验检测细胞生长情况,采用Transwell小室实验检测细胞迁移和侵袭能力,采用GraphPad软件(v6)的Mann-Whitney U检验分析CTCF基因在癌和癌旁组织间的mRNA差异表达.结果显示:过表达CTCF可显著促进HCCC-9810和RBE胆管癌细胞的生长、迁移和侵袭能力;敲低CTCF呈现相反的表型.通过通路富集分析发现:CTCF的表达水平在胆管癌中与p53信号通路活性显著负相关,过表达CTCF可显著下调p53及其靶基因p21的mRNA和蛋白表达水平,而敲低CTCF则显著促进p53和p21的mRNA和蛋白表达水平.综上,本研究揭示CTCF可能通过抑制p53信号通路而促进胆管癌细胞生长、迁移和侵袭而发挥促癌基因的功能.
The study aims to explore the functions and potential molecular mechanisms of CCCTC-binding factor(CTCF)in cholangiocarcinoma(CCA)cell lines. The CCA cell lines with stable overexpression or knockdown of CTCF were obtained by lentivirus infection. Western blotting(Wb)assays were used to detect the CTCF protein expression levels.The cell growth was detected by CCK-8 assays and plated cell clone formation assays. Cell migration and invasion was detected by Transwell. Mann-Whitney- DOI:10.3969/j.issn.1000-1565.2022.06.010CTCF通过抑制p53信号通路促进胆管癌细胞的生长、迁移和侵袭闫晗1,石培培2,王亚会3,高成明3,周钢桥1,3(1.河北大学 化学与环境科学学院,河北 保定 071002; 2.河北大学 生命科学学院,河北 保定 071002;3.军事科学院军事医学研究院 辐射医学研究所,北京 100850)摘 要:为了探索CCCTC结合因子(CCCTC-binding factor, CTCF)对人胆管癌细胞的生长、迁移和侵袭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CTCF的胆管癌细胞系,采用蛋白质免疫印迹法(Western blotting, Wb)检测CTCF蛋白表达水平,采用Cell Counting Kit-8(CCK-8)法和克隆形成实验检测细胞生长情况,采用Transwell小室实验检测细胞迁移和侵袭能力,采用GraphPad软件(v6)的Mann-Whitney U检验分析CTCF基因在癌和癌旁组织间的mRNA差异表达.结果显示:过表达CTCF可显著促进HCCC-9810和RBE胆管癌细胞的生长、迁移和侵袭能力;敲低CTCF呈现相反的表型.通过通路富集分析发现:CTCF的表达水平在胆管癌中与p53信号通路活性显著负相关,过表达CTCF可显著下调p53及其靶基因p21的mRNA和蛋白表达水平,而敲低CTCF则显著促进p53和p21的mRNA和蛋白表达水平.综上,本研究揭示CTCF可能通过抑制p53信号通路而促进胆管癌细胞生长、迁移和侵袭而发挥促癌基因的功能.关键词:胆管癌;CCCTC结合因子(CTCF);细胞生长;细胞迁移;细胞侵袭;p53通路中图分类号:R735.8 文献标志码:A 文章编号:1000-1565(2022)06-0625-11CTCF promotes the growth, migration and invasion of cholangiocarcinoma cells by inhibiting the p53 signaling pathwayYAN Han1, SHI Peipei2, WANG Yahui3, GAO Chengming3, ZHOU Gangqiao1,3(1.College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China; 2.College of Life Sciences, Hebei University, Baoding 071002, China; 3. Institute of Radiation Medicine, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 100850, China)Abstract: The study aims to explore the functions and potential molecular mechanisms of CCCTC-binding factor(CTCF)in cholangiocarcinoma(CCA)cell lines. The CCA cell lines with stable overexpression or knockdown of CTCF were obtained by lentivirus infection. Western blotting(Wb)assays were used to detect the CTCF protein expression levels.The cell growth was detected by CCK-8 assays and plated cell clone formation assays. Cell migration and invasion was detected by Transwell. Mann-Whitney- 收稿日期:2022-04-13 基金项目:国家自然科学青年基金资助项目(82002573) 第一作者:闫晗(1997—),女,河南焦作人,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:yanhancinada@126.com 通信作者:周钢桥(1972—),男,北京人,军事科学院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6期闫晗等:CTCF通过抑制p53信号通路促进胆管癌细胞的生长、迁移和侵袭U test of GraphPad software(v6)was used to analyze the mRNA differential expression of CTCF gene between cancer and adjacent tissues. The results show that overexpression of CTCF significantly promoted the growth, migration and invasion of HCCC-9810 and RBE cells, whereas knockdown of CTCF exhibited the opposite effects. Through pathway enrichment analysis, it was found that the expression level of CTCF was significantly negatively correlated with the activity of p53 signaling pathway in cholangiocarcinoma.Overexpression of CTCF significantly reduced the expression of p53 and its target gene p21, while knockdown of CTCF showed the opposite effects. In conclusion, this study revealed that CTCF function as an oncogene by inhibiting the p53 signaling pathway to promote the growth, migration and invasion of CCA cells.
[1] RONNEKLEIV-KELLY S M, PAWLIK T M. Staging of intrahepatic cholangiocarcinoma[J]. Hepatobiliary Surg Nutr, 2017, 6(1): 35-43. DOI:10.21037/hbsn.2016.10.02.
[2] SIRICA A E. Cholangiocarcinoma: Molecular targeting strategies for chemoprevention and therapy[J]. Hepatology, 2005, 41(1): 5-15. DOI:10.1002/hep.20537.
[3] LI H, CHEN L, ZHU G Y, et al. Interventional treatment for cholangiocarcinoma[J]. Front Oncol, 2021, 11: 671327. DOI:10.3389/fonc.2021.671327.
[4] TYSON G L, EL-SERAG H B. Risk factors for cholangiocarcinoma[J]. Hepatology, 2011, 54(1): 173-184. DOI:10.1002/hep.24351.
[5] BRIDGEWATER J, GALLE P R, KHAN S A, et al. Guidelines for the diagnosis and management of intrahepatic cholangiocarcinoma[J]. J Hepatol, 2014, 60(6): 1268-1289. DOI:10.1016/j.jhep.2014.01.021.
[6] LOBANENKOV V V, NICOLAS R H, ADLER V V, et al. A novel sequence-specific DNA binding protein which interacts with three regularly spaced direct repeats of the CCCTC-motif in the 5'-flanking sequence of the chicken c-myc gene[J]. Oncogene, 1990, 5(12): 1743-1753.
[7] KLENOVA E M, NICOLAS R H, PATERSON H F, et al. CTCF, a conserved nuclear factor required for optimal transcriptional activity of the chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in multiple forms[J]. Mol Cell Biol, 1993, 13(12): 7612-7624. DOI:10.1128/mcb.13.12.7612-7624.1993.
[8] SONG S H, KIM T Y. CTCF, cohesin, and chromatin in human cancer[J]. Genomics Inform, 2017, 15(4): 114-122. DOI:10.5808/gi.2017.15.4.114.
[9] LEE J S, RAJA P, PAN D, et al. CCCTC-binding factor acts as a heterochromatin barrier on herpes simplex viral latent chromatin and contributes to poised latent infection[J]. mBio, 2018, 9(1): e02372-e02317. DOI:10.1128/mbio.02372-17.
[10] KIM T G, KIM S, JUNG S, et al. CCCTC-binding factor is essential to the maintenance and quiescence of hematopoietic stem cells in mice[J]. Exp Mol Med, 2017, 49(8): e371. DOI:10.1038/emm.2017.124.
[11] KIM S, YU N K, KAANG B K. CTCF as a multifunctional protein in genome regulation and gene expression[J]. Exp Mol Med, 2015, 47(6): e166. DOI:10.1038/emm.2015.33.
[12] BURKE L J, ZHANG R, LUTZ M, et al. The thyroid hormone receptor and the insulator protein CTCF: two different factors with overlapping functions[J]. J Steroid Biochem Mol Biol, 2002, 83(1-5): 49-57. DOI:10.1016/S0960-0760(02)00256-X.
[13] FARRELL C M, WEST A G, FELSENFELD G. Conserved CTCF insulator elements flank the mouse and human beta-globin loci[J]. Mol Cell Biol, 2002, 22(11): 3820-3831. DOI:10.1128/mcb.22.11.3820-3831.2002.
[14] OH S, OH C, YOO K H. Functional roles of CTCF in breast cancer[J]. BMB Rep, 2017, 50(9): 445-453. DOI:10.5483/bmbrep.2017.50.9.108.
[15] LOUKINOV D I, PUGACHEVA E, VATOLIN S, et al. BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma[J]. Proc Natl Acad Sci U S A. 2002, 99(10):6806-6811.
[16] RECILLAS-TARGA F, PIKAART M J, BURGESS-BEUSSE B, et al. Position-effect protection and enhancer blocking by the chicken beta-globin insulator are separable activities[J]. PNAS, 2002, 99(10): 6883-6888. DOI:10.1073/pnas.102179399.
[17] YU X Q, LIU Q Y, HE J Y, et al. Vigilin interacts with CTCF and is involved in the maintenance of imprinting of IGF2 through a novel RNA-mediated mechanism[J]. Int J Biol Macromol, 2018, 108: 515-522. DOI:10.1016/j.ijbiomac.2017.11.109.
[18] HOLWERDA S J, DE LAAT W. CTCF: the protein, the binding partners, the binding sites and their chromatin loops[J]. Philos Trans R Soc Lond B Biol Sci, 2013, 368(1620): 20120369. DOI:10.1098/rstb.2012.0369.
[19] 吴洁,李鹏昌,庞钧译,等.CTCF在乳腺癌中的表达及其对细胞增殖的影响[J]. 基础医学与临床, 2015, 35(2): 157-161. DOI:10.16352/j.issn.1001-6325.2015.02.004.
[20] FILIPPOVA G N, LINDBLOM A, MEINCKE L J, et al. A widely expressed transcription factor with multiple DNA sequence specificity, CTCF, is localized at chromosome segment 16q22.1 within one of the smallest regions of overlap for common deletions in breast and prostate cancers[J]. Genes Chromosom Cancer, 1998, 22(1): 26-36. DOI:10.1002/(SICI)1098-2264(199805)22: 126: AID-GCC4>3.0.CO;2-9.
[21] ZHANG B, ZHANG Y J, ZOU X P, et al. The CCCTC-binding factor(CTCF)-forkhead box protein M1 axis regulates tumour growth and metastasis in hepatocellular carcinoma[J]. J Pathol, 2017, 243(4): 418-430. DOI:10.1002/path.4976.
[22] LAI Q, LI Q, HE C, et al. CTCF promotes colorectal cancer cell proliferation and chemotherapy resistance to 5-FU via the P53-Hedgehog axis[J]. Aging(Albany NY), 2020, 12(16): 16270-16293. DOI:10.18632/aging.103648.
[23] RAZUMILAVA N, GORES G J. Cholangiocarcinoma[J]. Lancet, 2014, 383(9935): 2168-2179. DOI:10.1016/S0140-6736(13)61903-0.
[24] SATO K, BAIOCCHI L, KENNEDY L, et al. Current advances in basic and translational research of cholangiocarcinoma[J]. Cancers(Basel), 2021, 13(13): 3307. DOI:10.3390/cancers13133307.
[25] DEBAUGNY R E, SKOK J A. CTCF and CTCFL in cancer[J]. Curr Opin Genet Dev, 2020, 61: 44-52. DOI:10.1016/j.gde.2020.02.021.
[26] WU J, LI P C, PANG J Y, et al. CCCTC-binding factor inhibits breast cancer cell proliferation and metastasis via inactivation of the nuclear factor-kappaB pathway[J]. Oncotarget, 2017, 8(55): 93516-93529. DOI:10.18632/oncotarget.18977.
[27] AUBREY B J, STRASSER A, KELLY G L. Tumor-suppressor functions of the TP53 pathway[J]. Cold Spring Harb Perspect Med, 2016, 6(5): a026062. DOI:10.1101/cshperspect.a026062. (
