CDX2基因通过NF-κB信号通路抑制结肠癌细胞增殖

doi:10.11904/j.issn.1002-3070.2018.05.003

摘要/Abstract

摘要： 目的观察过表达CDX2基因对人结肠癌细胞株增殖影响,并初步探讨其作用机制。方法通过基因转染技术将CDX2基因转染入结肠癌HT-29细胞株,经G418筛选后获得稳定转染细胞株,RT-PCR和Western blot检测CDX2基因表达。采用四甲基偶氮唑盐(MTT)法、流式细胞仪分析转染细胞的生物学行为。Western blot检测CDX2过表达对HT-29细胞内Cyclin D1和NF-κB磷酸化水平的影响。结果经脂质体转染和筛选,建立了稳定过表达CDX2人结肠癌HT-29细胞株。转染CDX2组与未转染组和空白质粒组相比,细胞的生长速度减慢(P＜0.05),细胞周期中G₁/G₀期比例增加(P＜0.05),而S期比例则减少(P＜0.05);与未转染组和空白质粒组比较,转染CDX2基因组HT-29细胞的Cyclin D1和p-NF-κB活性明显降低。结论 CDX2基因可能通过抑制NF-κB通路抑制Cyclin D1活性,从而抑制人结肠癌HT-29细胞增殖。

关键词: 结肠癌, 细胞增殖, CDX2, NF-κB

Abstract: Objective The aim of this study was to observe the effects of overexpressing CDX2 gene on proliferation of human colon cancer HT-29 cell line and to explore its mechanism.Methods CDX2 was transfected into HT-29 cells by gene transfection technology,and stable transfected cell clones with stably expressing CDX2 gene were obtained by G418 selection.The expression of CDX2 was detected by RT-PCR and Western blotting.The biological behavior of transfected cells were analyzed by methythiazoletertraolium(MTT)assay and flow cytometry.Western blot assay was used to detect the expression of cyclin D1 and NF-κB phosphorylation level in HT-29 cells with overexpressing CDX2 gene.Results A system for stably overexpressing CDX2 human colon cancer HT-29 cell line was established.Compared with the nontransfected and empty vector transfected HT-29 cells,the growth rate of the cells in the transfected CDX2 group was significantly slower(P＜0.05).The proportion of G₁/G₀ phase of cell cycle was significantly increased(P＜0.05),and the proportion of S phase was significantly decreased(P＜0.05).Compared with the non-transfected and empty vector transfected cells,the expression of cyclin D1 and p-NF-κB activity were significant low in overexpressing CDX2 cells.Conclusion CDX2 gene may inhibit the proliferation of HT-29 cells through blocking the activity of NF-κB pathway and down-regulation of cyclin D1 protein expression.

Key words: Colorectal carcinoma, Proliferation, CDX2, NF-κB

中图分类号:

R735.3

陈鳞, 王恩湘, 刘群友, 钟鹰, 周国良, 赵喜雁. CDX2基因通过NF-κB信号通路抑制结肠癌细胞增殖[J]. 实用肿瘤学杂志, 2018, 32(5): 398-403.

CHEN Lin, WANG Enxiang, LIU Qunyou, ZHONG Ying, ZHOU Guoliang, ZHAO Xiyan. CDX2 gene inhibits of colon cancer cell proliferation by NF-κB signaling pathway[J]. PRACTICAL ONCOLOGY JOURNAL, 2018, 32(5): 398-403.

参考文献

1 Chen W,Zheng R,Baade PD,et al.Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66(2):115-132.
2 Eda A,Osawa H,Yanaka I,et al.Expression of homeobox gene CDX2 precedes that of CDX1 during the progression of intestinal metaplasia[J].J Gastroenterol,2002,37(2):94-100.
3 Shah SS,Wu TT,Torbenson MS,et al.Aberrant CDX2 expression in hepatocellular carcinomas:an important diagnostic pitfall[J].Hum Pathol,2017,64:13-18.
4 Masood MA,Loya A,Yusuf MA.CDX2 as a prognostic marker in gastric cancer[J].Acta Gastroenterol Belg,2016,79(2):197-200.
5 Fang YH,Lai MD.CDX2,a new marker for colorectal cancer[J].International Journal of Genetics,2006,29(4):311-316.
6 Blache P,van de Wetering M,Duluc I,et al.SOX 9 is an intestine crypt transcription factor,is regulated by the Wnt pathway,and represses the CDX2 and MUC 2genes[J].J Cell Biol,2004,166(1):37-47.
7 Kawai H,Tomii K,Toyooka S,et al.Promoter methylation downregulates CDX2 expression in colorectal carcinomas[J].Oncol Rep,2005,13(3):547-551.
8 Yamamoto H,Bai YQ,Yuasa Y.Homeodomain protein CDX 2 regulates goblet-specific gene expression[J].Biochem Biophys Res Commun,2003,300(4):813-818.
9 Subtil C,Guérin E,Schneider A et al.Frequent rearrangements and amplification of the CDX2 homeobox gene in human sporadic colorectal cancers with chromosomal instability[J].Cancer Lett,2007,247(2):197-203.
10 杨周亮,应伟奡,胡清涛.CDX2、PTEN在结直肠癌中的表达及其与Ki67相关分析[J].实用肿瘤学杂志,2007,21(1):13-15.
11 Li XG,Xu GF,Zhai ZY,et al.CDX2 increases SLC7A7 expression and proliferation of pig intestinal epithelial cells[J].Oncotarget,2016,7(21):30597-30609.
12 Aranha MM,Borralho PM,Ravasco P,et al.NF-kappaB and apoptosis in colorectal tumourigenesis[J].Eur J Clin Invest,2007,37(5):416-424.
13 Yamamoto Y,Gaynor RB.IkappaB kinases:key regulators of the NF-kappaB pathway[J].Trends Biochem Sci,2004,29(2):72-79.
14 Chen BJ,Zeng S,Xie R,et al.hTERT promotes gastric intestinal metaplasia by upregulating CDX2 via NF-κB signaling pathway[J].Oncotarget,2017,8(16):26969-26978.
15 He S,Sun XJ,Zheng JB,et al.Recombinant lentivirus with enhanced expression of caudal-related homeobox protein 2 inhibits human colorectal cancer cell proliferation in vitro[J].Mol Med Rep,2015,12(2):1838-1844.
16 Karin M.NF-kappaB and cancer:mechanisms and targets[J].Mol Carcinog,2006,45(6):355-361.
17 Karin M,Ben-Neriah Y.Phosphorylation meets ubiquitination:the control of NF-kappa B activity[J].Annu Rev Immunol,2000,18:621-663.
18 Park SY,Shin JH,Kee SH.E-cadherin expression increases cell proliferation by regulating energy metabolism through nuclear factor-κB in AGS cells[J].Cancer Sci,2017,108(9):1769-1777.
19 Karin M,Cao Y,Greten FR et al.NF-kappa B in cancer:from innocent by stander to major culprit[J].Nat Rev Cancer,2002,2(4):301-310.
20 Ren L,Li Z,Dai C,et al.Chrysophanol inhibits proliferation and induces apoptosis through NF-κB/cyclin D1 and NF-κB/Bcl-2 signaling cascade in breast cancer cell lines[J].Mol Med Rep,2018,17(3):4376-4382.
21 Ma L,Feng L,Ding X,et al.Effect of TLR4 on the growth of SiHa human cervical cancer cells via the MyD88-TRAF6-TAK1 and NF-κB-cyclin D1-STAT3 signaling pathways[J].Oncol Lett,2018,15(3):3965-3970.
22 Inaba T,Matsushime H,Valentine M,et al.Genomic organization,chromosomal localization,and independent expression of human cyclin D genes[J].Genomics,1992,13(3):565-574.
23 Kumar Mongre R,Sharma N,Singh Sodhi S,et al.Novel phyto-derivative BRM270 inhibits hepatocellular carcinoma cells proliferation by inducing G2/M phase cell cycle arrest and apoptosis in xenograft mice model[J].Biomed Pharmacother,2017,87:741-754.
24 Oakley GJ,Denning KL,Graffeo V,et al.Same difference:A pilot study of cyclin D1,bcl-2,AMACR,and ALDH-1 identifies significant differences in expression between primary colon adenocarcinoma and its metastases[J].Pathol Res Pract,2016,212(11):995-1003.
25 Cai X,Hu X,Tan X,et al.Metformin induced AMPK activation,G0/G1 phase cell cycle arrest and the inhibition of growth of esophageal squamous cell carcinomas in vitro and in vivo[J].PLoS One,2015,10(7):e0133349.

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