ZIC1过表达对结直肠癌HCT-116细胞增殖及对苦参碱类生物碱药物敏感性的影响
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摘要
目的:检测ZIC1基因在结直肠癌细胞内过表达后对癌细胞增殖能力的影响,以及对苦参碱类生物碱药物敏感性的改变情况。方法:首先从五种结直肠癌细胞株中筛选出ZIC1基因表达最低的一株,对其进行慢病毒转染,使其稳定表达ZIC1基因或空载体,再利用Western blot技术和qRT-PCR技术鉴定ZIC1蛋白/mRNA表达情况。通过CCK-8法和平板克隆形成实验检测过表达ZIC1基因后细胞增殖能力的改变,并利用CCK-8法分析稳转细胞株对苦参碱类生物碱药物敏感性的改变。结果:HCT116细胞株中ZIC1 mRNA/蛋白表达均显著低于其他四株(P <0. 001);慢病毒转染后,含r LV-ZIC1-PGK-Puro的重组慢病毒转染HCT116细胞内ZIC1 mRNA/蛋白表达水平显著高于对照组(P <0. 001)。CCK-8实验结果示,ZIC1基因过表达后HCT116细胞增殖能力明显降低(P <0. 01);实验组克隆形成率明显低于对照组(P=0. 003)。CCK-8法发现ZIC1过表达后HCT116结直肠癌细胞对苦参碱及槐定碱的药物敏感性得到明显的升高,而对氧化苦参碱及槐果碱的药物敏感性无明显改变。结论:HCT116结直肠癌细胞内ZIC1基因过表达后能有效抑制癌细胞的增殖,并增敏苦参碱及槐定碱对结直肠癌HCT116细胞的药理作用。
Objective: To evaluate the effect of ZIC1 gene on the proliferation ability of cancer cells in colorectal cancer cells and changes of the sensitivity of matrine alkaloid drugs. Methods: Firstly,one cell line with lowest expression of ZIC1 was selected from the five kinds of colorectal cancer cell lines,and was infected by r LV-ZIC1-PGK-Puro or r LV-PGK-Puro followed by Western blot and qRT-PCR to assess ZIC1 protein/mRNA expression.Through CCK-8 and colony formation assay,the cell proliferation was detected. CCK-8 was also used to investigate changes of the sensitivity of matrine alkaloid. Results: The expression of ZIC1 mRNA/protein in HCT116 cell lines was significantly lower than that of the other four( P < 0. 001). The expression level of ZIC1 mRNA/protein in HCT116 cells with r LV-ZIC1-PGK-Puro was significantly higher than that in the control group( P < 0. 001). The results of CCK-8 showed that the proliferation of HCT116 cells was significantly reduced after ZIC1 gene overexpression( P < 0. 01). The clone formation rate of the experimental group was significantly lower than that in the control group( P = 0. 003). CCK-8 also found that HCT116 colorectal cancer cell with over-expressed ZIC1 had higher sensitivity of matrine and sophoridine. Conclusion: Over-expressed ZIC1 in HCT116 colorectal cancer cell can effectively inhibit the proliferation of cancer cells,and increase the sensitivity of matrine and sophoridine.
Objective: To evaluate the effect of ZIC1 gene on the proliferation ability of cancer cells in colorectal cancer cells and changes of the sensitivity of matrine alkaloid drugs. Methods: Firstly,one cell line with lowest expression of ZIC1 was selected from the five kinds of colorectal cancer cell lines,and was infected by r LV-ZIC1-PGK-Puro or r LV-PGK-Puro followed by Western blot and qRT-PCR to assess ZIC1 protein/mRNA expression.Through CCK-8 and colony formation assay,the cell proliferation was detected. CCK-8 was also used to investigate changes of the sensitivity of matrine alkaloid. Results: The expression of ZIC1 mRNA/protein in HCT116 cell lines was significantly lower than that of the other four( P < 0. 001). The expression level of ZIC1 mRNA/protein in HCT116 cells with r LV-ZIC1-PGK-Puro was significantly higher than that in the control group( P < 0. 001). The results of CCK-8 showed that the proliferation of HCT116 cells was significantly reduced after ZIC1 gene overexpression( P < 0. 01). The clone formation rate of the experimental group was significantly lower than that in the control group( P = 0. 003). CCK-8 also found that HCT116 colorectal cancer cell with over-expressed ZIC1 had higher sensitivity of matrine and sophoridine. Conclusion: Over-expressed ZIC1 in HCT116 colorectal cancer cell can effectively inhibit the proliferation of cancer cells,and increase the sensitivity of matrine and sophoridine.
引文
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[2]Chen X,Lin Z,Xue M,et al.ZIC1 promoter hypermethylation in plasma dna is a potential biomarker for gastric cancer and intraepithelial neoplasia[J].PLo S One,2015,10(7):e0133906.
[3]Gan L,Chen S,Zhong J,et al.ZIC1 is downregulated through promoter hypermethylation,and functions as a tumor suppressor gene in colorectal cancer[J].PLo S One,2011,6(2):e16916.
[4]Wang JF,Li PF,Cao F,et al.ZIC1 expression in colorectal cancer tissue and its effect on colon cancer cell proliferation[J].Journal of Jiangsu University(Medicine Edition),2015,25(01):10-13,18.[王杰锋,李鹏飞,曹方,等.ZIC1在结直肠癌中的表达及其对结肠癌细胞增殖能力的影响[J].江苏大学学报(医学版),2015,25(01):10-13,18.]
[5]Han H,Cao XK,Yin X,et al.Advance in studies on anti-tumor mechanism of oxymatrine[J].Chinese Medicine Journal of Research and Practice,2017,31(02):78-81.[韩华,曹苋恺,殷鑫,等.氧化苦参碱抗肿瘤作用机制的研究进展[J].现代中药研究与实践,2017,31(02):78-81.]
[6]Qian LQ,Gao QG,Shen GH,et al.Research progress of matrine alkaloids and their derivatives in the anti-hepatoma[J].Journal of Practical Oncology,2015,30(1):97-100.[钱利强,高泉根,沈根海,等.苦参碱类生物碱及其衍生物抗肝癌作用研究进展[J].实用肿瘤杂志,2015,30(1):97-100.]
[7]Yu Min,Cao Fang,Zhu Rong,et al.Effect of ZIC1 associated with matrine on proliferation,migration and apoptosis in MDA-MB-231 human breast cancer cell line[J].Modern Oncology,2016,24(10):1528-1533.[于民,曹方,朱荣,等.ZIC1联合苦参碱对人乳腺癌MDA-MB-231细胞增殖、迁移及凋亡的影响[J].现代肿瘤医学,2016,24(10):1528-1533.]
[8]Liu SN,Liu R,Fan TT,et al.Advances in diagnosis and treatment of colorectal cancer[J].Shandong Medical Journal,2017,57(40):110-112.[刘盛楠,刘瑞,范婷婷,等.结直肠癌的诊断和治疗研究进展[J].山东医药,2017,57(40):110-112.]
[9]Aruga J.ZIC family proteins in emerging biomedical studies[J].Adv Exp Med Biol,2018,1046:233-248.
[10]Zhong J,Chen S,Xue M,et al.ZIC1 modulates cell-cycle distributions and cell migration through regulation of sonic hedgehog,PI(3)K and MAPK signaling pathways in gastric cancer[J].BMCCancer,2012,12:290.
[11]Gan LH.The molecular mechanism of ZIC1 gene in colon cancer and its inhibitory effect[D].Hangzhou:Zhejiang University,2011.[甘丽虹.ZIC1基因在结肠癌中的表观调控及其抑癌作用的分子机制研究[D].杭州:浙江大学,2011.]
[12]Yang LP,Wu ZH,Liu W.Curative effect of compound matrine injection in combination with FOLFOX-4 adjuvant chemotherapy for sub-operation colorectal cancer patients:A clinical observation[J].Journal of Clinical and Experimental Medicine,2010,9(16):1224-1225.[杨立平,吴兆华,刘伟.复方苦参素联合FOLFOX-4方案对结直肠癌术后辅助化疗的疗效观察[J].临床和实验医学杂志,2010,9(16):1224-1225.]
[13]Ren HT,Wang ZW,Wang YL,et al.Effects of matrine on the efficacy and immunity of postoperative chemotherapy in patients with colorectal cancer[J].Modern Chinese and Western Medicine Journal,2016,25(27):3066-3068.[任宏涛,王中卫,王亚利,等.苦参碱对结直肠癌患者术后化疗减毒增效及增强免疫力的影响[J].现代中西医结合杂志,2016,25(27):3066-3068.]