CBX3在细胞周期中通过CDK6独立于激酶的作用促进结肠癌细胞增殖
发布时间：2018-10-29 11:45
【摘要】：目的异染色质蛋白1γ(CBX3)连接组蛋白甲基化从而发挥转录沉默、DNA修复、RNA剪切等功能。研究CBX3在结肠癌中的生物学作用及其相关机制有利于为肿瘤诊断、治疗提供新的靶点。方法运用IHC和Western Blot方法分别检测人结肠癌组织及其癌旁组织CBX3的表达;利用CRISPR/Cas9基因编辑技术敲出HCT116细胞中的CBX3并构建稳定细胞系;通过MTS、EDU、克隆形成实验及流式细胞仪检测CBX3对结肠癌细胞增殖、周期、凋亡的影响;qRT PCR筛选CBX3影响结肠癌细胞周期G1-S期相关靶点,Western Blot和免疫荧光进一步在蛋白水平上验证;运用慢病毒质粒干扰及过表达实验、激酶抑制实验分析CBX3、CDK6、p21三者之间的关系;通过裸鼠成瘤实验在体内验证CBX3对结肠癌发生发展中的作用;IHC和Western Blot进一步分析CDK6与p21在人结肠癌中的表达关系。结果CBX3在结肠癌组织中显著高表达;敲出CBX3明显抑制结肠癌细胞增殖及细胞周期G1-S期进展,促进下游靶点CDK6和p21上调;CDK6在缺乏CBX3时能通过独立于激酶的活性调控p21表达限制细胞过度增殖;CBX3在裸鼠体内能够影响结肠癌细胞发生发展。结论CBX3通过抑制CDK6/p21的表达促进结肠癌发生发展,从而破坏了CDK6的负反馈调节机制。
[Abstract]:Objective to methylate heterochromatin 1 纬 (CBX3) binding histone to play a role in transcription silencing, DNA repair and RNA shearing. The study of the biological role of CBX3 in colon cancer and its related mechanisms may provide a new target for tumor diagnosis and treatment. Methods IHC and Western Blot were used to detect the expression of CBX3 in human colon cancer tissues and adjacent tissues respectively, and CRISPR/Cas9 gene editing technique was used to knock out CBX3 in HCT116 cells and construct stable cell lines. The effects of CBX3 on the proliferation, cycle and apoptosis of colon cancer cells were detected by MTS,EDU, clone forming assay and flow cytometry. QRT PCR screening of CBX3 related targets for G1-S phase of colon cancer cell cycle, Western Blot and immunofluorescence were further verified at protein level. The relationship among CBX3,CDK6,p21 was analyzed by plasmid interference and overexpression of lentivirus and kinase inhibition assay, and the role of CBX3 in the development of colon cancer in vivo was verified by tumorigenesis test in nude mice. IHC and Western Blot were used to analyze the expression of CDK6 and p21 in human colon cancer. Results CBX3 was significantly overexpressed in colon cancer tissues, knockout CBX3 significantly inhibited the proliferation of colon cancer cells and progression of cell cycle G1-S phase, and promoted downstream target CDK6 and p21 upregulation. In the absence of CBX3, CDK6 could restrict the proliferation of human colon cancer cells by regulating the expression of p21 independent of kinase activity, and CBX3 could influence the development of colon cancer cells in nude mice. Conclusion CBX3 can promote the development of colon cancer by inhibiting the expression of CDK6/p21, thus destroying the negative feedback regulation mechanism of CDK6.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.35
[Abstract]:Objective to methylate heterochromatin 1 纬 (CBX3) binding histone to play a role in transcription silencing, DNA repair and RNA shearing. The study of the biological role of CBX3 in colon cancer and its related mechanisms may provide a new target for tumor diagnosis and treatment. Methods IHC and Western Blot were used to detect the expression of CBX3 in human colon cancer tissues and adjacent tissues respectively, and CRISPR/Cas9 gene editing technique was used to knock out CBX3 in HCT116 cells and construct stable cell lines. The effects of CBX3 on the proliferation, cycle and apoptosis of colon cancer cells were detected by MTS,EDU, clone forming assay and flow cytometry. QRT PCR screening of CBX3 related targets for G1-S phase of colon cancer cell cycle, Western Blot and immunofluorescence were further verified at protein level. The relationship among CBX3,CDK6,p21 was analyzed by plasmid interference and overexpression of lentivirus and kinase inhibition assay, and the role of CBX3 in the development of colon cancer in vivo was verified by tumorigenesis test in nude mice. IHC and Western Blot were used to analyze the expression of CDK6 and p21 in human colon cancer. Results CBX3 was significantly overexpressed in colon cancer tissues, knockout CBX3 significantly inhibited the proliferation of colon cancer cells and progression of cell cycle G1-S phase, and promoted downstream target CDK6 and p21 upregulation. In the absence of CBX3, CDK6 could restrict the proliferation of human colon cancer cells by regulating the expression of p21 independent of kinase activity, and CBX3 could influence the development of colon cancer cells in nude mice. Conclusion CBX3 can promote the development of colon cancer by inhibiting the expression of CDK6/p21, thus destroying the negative feedback regulation mechanism of CDK6.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.35
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