染色盒同源物3对人结直肠癌细胞增殖的影响及其机制研究

The effect of CBX3 on the biological behavior of human colorectal cancer cells and its mechanism

目的探讨染色盒同源物3(CBX3)在结直肠癌组织和细胞中的表达,及其对结直肠癌细胞增殖能力的影响,并初步探讨其机制。方法运用生物信息学方法对CBX3的表达情况、生存期以及共表达网络进行分析。应用免疫组织化学染色法检测CBX3在结直肠癌组织和配对癌旁组织中的差异表达。应用Western blot检测正常结肠上皮细胞NCM460以及结直肠癌细胞HCT116、HT29、SW620中CBX3的表达水平。将CBX3过表达质粒和特异性小干扰分别转染至HCT116细胞中,采用磺酰罗丹明B(SRB)染色实验和克隆形成实验检测细胞的增殖能力。应用转录组测序技术检测差异表达基因。应用qRT-PCR及Western blot实验对差异基因进行验证。应用生物信息学方法对差异基因及CBX3进行相关性分析。结果生物信息学分析显示CBX3在结直肠癌组织中表达高于癌旁组织(P<0.001),CBX3高表达组患者无病生存期低于低表达组(P<0.05),CBX3对转录组的影响广泛且复杂。CBX3在结直肠癌组织中表达高于配对癌旁组织(P<0.0001)。CBX3在结直肠癌细胞中表达高于正常结肠上皮细胞(P<0.01)。SRB实验及克隆形成实验证实,过表达CBX3增强了HCT116细胞的增殖能力,敲低CBX3增殖能力降低(P<0.01)。转录组测序结果显示,敲低CBX3后,CCAAT/增强子结合蛋白delta(CEBPD)mRNA表达升高(P<0.001),蛋白表达相应升高(P<0.05),且两者存在负性相关(P<0.001,R=-0.2)。结论CBX3在结直肠癌组织及细胞中高表达,能够在体外促进细胞增殖。CBX3可能通过抑制CEBPD转录因子的表达来发挥促癌作用。

Objective To investigate the expression of Chromebox3(CBX3)in colorectal cancer tissues and cells.To explore the effect of CBX3 on the proliferation ability of colorectal cancer cells and its mechanism.Methods The bioinformatics method was used to analyze the expression,survival time and co-expression network of CBX3.Immunohistochemical staining was used to detect the differential expression of CBX3 in colorectal cancer tissues and paired adjacent tissues.Western blot was used to detect the expression level of CBX3 in normal colon epithelial cells NCM460 and colorectal cancer cells HCT116,HT29 and SW620.The CBX3 expression plasmid and specific siRNA were transfected into HCT116 cells,respectively.Sulfhodamine B(SRB)staining experiment and clone formation experiment were used to detect the cell proliferation ability.RNA-seq was used to detect differentially expressed genes.qRT-PCR and Western blot were used to verify the differential genes.Bioinformatics method was used to analyze the correlation between differential genes and CBX3.Results Bioinformatics analysis showed that the expression of CBX3 in colorectal cancer tissues was higher than that in adjacent tissues(P<0.001).The disease-free survival time of patients with high expression of CBX3 was lower than that of patients with low expression of CBX3(P<0.05).CBX3 had extensive and complex effects on the transcriptome.The expression of CBX3 in colorectal cancer tissues was higher than that in matched adjacent tissues(P<0.0001).The expression of CBX3 in colorectal cancer cells was higher than that in normal colon epithelial cells(P<0.01).SRB experiment and clone formation experiment confirmed that overexpression of CBX3 enhanced the proliferation of HCT116 cells,while knockdown of CBX3 decreased the proliferation of HCT116 cells(P<0.01).Transcriptome sequencing results showed that the mRNA expression of CCAAT/enhancer binding protein Delta(CEBPD)was increased after knocking down of CBX3(P<0.001),and the protein expression was correspondingly increased(P<0.05),and there was a negative correlation between CEBPD and CBX3(P<0.001,R=-0.2).Conclusion CBX3 is highly expressed in colorectal cancer tissues and cells,and can promote cell proliferation in vitro.CBX3 may play a role in promoting tumor progression by inhibiting the expression of CEBPD transcription factors.