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目的:利用小分子干扰RNA(siRNA)技术抑制人直肠癌Colo320细胞c-Myc基因的表达。为研究c-Myc基因在人直肠癌Colo320细胞中的作用提供一个新的方法。方法:设计人直肠癌Colo320细胞基因特异性小分子干扰RNA,用体外转录方法合成人直肠癌Colo320细胞基因的小分子干扰RNA并转染人直肠癌Colo320细胞,培养48~96h后,收集细胞RNA应用实时荧光定量RT-PCR方法检测转染细胞中c-Myc基因mRNA水平变化,Westernblot检测C-MYC蛋白的表达,四甲基偶氮唑蓝(MTT法)和集落形成实验检测细胞增殖活性。结果:转染siRNA后,与对照组相比,实验组pGensil-c-Myc-1-4的c-Myc基因mRNA水平明显降低,MTT法和集落形成实验表明实验组的增殖速率明显低于对照组的增殖速率。结论:在人直肠癌Colo320细胞中存在RNA干扰的机制,特异性siRNA能够有效的抑制c-Myc基因的表达,为研究c-Myc基因在肿瘤细胞中的调节途径提供了一个新的方法。
OBJECTIVE: To inhibit the expression of c-Myc gene in human colorectal cancer cell line Colo320 by using small interfering RNA (siRNA) technique. To provide a new method for studying the role of c-Myc gene in human colorectal cancer Colo320 cells. Methods: Human colorectal cancer cell line Colo320 was designed and transfected into human Colorectal Carcinoma Colo320 cells by in vitro transcription. Small interfering RNA was transfected into human Colorectal Carcinoma Colo320 cells and cultured for 48-96 hours. Cell RNA The mRNA level of c-Myc in transfected cells was detected by real-time fluorescence quantitative RT-PCR, the expression of C-MYC protein was detected by Western blot, MTT assay and colony formation assay. Results: Compared with the control group, the mRNA level of c-Myc in pGensil-c-Myc-1-4 was significantly decreased in the experimental group. The MTT assay and colony formation assay showed that the proliferation rate of the experimental group was significantly lower than that of the control group Group proliferation rate. CONCLUSION: The mechanism of RNAi interference in human colorectal cancer cell line Colo320 and the specific siRNA can effectively inhibit the expression of c-Myc gene provide a new method for studying the regulation of c-Myc gene in tumor cells.