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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-my基因的表达,探讨c-myc基因在人直肠癌Colo320细胞中的作用。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细胞的小分子干扰RNA并转染该细胞,培养48~96h后,收集细胞RNA,应用实时荧光定量PCR方法检测转染细胞中c-myc基因mRNA水平变化,Westernblotting检测c-myc表达的蛋白,四甲基偶氮唑蓝(MTT)法和集落形成试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、2、3、4的c-myc基因mRNA水平明显降低,c-myc的蛋白表达也明显降低。MTT法和集落形成试验检测表明,实验组的细胞增殖速率明显低于对照组。结论在人直肠癌Colo320细胞中存在RNA干扰的机制,特异性siRNA能够有效地抑制c-myc基因的表达,从而抑制Colo320细胞的增殖。
Objective To inhibit the expression of c-my gene in human colorectal cancer cell line Colo320 by using small interfering RNA (siRNA) technique and to investigate the role of c-myc gene in human colorectal cancer cell line Colo320. Methods Human colorectal cancer cell line Colo320 gene-specific small interfering RNA was designed and synthesized. Small interfering RNA (RNAi) of Colo320 cells was transfected into human colorectal cancer cell line Colo320 by in vitro transcription method. After culturing for 48-96 h, RNA was extracted and quantified by real- The mRNA levels of c-myc in transfected cells were detected by PCR. The protein expression of c-myc was detected by Western blotting. MTT and colony formation assay were used to detect the cell proliferation. Results Compared with the control group, the mRNA level of c-myc gene and the protein expression of c-myc in pGensil-c-myc-1,2,3,4 were significantly decreased. MTT assay and colony formation assay showed that the proliferation rate of the experimental group was significantly lower than the control group. Conclusion The mechanism of RNA interference exists in human colorectal cancer Colo320 cells. Specific siRNA can effectively inhibit the expression of c-myc gene and thus inhibit the proliferation of Colo320 cells.