摘要
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，Western blotting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 c-myc gene expression in human rectal cancer cell line Colo320 by small interfering RNA technique, so as to assess the role of c-myc in Colo320 cells. Methods： The c-myc gene specific siRNA was designed and prepared by in vitro transcription. The prepared c-myc siRNA was transfected into Colo320 cells. After cultured for 48-96 hours, the cells were harvested, c-myc mRNA and protein level was monitored by fluorescence real time reverse transcription-poly merase chain reaction, c-myc protein expression was detected by Western blotting, and the cell proliferation activities were assayed by tetrazolium bromide （MTT） colorimetry and clone test. Results： Compared with control group, the mRNA of pGensil-c-myc-1, 2, 3, and 4 in Colo320 cells was obviously decreased in c-myc siRNA transfected ceils. C-myc protein expression was also decreased obviously. MTT assay and clone test showed that c-myc siRNA apparently slowed down the proliferation of Colo320 cells. Conclusion： Our results suggest that RNA interference exists in Colo320 cell line; c-myc siRNA can specifically inhibit the expression of c-myc gene in Colo320 cells, subsequently inhibits the proliferation of Colo320 cells.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
2006年第5期367-370,共4页
Chinese Journal of Cancer Biotherapy
基金
湖北省技攻关计划项目(编号2006AA301B66-3)
