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目的观察针对单位点与双位点的RNA干扰技术抑制大肠癌LoVo细胞表皮生长因子受体(EGFR)的表达,诱导细胞凋亡以及对5-氟尿嘧啶(5-FU)敏感性的差异。方法构建质粒表达载体pU6-EGFR-shRNA-1和pU6-EGFR-shRNA-2,脂质体转染人大肠癌LoVo细胞,G418筛选4周,分5组:1组为LoVo细胞,2组为对照质粒载体HK,3组为pU6-EGFR-shRNA-1,4组为pU6-EGFR-shRNA-2,5组为pU6-EGFR-shRNA-1和pU6-EGFR-shRNA-2各半量。实时荧光定量PCR和Westernblot检测mRNA和蛋白的表达,流式细胞仪检测细胞凋亡率,细胞増殖分析试剂CCK-8(CellCountingKit-8)测定5-FU不同浓度和时间对LoVo细胞的抑制率和半数抑制浓度(IC50)。结果正确构建了短发夹状RNA(shRNA)质粒表达载体,成功转染;3、4、5组mRNA表达分别下降了(80·2±3·4)%、(81·3±2·8)%和(90·6±2·8)%,蛋白表达分别下降了(74·1±4·0)%、(73·4±2·3)%和(90·4±3·3)%,凋亡率增加了(10·4±0·5)%、(10·1±0·4)%和(14·2±0·5)%,同时对5-FU的IC50和细胞抑制率作统计学分析,5组,3、4组和1、2组三者之间比较差异有统计学意义,而1组2组间、3组4组间各项比较差异无统计学意义。结论两条shRNA均可有效抑制LoVo细胞EGFR表达,促进细胞凋亡,提高5-FU对癌细胞的杀伤作用,靶向联合位点的RNA干扰技术较单一位点的干扰效果更显著,为大肠癌的基因治疗联合化疗提供了新的思路。
Objective To observe the effect of single and double site RNA interference on the expression of epidermal growth factor receptor (EGFR), the induction of apoptosis and the sensitivity to 5-fluorouracil (5-FU) in colorectal cancer LoVo cells. Methods The plasmid vector pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2 were constructed and transfected into human LoVo cells by lipofectamine 2000. G418 cells were screened for 4 weeks and divided into 5 groups: group 1 was LoVo cells, group 2 was Control plasmid vector HK, pU6-EGFR-shRNA-3 in group 3, pU6-EGFR-shRNA-1,4 in group 1, and pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA- Real-time quantitative PCR and Western blot were used to detect the expression of mRNA and protein. Flow cytometry was used to detect the apoptosis rate. Cell proliferation assay kit CCK-8 (CellCounting Kit-8) was used to determine the inhibitory rate of LoVo cells with different concentrations and time of 5-FU Half inhibitory concentration (IC50). Results The plasmid vector of short hairpin RNA (shRNA) was correctly constructed and successfully transfected. The expression of mRNA in groups 3, 4, and 5 decreased by (80.2 ± 3.4)%, (81.3 ± 2.8) (74.4 ± 2.3)% and (90.4 ± 3.3)%, (90.6 ± 2.8)% and (74.4 ± 2.3)%, (10.4 ± 0.5)%, (10.1 ± 0.4)% and (14.2 ± 0.5)%, respectively, while the IC50 of 5-FU and cell inhibition Rates for statistical analysis, 5 groups, 3,4 groups and 1,2 groups were statistically significant differences between the three groups, but 1 group 2 groups, 3 groups 4 groups between the comparison was not statistically significant. Conclusion Both shRNAs can effectively inhibit the expression of EGFR in LoVo cells, promote the apoptosis and increase the cytotoxicity of 5-FU on cancer cells. The siRNA targeting the combined site is more effective than the single site in interfering with EGFR expression. Cancer gene therapy combined with chemotherapy provides a new way of thinking.