【作者】 周雪峰；

【导师】 王建军；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 目的：观察VEGF-C和RAF-1基因共沉默对人肺癌细胞A549增殖、侵袭、细胞周期等的影响，并初步探讨其机制。方法：构建靶向VEGF-C和RAF-1基因的shRNA，酶切鉴定、测序正确后，转染A549细胞，分别采用RT-PCR和Western blot方法检测mRNA和蛋白表达，选择有效干扰序列，并筛选出稳定表达shRNA的细胞系。将VEGF-C和RAF-1构建到同一个质粒载体，筛选出稳定表达双shRNA的细胞系，并观察其抑制效果。MTT法检测细胞增殖能力，Boyden小室检测细胞侵袭能力，流式细胞仪(FCM)检测蛋白表达、细胞周期及细胞凋亡水平。Western blot方法检测相关信号通路Caspase3及PDK1、AKT、RAF和ERK磷酸化水平情况。结果：成功构建靶向VEGF-C和RAF-1基因的有效shRNA，并筛选出稳定转染细胞系A549-VEGF-Ce和A549-RAF-1e，检测mRNA和蛋白表达明显受到抑制。成功构建联合表达VEGF-C和RAF-1基因的有效shRNA表达载体，并筛选出稳定转染细胞系A549-RAF-1-VEGF-Ce，检测mRNA和蛋白表达与单独VEGF-C和RAF-1shRNA作用一致。MTT法检测结果表明A549-Raf-1-VEGF-Ce与A549-Raf-le以及A549-VEGF-Ce的增殖能力与对照组细胞A549相比明显降低。A549-Raf-le增殖的平均值较A549-VEGF-Ce略低，但无统计学差异。A549-Raf-1-VEGF-Ce的增殖能力与A549-VEGF-Ce相比显著下降。体外趋化侵袭实验显示，A549-Raf-le组、A549-VEGF-Ce组以及A549-Raf-1-VEGF-Ce组的侵袭细胞数比A549组和各无效shRNA组侵袭细胞数减少，各实验组之间差异无显著性。检测细胞周期分布发现，A549-VEGF-Ce细胞其G1期细胞比例高于其他各组细胞，而A549-Raf-le组细胞出现了凋亡峰。A549-Raf-1-VEGF-Ce组细胞G1期的细胞与A549-VEGF-Ce组相比减少的，其凋亡明显增高。进一步检测各实验组细胞的凋亡水平，A549-Raf-1-VEGF-Ce细胞的凋亡率(24％)高于A549-Raf-le细胞的凋亡率(17％)，其他各组细胞凋亡率无明显变化，与细胞周期分析结果相一致。Western blot结果显示在A549-Raf-1-VEGF-Ce和A549-Raf-le中能检测到活化的Caspase3大亚基片段，各组细胞中AKT、PDK1、RAF的磷酸化水平无明显变化，A549-Raf-1-VEGF-Ce细胞ERK的磷酸化水平有较明显的下降。结论：稳定转染靶向VEGF-C和RAF-1基因的shRNA能够抑制A549细胞的增殖与体外侵袭能力，并调控细胞周期分布及细胞凋亡。同时干扰RAF-1和VEGF-C有一定的协同效应，与Caspase3途径及ERK的磷酸化水平增高有关。更多还原

【Abstract】 Objective To explore the effects and possible mechanisms of RNA interference targeting VEGF-C and RAF-1 mRNA on the proliferation, invasion and cell cycle of human lung cancer cell line A549.Methods According to the basic principle of RNA interference, plasmid which expressed short hairpin RNA (shRNA) corresponding to VEGF-C and RAF-1 were respectively constructed by DNA recombining technology. After restriction endonuclease digestion and sequence were identified, the plasmid was transfected into A549 cell. Then the expression of mRNA and protein was detected by RT-PCR and Western blot. According to the result, the effective sequence was selected and the stable cell line which expressed shRNA was screened out. Plasmid vector with both VEGF-C and RAF-1 were constructed, and the stable cell line transfected with that were built. MTT was used to test the cell growth, Boyden methods was used to dectect the the invasion ability of A549 cell, flowcytometry was used to observe the expression level of protein, cell cycle and apoptosis, and Western blot was using to test the the level of associated pathway such as Caspase3, PDK1, AKT, RAF and ERK phosphorylation.Results The expressions of mRNA and protein of A549-VEGF-Ce and A549-RAF-le cell line which was transfected with shRNA targeting VEGF-C or RAF-1 gene were significantly inhibited. The inhibited effect of A549-RAF-1-VEGF-Ce cell line which contained two kinds of shRNA was same as A549-VEGF-Ce or A549-RAF-le cell line. According to MTT results, the cell proliferation ability of A549-Raf-1-VEGF-Ce, A549-Raf-le and A549-VEGF-Ce cell lines were significantly lower than the control group.The average number of A549-RAF-le cell line was a little lower than that of A549-VEGF-Ce, but there was no significant difference between them. Nevertheless, the proliferation ability of A549-Raf-1-VEGF-Ce cell was dramaticly lower than that of A549-VEGF-Ce. In the invasion test in vitro, the invasion cell number of A549-Raf-le, A549-VEGF-Ce and A549-Raf-1-VEGF-Ce was lower than control cell group, but there were no significant difference among them. In cell cycle tested by flowcytometry, the proportion of cell number in G1 phase of A549-VEGF-Ce was higher than other cell groups and a apoptosis peak was emerged in A549-Raf-le group.The A549-Raf-1-VEGF-Ce cell line with lower proportion of G1 phase cell than A549-VEGF-Ce had obviously more apoptosis cell. Further examination about the apoptosis level of all cell lines showed that the apoptosis ratio of A549-Raf-1-VEGF-Ce (24%) was higher than that of A549-Raf-le (17%) and the ration of other groups which kept in line with cell cycle test had no significant changes. Western blot results showed that actived large subunit of Caspase3 was caught both in 549-Raf-1-VEGF-Ce and A549-Raf-le group, and the phosphorylation level of AKT, PDK1 and RAF in all cell groups had no significant difference exception of the obvious decrease of phosphorylation level of ERK in A549-Raf-1-VEGF-Ce cell line.Conclusion shRNA targeting VEGF-C and RAF-1 gene could inhibit the proliferation and invasion abilities of A549 cell and regulate the cell cycle and apoptosis of tumor cell. And RNA interference targeting on VEGF-C and RAF-1 gene simultaneously had a cooperative effect mainly due to the increased level of Caspase3 and phosphorylation form of ERK.更多还原