c-Myc和VEGF基因的小分子干扰RNA对人结直肠癌细胞的影响

Effect of siRNA targeting c-Myc and VEGF on human coiorectal cancer cells

目的研究靶向c-Myc和血管内皮生长因子（VEGF）基因的小分子干扰RNA（siRNA）表达质粒对人结直肠癌细胞株的影响。方法设计靶向c-Myc和VEGF的特异性siRNA。应用体内载体表达法合成其表达质粒．转染人结直肠癌细胞株Volo后．以Westernblot检测c-Myc和VEGF的蛋白表达；以MTT、流式细胞术、TUNEL染色分析、基质胶侵袭实验检测细胞增殖活性、凋亡特性、细胞周期分布及侵袭能力的改变。结果酶切及DNA测序证实c—Myc和VEGF的siRNA表达质粒均构建成功。转染细胞后，与对照组相比较。重组质粒组细胞c-Myc和VEGF的蛋白表达水平显著下调明显（均P〈0．01）。重组质粒c—Myc组、VEGF组和c—Myc加VEGF组细胞增殖抑制率分别为（59．20±5．05）％、（32．31±3．48）％和（75．81±7．89）％：细胞凋亡率分别为（40．50±4．37）％、（21．30±2．98）％和（62．59±9．66）％，均显著高于对照组[细胞增殖抑制率（6．80±1．45）％；细胞凋亡率（2．90±0．36）％；均P〈0．05]。重组质粒VEGF组和c—Myc加VEGF组的细胞侵袭率分别为（7．34±3．65）％和（2．80±1．02）％，明显低于对照组的（18．57±7．46）％（P〈0．05）。其中，重组质粒c-Myc加VEGF组细胞增殖、调亡及侵袭性的改变最为显著，明显高于另外两个重组质粒组（均P〈0．05）。结论特异性siRNA有效抑制了人结直肠癌Volo细胞c—Myc和VEGF的表达．从而抑制了肿瘤细胞的增殖活性和侵袭性．并诱导细胞凋亡．两者具有协同作用。

Objective To investigate the biological behavioral effects of specific siRNA expression plasmids targeted against c-Myc and vascular endothelial growth factor （VEGF） on human colorectal cancer cell line Volo. Methods The expression plasmids with small interfering RNA （siRNA） aiming at c-Myc and VEGF were designed and constructed respectively, then transfected into Volo cells by eukaryocyte transfection technique. The protein expressions of c-Myc and VEGF were detected by Western blotting. Cellular proliferation, apoptosis, cycle distribution and invasion character were analyzed by tetrazolium bromide colorimetry（MTT）, flow cytometry （FCM）, TUNEL assay and matrigel invasion assay respectively. Results Enzymatic digestion and DNA sequencing confirmed that the c-Myc and VEGF specific siRNA expression plasmids were constructed successfully. After plasmids were transfected into cells, the protein expressions of c-Myc and VEGF were significantly downregulated respectively as compared with control group （P〈0.01）. The cellular proliferation inhibitory rates in c-Myc siRNA group, VEGF siRNA group and c-Myc＋VEGF group were （59.20±5.05）%, （32.31±3.48）% and （75.81±7.89）% respectively, which were higher than that in control group [（6.80±1.45）%]（all P〈0.05）. The cell apoptosis rate in above 3 groups were （40.50±4.37）%, （21.30±2.98）% and （62.59±9.66）% respectively, which were higher than that in control group [ （2.90±0.36）%] （all P〈0.05）. The cell invasion rates in VEGF siRNA group and c-Mye＋VEGF siRNA group were（7.34±3.65）% and（2.80±1.02）%, which were lower than that in control group[（18.57±7.46）%]（P〈0.05）. The effect of c-Myc＋VEGF siRNA group was greater. Conclusions The specific siRNA efficiently silences the expression of c-Myc and VEGF, subsequently, suppresses the cell proliferation, triggers the cell apoptosis and inhibits the cell invasiveness in these transfected colorectal cancer Volo cells. In addition, the synergism of siRNA-c-Myc and siRNA-VEGF in transfected cells can be found.