慢病毒载体介导半乳凝素-3-shRNA沉默基因对肿瘤细胞的影响

Effects of galectin-3 gene silencing by galectin-3-shRNA mediated with lentiviral vector on tumor cells

目的:构建干扰半乳凝素-3(Galectin-3)的重组U6慢病毒质粒,体外评价和观察内源性小RNA干扰效果及对细胞增殖和凋亡的影响.方法:根据siRNA设计原则,设计合成短发夹DNA,用于体内转录合成干扰Galectin-3编码序列的发夹RNA.连接,筛选,酶切,鉴定pGCL-GFP/U6 Gal-3shRNA-1;以含无关序列发夹结构的质粒载体为对照,感染乳腺癌细胞系,采用RT-PCR和Western Blot检测mRNA和蛋白表达水平与对照组的差别;MTT法和流式细胞检测其对细胞增殖、凋亡的干扰情况.结果:测序证实重组质粒构建成功,体外试验表明,Galectin-3的mRNA和蛋白表达均一致降低,转染组为(0.028%),阴性对照组为(0.617%),空白对照组为(0.992%),转染组mRNA干扰效率达95%(P<0.05);Galectin-3蛋白表达明显下调,与阴性对照组和空白对照组相比较,差异有统计学意义(P<0.05).MTT检测结果转染组为(0.40±3.69)%,阴性对照组(0.71±0.16)%;转染组和阴性对照组间差异均有统计学意义(P<0.05).细胞凋亡百分率为68.78%.结论:慢病毒介导的Galectin-3-shRNA成功在体外敲减了肿瘤细胞的目的蛋白,影响了肿瘤的发生与发展.

AIM： To construct a recombinant lentiviral U6 plasmids of RNA interference （RNAi） of galectin-3 gene, and evaluate and observe the interference vector on cell proliferation and effects of galectin-3-shRNA/U6 apoptosis by endogenous small RNAi in vitro. METHODS： According to the target sequence and design principle of siRNA, the short hairpin DNA was designed and synthesized to interfere with gene Galectin-3. The shDNA duplex was ligated into the recombinant vector pGCL- GFP/U6 plasmid. The positive colonies of pGCL-GFP/U6 Gal-3 shDNA-1 were selected by double restriction enzyme digestion with Hpa I and Xho I. Cell line was transfected with pGCL- GFP/U6 Gal-3 shDNA-1 using pGCL-GFP/U6-scrambled shDNA as control. The expression level of galectin-3 was detected by RT- PCR and Western Blot; then PI-labeled flow cytometric analysis was performed for detecting cell apoptosis, and MTF assay for cell proliferation. RESULTS： The recombinant vector was successfully constructed which was confirmed by sequencing. The in vitro experiment indicated that the expression level of Galectin-3 was down-regulated as Western Blot and RT-PCR analyses demonstrated （P 〈 0.05 vs control group） ;The interfering efficiency of lentiviral vector was 95%. MTT assay showed that the proliferation of MCF- 7 were suppressed to （0.40 ± 3.69） % in transfected group and to （0.71±0. 156）% in control group; the difference between the 2 groups was significant statistically （ P 〈 0. 05 ）. FCM assay indicated that lentiviral vector induced cell apoptosis by about 68.78%. CONCLUSION： RNA interference can successfully interfere the expression of galectin-3 in cultured tumor cells and may affect the occurrence and development of the tumor.