mir-7-3基因慢病毒表达载体的构建及对人胶质瘤细胞生长抑制作用的研究

Construction of recombinant lentivirus containing human mir-7-3 like sequence and its inhibitory effects on human glioma proliferation

目的构建mir-7-3基因慢病毒表达载体，研究mir-7-3基因的功能及其在胶质瘤基因治疗中的应用。方法将Lenti—GFP-mir-7-3质粒和包装质粒pRsv—REV、pMDlg-pRRE、pMD2G共同转染至人胚胎肾上皮细胞系293T细胞，获得携带mir-7-3基因和绿色荧光蛋白（GFP）基因的重组慢病毒FIV—CMV—GFP-mir-7-3．荧光显微镜下观察293T细胞的荧光表达：取浓缩纯化后的病毒上清感染人胶质瘤细胞U251作为转染组，同时设转染空载病毒的阴性对照组和空白对照组，RT—PCR鉴定U251细胞中mir-7-3基因的表达水平。Western blotting检测转染后48h细胞表皮生长因子受体（EGFR）及丝氨酸／苏氨酸蛋白激酶（AKT2）蛋白的表达。MTT法检测转染后1-6d细胞存活率的变化。流式细胞术检测转染后48h细胞周期的变化。结果Lenti—GFP—mir-7-3共转染包装细胞293T能产生高浓度的重组慢病毒F1V—CMV-GFP-mir-7—3，荧光显微镜下能直接观察到GFP，FIV-CMV—GFP-mir-7-3中携有正确的mir-7-3基因，目的基因mir-7-3能被重组慢病毒高效地导入U251。与空白对照组和阴性对照组比较，转染后48h转染组细胞EGFR及AKT2蛋白的表达下降（AKT2表达下调42％，EGFR表达下调38％）。G0／G1期细胞增加，S期细胞数目下降，转染3、4、5、6d后转染组细胞存活率降低，差异均有统计学意义（P〈0．05）。结论成功构建了携带mir-7-3基因的重组慢病毒载体；mir-7-3有效地抑制EGFR通路表达，抑制细胞周期G1期向S期转化，抑制胶质瘤增殖，因此mir-7—3有可能成为胶质瘤基因治疗的候选药物。

Objective To construct a lentiviral vector containing mir-7-3 gene and study the function of mir-7-3 gene and its role in glioma gene therapy. Methods The plasmid Lenti-GFP-mir-7-3 and packaging plasmids pRsv-REV, pMDlg-pRRE and pMD2G were co-tmnsfected into the human embryonic kidney epithelial cell line 293T cells, and then, recombinant lentiviral F1V-CMV-GFP-mir-7-3 carried mir-7-3 gene and GFP gene was obtained; fluorescence expression of 293T cells was observed under fluorescence microscope. The supernatant was collected, concentrated and identified, and then, it was used to transfect into the U251 glioma cells （positive transfection group）; and blank control group （cells transfected with empty vector） and negative control group （parental cells） were also employed. Real time-PCR was used to examine the relative contents of mir-7-3 in U251 ceils; Western blotting was employed to detect the epidermal growth factor （EGFR） and serine/threonine kinase （AKT2） protein expressions; cell cycle was analyzed by flow cytometry and cell proliferative activities were measured by MTT method. Results Recombinant lentiviral FIV-CMV-GFP-mir-7-3 carried mir-7-3 gene and GFP gene was successfully constructed in 293T cells; electrophores showed that the sequence of RT-PCR product was consistent with the data ofmir-7-3 by DNA sequence analysis, indicating that the mir-7-3 gene was successfully cloned; strong green fluorwscence was observed by fluorwscent microscopy. The supematant of lentivirus-transfected 293T cells was effectively infected into U251 cells and the relative content of mir-7-3 could be observed in the transfected U251 cells. As compared with those in the parental cells and the cells transfected with empty vector, the protein expressions of EGFR and AKT2 in the transfected group decreased significantly, reaching 38% and 42%, respectively （P〈0.05）. As compared with those in the parental cells and the cells transfected with empty vector, the cells at G0/G1 phase increased, the S phase fraction was lower and the survival rates dramatically dropped in the mir-7-3 transfected cells 3, 4, 5 and 6 d after implanation. Conclusions The lentiviral vector containing mir-7-3 gene was constructed successfully. Mir-7-3 could specifically suppress EGFR and AKT2 expressions, induce gene silencing, inhibit cell growth, indicating that this way should be a new strategy in glioma gene therapy.