重组慢病毒CD133-miR30-shRNA的构建及抗肝癌细胞生长的作用

Construction of lentiviral vector mi R30-CD133 targeting CD133 gene and its inhibition of liver cancer cell growth

目的:构建沉默CD133基因的mi R30-CD133慢病毒,探讨其对肝癌细胞株SMMC7721生物学行为的影响。方法:针对已经筛选确定的CD133基因干扰有效序列,合成靶序列的Oligo DNA,与p PRIME载体连接。用p PRIME-mi R30-CD133、ps PAX2和p MD2G 3种质粒共转染293T细胞,包装产生慢病毒,测定病毒滴度;荧光显微镜下测定病毒感染效率;q RT-PCR、Western blot检测CD133表达;CCK-8法检测细胞生长;annexin V/PI检测细胞凋亡。根据实验要求(1)Blank组;(2)sh NC组;(3)sh CD133组。结果:病毒滴度为6.58×109 PFU/m L,病毒感染效率为(80.8±9.1)%。q RT-PCR结果显示sh CD133组相对Blank组的CD133表达量的平均值为0.18±0.04,sh CD133组相对Blank组和sh NC组有显著的CD133敲减作用(P<0.05)。Western blot结果显示sh CD133组的CD133表达明显下降,约为sh NC组的10%(P<0.05)和Blank组的8%(P<0.05)。重组慢病毒抑制肝癌细胞生长,与Blank组(25.3%)、sh NC组(24.3%)相比,sh CD133组凋亡细胞比例(41.3%)显著增加(P<0.05)。结论:成功构建慢病毒PRIME-mi R30-CD133,为CD133基因的功能研究提供了工具。

Objective To construct lentiviral vector mi R30-CD133 targeting CD133 gene and investigate its effects on SMMC7721 cells. Methods Thespecific sequence of DNA which containing both sense and antisense Oligo DNA of the targeting sequence were designed, synthesized and cloned into the p PRIME vector. p PRIMEmi R30-CD133, ps PAX2 and p MD2 G were co-transfected on 293 T cells to get the lentivirus the transfection efficiency was investigated by confocal laser scanning microscope. The expression of mi R30-CD133 on CD133 were detected by q RT-PCR and Western blott. The proliferation and apoptosis effect of mi R30-CD133 was respectively evaluated by CCK-8 assay and Annexin V / PI. Results Functional PFU titers of PRIME-mi R30-CD133 were 6.58 ×109PFU / m L. The expression of CD133 m RNA in sh CD133 group（0.18 ± 0.04） was less than Blank group and sh NC group（P〈0.05）. The expression of CD133 protein in sh CD133 group was less than Blank group（10%） and sh NC group（8%）（P〈0.05）. Cells proliferation activity were significantly inhibited in sh CD133 group compared with control group. Apoptosis rate were significantly increased in sh CD133 group（41.3%） compared with Blank group（25.3%）and sh NC group（24.3%）. Conclusion The lentivirus mi R30 vector targeting CD133 gene was successfully constructed, which will be a basis to the further CD133 gene studies.