Construction of lentiviral vector miR30-CD133 targeting CD133 gene and its inhibition of liver cancer cell growth
10.3969/j.issn.1006-5725.2016.10.006
- VernacularTitle:重组慢病毒CD133-miR30-shRNA的构建及抗肝癌细胞生长的作用
- Author:
Jian NIU
;
Yue WANG
;
Bin LIU
- Publication Type:Journal Article
- Keywords:
Liver neoplasms;
Stem cell;
CD133;
Cell proliferation;
Gene expression;
Lentivirus
- From:
The Journal of Practical Medicine
2016;32(10):1571-1574
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct lentiviral vector miR30-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 pPRIME vector. pPRIME-miR30-CD133, psPAX2 and pMD2G were co-transfected on 293T cells to get the lentivirus the transfection efficiency was investigated by confocal laser scanning microscope. The expression of miR30-CD133 on CD133 were detected by qRT-PCR and Western blott. The proliferation and apoptosis effect of miR30-CD133 was respectively evaluated by CCK-8 assay and AnnexinV/PI. Results Functional PFU titers of PRIME-miR30-CD133 were 6.58 × 109 PFU/mL. The expression of CD133 mRNA in sh CD133 group (0.18 ± 0.04) was less than Blank group and shNC group (P<0.05). The expression of CD133 protein in sh CD133 group was less than Blank group (10%) and shNC group (8%) (P < 0.05). Cells proliferation activity were significantly inhibited in shCD133 group compared with control group. Apoptosis rate were significantly increased in shCD133 group (41.3%) compared with Blank group (25.3%)and shNC group (24.3%). Conclusion The lentivirus miR30 vector targeting CD133 gene was successfully constructed, which will be a basis to the further CD133 gene studies.
