Construction of lentiviral vector-mediated Cx32 stably over-expressed Huh7 cell line and its effect on cell proliferation
10.3969/j.issn.1001-1978.2018.02.026
- VernacularTitle:慢病毒介导稳定高表达Cx32的Huh7细胞系建立及其对细胞增殖的影响
- Author:
Yu-Ting JI
1
;
Yan YANG
;
Rong-Sheng ZHENG
;
Wu-Qiong ZHANG
;
Jing LIU
Author Information
1. 蚌埠医学院第一附属医院肿瘤内科
- Keywords:
hepatocellular carcinoma;
connexin32;
lentivirus;
vector construction;
stable transfection;
therapeutic target
- From:
Chinese Pharmacological Bulletin
2018;34(2):284-289
- CountryChina
- Language:Chinese
-
Abstract:
Aim To construct a lentiviral vector for stable delivery of the connexin32 (Cx32) gene in human hepatocellular carcinoma cell line Huh7,and also to detect its effect on cell proliferation.Methods Human Cx32 gene sequence was obtained by whole gene synthesis and amplified by PCR,and was then inserted into LV5-GFP lentiviral vector to construct the recombinant plasmid LV5-GFP-hCx32.Following identified by restriction endonuclease digestion and DNA sequencing,this plasmid together with lentiviral package plasmid was transfected into 293T cells to produce the lentiviral particles,and the viral titer was assessed under fluorescent microscope.Targeted Huh7 cells were infected with the lentivirus (LV5-hCx32 and LV5-NC),and the infected cells after selection with puromycin were amplified and cultured.The expression and localization of Cx32 were detected by real-time RT-PCR,Western blot and immunofluorescence assay,respectively.The gap junction (GJ) function between adjacent cells was measured by dye transfer assay.The Huh7 cell proliferation capacity was determined by MTT and colony formation assays.Results The results of double enzyme digestion and DNA sequencing proved that the recombinant lentiviral vector LV5-GFP-hCx32 was successfully constructed.After packing in 293T cells,the recombinant lentivirus LV5-GFP-hCx32 with virus drops to 3 × 1011 TU · L-1 was obtained.Huh7 cells transiently infected with the lentivirus LV5-GFP-hCx32 remarkably over-expressed Cx32 at both mRNA and protein levels.Moreover,Cx32 expression was also significantly up-regulated in stably transfected Huh7 cells,and the presence of enhanced functional GJ in intact cells could be detected due to an increased amount of Cx32 protein along the plasma membrane at cell-cell contacts.Compared to LV5-NC group,the proliferation ability in Huh7 cells with recombinant Cx32 declined (P ＜ 0.05).Conclusions The lentiviral vector over-expressing Cx32 gene is successfully constructed,and can stably transfect Huh7 cells to yield sustained over-expression of exogenous Cx32 gene,thus eventually inhibits cell proliferation.
