Construction of Smac siRNA lentiviral vector and its silencing effect on Smac gene in human LECs

Authors: Li Yun,  Zheng Guangying,  Jiang Yu

DOI: 10.3760/cma.j.issn.2095-0160.2016.03.002
Published 2016-03-10
Cite as Chin J Exp Ophthalmol, 2016,34(3): 199-204.

Abstract

Background

Research comfirmed that second mitochondrial activator of caspase (Smac) is a promoting tumor cell apoptosis protein.Our previous study showed that the expression level of Smac in LECs is obviously higher in cataract than that in normal eyes.We assumed that silencing Smac gene in LECs can inhibit the apoptosis of LECs.The way to transfect Smac siRNA into LECs is a key step.

Objective

This study was to construct siRNA lentiviral vector of Smac and identify its silencing efficiency in human lens epithelial B3 cell line(HLE-B3) and establish low-expressed Smac HLE-B3 line.

Methods

Based on the genebank and our previous study, siRNA sequence of Smac was designed and composed.The synthetic double-stranded DNA was linked to the lentiviral vector GV118 by T4 DNA ligase and then transformed DH5α competent cells.The plasmids were transformed into the DH5α competent cells.Recombinant colonies were screened by PCR and sequenced.Recombinant plasmids and two other auxiliary plasmids were used to infect 293T cells.Cell culture supernatant was collected for the measurement of viral titer.Recombinant lentiviral vector was used to infect HLE-B3 cells to calculate the viral multiplicity of infection (MOI) under the fluorescence microscope.Transfection efficiency was examined by calculating the GFP-positive cells.HLE-B3 cells were divided into negative control group, siRNA plasmid tranfected group and GV118-Smac-siRNA1 tranfected group.The relative expression levels of Smac mRNA in the cells were detected and compared among the three groups by real-time fluorescent quantitative PCR.

Results

GV118-Smac-siRNA was successfully constructed with the positive colonies 340 bp and blank vector colonies 299 bp, and viral titer was 3.0×108 TU/ml.At a MOI of 100, the infecting efficiency of the vector on HLE-B3 cells was about 82% and the cytotoxicity was low.The relative expression levels of Smac mRNA were (101.290±8.349)%, (92.330±6.320)% and (32.540±4.221)% in the negative control group, siRNA plasmid tranfected group and GV118-Smac-siRNA1 tranfected group, respectively, showing a significant difference among the three groups(F=32.871, P<0.01), and the relative expression level of Smac mRNA was significantly lower in the GV118-Smac-siRNA1 tranfected group than that in the negative control group (P=0.000). However, no significant difference was found in the Smac mRNA expression between the blank plasmid group and the negative control group (P=0.535).

Conclusions

GV118-Smac-siRNA lentiviral vector is successfully constructed.Smac-siRNA can effectively inhibit the expression of Smac mRNA in human LECs.

Key words:

Mitochondrial proteins; Apoptosis; RNA, small interfering; Genetic vectors; Lentivirus/genetics; Gene transfer techniques; Humans; Lens epithelial cells

Contributor Information

Li Yun
Department of Ophthalmology, the First Affiliated Hospital of Zhengzhou University, the Key Discipline Open Laboratory of Clinical Medicine for Institutions of Higher Learning in Henan Province, Zhengzhou 450052, China
Zheng Guangying
Jiang Yu
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Updated: October 9, 2019 — 9:17 am