Construction and identification of the recombinant vector silencing peroxisome proliferator-activated receptor-γand expressing calcitonin gene-related peptide gene

Abstract

Objective To construct the recombinant vector silencing peroxisome proliferator-activated receptor-γ (PPARγ) and expressing calcitonin gene-related peptide (CGRP) gene.Methods The pGFP-V-RS vector we selected contains a small interfering RNA (siRNA) expressing unit,and also can express exogenous gene inserted.The PPARγ siRNA oligonucleotides sequence was ligated into pGFP-VRS vector after annealling,and then PPAR siRNA recombinant vector (pGFP-V-RS-siPPARγ) was obtained by restriction analysis and DNA sequencing.The CGRP gene fragment was ligated into linear plasmids pGFP-V-RS-siPPARγrecombinant vector,and then the recombinant vector pGFP-V-RS-siPPARγ-exCGRP was obtained by restriction analysis and DNA sequencing.The HEK-293 cells were collected and divided into 4 groups at random.In control group,the cells were only transfected with vector pGFP-V-RS plasmid.In SI group,the cells were transfected with recombinant vector pGFP-V-RS-siPPARγplasmid.In EX group,the cells were transfected with recombinant vector pGFP-V-RS-exCGRP plasmid.In DOU group,the cells were transfected with pGFP-V-RS-siPPARγ-exCGRP plasmid.There were 5 × 106 HEK-293 cells in electrode cup of each group,and 2.5 μl above-mentioned plasmids were added respectively.The cells were given DC voltage pulse one time (15 ms).The cells were harvested in each group after culture for 48 h,and CGRP and PPARγmRNA and protein expression levels were detected by using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.Results The results of restriction analysis and DNA sequencing were completely matched with those of the designed sequences after the PPARγsiRNA oligonucleotides sequence and CGRP gene fragment were cloned into pGFP-V-RS vector.After recombinant vector pGFP-V-RS-siPPARγ-exCGRP was transfected into HEK-293 cells,the expression levels of PPARγ mRNA and protein were reduced in DOU group DOU (0.036 ±0.003 and 0.108 ±0.031)as compared with EX group (0.156 ±0.014 and 0.778 ±0.103) and control group (0.148 ±0.014 and 0.742 ± 0.143) (P ＜ 0.05),but showed no significant difference in comparison to SI group (0.054 ± 0.005and 0.135±0.039) (P＞0.05).The expression level of CGRP mRNA and protein was significantly increased in DOU group (1.144 ±0.106 and 1.244 ±0.184) as compared with IS group (0.320 ±0.030 and 0.370 ±0.089) and control group (0.444 ±0.041 and 0.274 ±0.062) (P ＜0.05),but there was no significant difference in comparison to EX group (1.021 ±0.095 and 1.221 ±0.181) (P ＞0.05).Conclusion We successfully constructed the recombinant vector pGFP-V-RS-siPPARγ-exCGRP that silencing PPARγand expressing CGRP gene,and the recombinant vector can efficaciously suppress PPARγgene expression and promote CGRP gene expression simultaneously. Key words: RNA interference; Peroxisome proliferator-activated receptor-γ; Calcitonin generelated peptide; Recombinant vector