The Influence of pcDNA 3.0-WWOX Recombinant Vector on the Expression of WWOX and △Ψm in Gallbladder Carcinoma Cells

［Abstract］Objective　To explore the influence of PcDNA3.0-WWOX eukaryotic expression vector on the expression of WWOXmRNA，protein and transmembrane potential of mitochondrion（△Ψm） in human gallbladder carcinoma cells. Methods Total RNA in the human gallbladder carcinoma cells was extracted. Then the cDNA of WWOX gene was synthesized by RT-PCR and PCR products were reclaimed and purified. Both of WWOX gene and pcDNA3.0 vector were cut by restriction enzymes ECOR Ⅰ and XhoⅠ. The double enzyme WWOX fragments and pcDNA 3.0 vector were restructured under the action of T4 DNA ligase. After PcDNA 3.0-WWOX recombinant plasmid transformation，the extraction of the plasmid by double enzyme cutting was sent to identification and sequencing identification. Then the pcDNA 3.0-WWOX recombinant plasmid was used to transfect gallbladder cancer GBC-SD cells. RT-PCR and Western Blot were used to detect the expression level of WWOX gene and JC-1 dyeing was used to detect the transmembrane potential （△Ψm） of gallbladder cancer cells' mitochondria．Results　We successfully construct pcDNA3.0 - WWOX recombinant plasmid，and the sequencing analysis showed that the DNA sequence was the same to WWOX sequence in Gene Bank. pcDNA3.0 - WWOX recombinant plasmid was successfully transfected to gallbladder cancer GBC- SD cells. RT-PCR and Western blot results showed that 48 hours after plasmid transfection in gallbladder cancer GBC-SD cells，the mRNA gray ratio of pcDNA-3.0-WWOX group was obviously higher than that of the control groups（P＜0.05），the amount of mRNA expression between the control groups had no significant difference（P＞0.05）. The protein gray ratio of pcDNA-3.0-WWOX group was obviously higher than that of the control group（P＜0.05）， and the amount of protein expression between the control groups had no significant difference（P＞0.05）. The green fluorescent signals in mitochondria in cells in the pcDNA-3.0-WWOX group was higher than the control groups（P＜0.05），while had no significant difference between the control groups（P＞0.05）. Conclusions We have successfully constructed PcDNA3.0-WWOX recombinant vector and performed transformation and transfection of this vector in vitro. Targeted PcDNA3.0 - WWOX recombinant vector can effectively inhibit WWOX mRNA degradation in the gallbladder cancer cells， effectively promote the expression of the protein and early apoptosis of the cells. WWOX gene may participate in mitochondria dependent apoptotic pathways which regulates gallbladder cancer cells growth.