Construction Of Pin1shRNA Lentiviral Vector And It’s Effect On The Human Alveolar Epithelial Cells Apoptosis Induced By Hyperoxia

Objective: The peptidyl prolyl cis/trans isomerase (Pin1) has beenaroused widespread interest as an inhibitor of tumor cell targets. Pin1couldplay an important role in the oxidative stress pathway, but it is seldom reportedif the inhibition of it’s expression could reduce the hyperoxia lung injury. RNAiare regarded as a fast and efficient way of gene silence, which can regulatetarget gene expression. The research of gene function and signal transductionpathway had attracted more and more attention. To construct a Pin1shRNAlentiviral vector and tranfect stably A549cells. The role of Pin1in oxidativestress signal pathway mediating the alveolar epithelial apoptosis induced byhyperoxia were investigated. Methods: The gene sequences were cloned intopLenR-GPH-shRNA lentiviral vector, which was selected by Genebanksearchers. The pLenR-GPH-shRNA and lentiviral vector packaging plasmidmix were cotransfected into293T cells to package lentivirus particles.Culturevirus supernatant was harvested, and then the virus titer was determined byserial dilution assay. A549cells were transduced with the constructed lentiviralvectors, and real-time polymerase chain reaction (qPCR) and Western blot wereused to evaluate the Pin1expression. The study was divided into a controlgroup, a hyperoxia group, an A549-Pin1shRNA hyperoxia group and a negativelentivirus group. The hyperoxia group, A549-Pin1shRNA hyperoxia group andnegative lentivirus group were exposed to a mixture of O2(900ml/L)and CO2(50ml/L) for10minutes, then cultured in a closed environment for24h.The control group was cultured with10%FBS and1%streptomycin1640medium. The change of morphology were observed under inverted microscopeafter exposure to oxygen or room air for24hours; Cell apoptosis was detectedby flow cytometry (FC) after24hours; the expression of Caspase-9and XIAPwere detected by immunohistochemistry; the production of ROS (reativeoxygen species), and cellular mitochondria membrane potential (△Ψm) weredetermined by fluorescence microscopy. Results:(1) Restriction enzymeanalysis and sequencing demonstrated that the recombinant plasmid namedpLenR-GPH-Pin1shRNA was successfully prepared and had a higher titer.qPCR and Western blot revealed that the significant down-regulation of Pin1expression in A549cells.(2)Under the inverted microscopy, A549cells in thecontrol group were grew in good condition and sticked to each other tightly.The suspension cells were less. Compared with the control group, the changesin morphology of A549were most obvious in the hyperoxia group and negativelentivirus group. It were grew showly, the living cells decreased and had alarge number of suspension cells. The gap between cells were increased.However, the changes in morphology of A549were obviously improved in theA549-Pin1shRNA hyperoxia group.(3) Flow cytometry showed that comparedto the hyperoxia group and the negative lentivirus group, the apoptosis rate ofA549cells had not statistical significant difference(P>0.05). The control groupwas significantly decreased; But compared with the hyperoxia group,the apoptosis rate of A549cells were obviously decreased in the A549-Pin1shRNAhyperoxia group (P<0.05), and not reached the control group(P<0.05).(4)Immunohistochemistry results showed the followings, the expression ofCaspase-9and XIAP in the hyperoxia group and the negative lentivirus groupwere no statistical significant difference(P>0.05). Compared with the controlgroup, the expression of Caspase-9were significantly increased and XIAP weredecreased (P<0.05); But the expression Caspase-9and XIAP of theA549-Pin1shRNA hyperoxia group were between them (P<0.05).(5)Fluorescence microscopy showed that mitochondrial ROS were redfluorescence, and the nuclei were blue fluorescence. Only a faint redfluorescence in the control group. There were no difference between thehyperoxia group and the negative lentivirus group (P>0.05). Compared withthe control group, the production of mitochondrial ROS were increasedsignificantly （P＜0.05). The production of mitochondrial ROS in theA549-Pin1shRNA hyperoxia group were decreased（P＜0.05）, but not reachedthe control group(P<0.05).(6) Fluorescence microscopy showed thefollowings, compared with the control group, the mitochondrial membranepotential were decreased significantly（P＜0.05）, but no difference between thehyperoxia group and the negative lentivirus group(P>0.05). Compared with thehyperoxia group, the mitochondrial membrane potential were increased in theA549-Pin1shRNA hyperoxia group(P<0.05), but not reached the controlgroup(P<0.05). Conclusion:1. We established a A549-Pin1shRNA and inhibited itsexpression. Both qPCR and Western blot demonstrated down-regulation of Pin1expression in A549cells.2. The hyperoxia was thought to activate PKCβ,resulting in phosphorylation of p66shc. This is turn activates the Pin1resultingin isomerization of p66shc and chondrosome transposition. It could increase theROS production, decrease the mitochondrial membrane potential and increaseapoptosis rate of A549cells. In the A549-Pin1shRNA hyperoxia group, wefound dampened oxidative stress. It is expected to become a new treatment ofhyperoxia-induced acute lung injury.