Effect Of Lentivirus-mediated ZnT1RNAi On BDNF Level In Neurons And The Dynamic Associated Changes Between Autophagy And Apoptosis After Spinal Cord Injury

ObjectiveTo construct a lentiviral vector of RNA interference (RNAi) of ZnT1gene anddetect the effect of the vector in rat neurons and the best affect conditions. Atthe different affection of the vector in rat neurons on ZnT1, we detected theespression of ZnT1and BDNF of neurons to promulgate the regulatorymechanism of ZnT1on BDNF/Trkb pathway. In addition, we detected thecorrelation among the dynamic change of autophagy and apoptosis as well asZnT1and autophagy after SCI in order to announce the relation betweenautophagy and apoptosis as well as the effect of autophagy in zinc metabolismafter SCI.Methods1. Disigned and synthesized siRNA oligonucleotides which targeted ZnT1gene of Sprague-Dawley rats. At the same time, the negative controloligonucleotides were systhesized. The synthesized sequences of theoligonucleotides were formed a double-stranded DNA through annealing. ThepFU-GW-iRNA plasmid was digested by Hpa I and Xho I, and the linear plamidwas produced. Then, the linear plamids were confirmed by DNA sequencing.2. HEK293T cells were cultured and cotransfected with lentiviral vectorpFU-GW-iRNA and packing plasmidspHelper1.0, pHelper2.0to producelentiviral vector which can infect other cells. The lentiviral vectors were namedas LV-NC/shRNA, LV-ZnT1/shRNA-A, LV-ZnT1/shRNA-B andlV-ZnT1/shRNA-C according to the different siRNA sequences. And then thetiter of the lentiviral vector was detected. 3. Neurons of rat were prepared from Sprague-Dawley rats according to thepreviously described methods. And NSE staining was used to detected that thecells were certainly neurons. The neurons were transfected under the differentMOI conditions (MOI=1,3,6,8) to confirm the best transfect conditions. Afterthat, the transfected neurons under the best transfect consider was divided intoZnT1-siRNA-A group, ZnT1-siRNA-B group, ZnT1-siRNA-C group, controlgroup and negative control group. The changes of ZnT1protein was detectedby Western blot.4. The neurons were transfected with the lentiviral vectors under the bestMOI conditions.72h after transfection, neurons were collected. RT-PCR andWestern blot were used to detect the expression of ZnT1and BDNF. Elisamethod was used to detect the BDNF release in culture supernatant, at thesame time, cell vitality of neurons after ZnT1-RNAi was determined by MTTmethod.5. Total192male Sprague-Dawley rats were divided into two groups:sham-operated group and SCI group. SCI was induced by impact-contusion ofthe spinal cord at T10. Transmission electron microscope (TEM) was used todeterminate the confirmation of autophagy. We traced LC3, Caspase-3, Bax andBcl-2with RT-PCR and Western blot as well as ZnT1and LC3with western blotat1h,2h,6h,12h,24h,3d,7d after SCI.Results1. The target oligonucleotide was accurately cloned into pFU-GW-iRNAplasmid. The lentiviral particles were harvested. The titer was8×108TU/mL. AtMOI=6, the transfection was about93%and the neurons were at a goodcondition.The ZnT1expression was inhibited in the three groups, the inhibitioneffects were about47.74±1.40%，81.19±1.36%，94.10±2.41%.2. The inhibition on ZnT1of A, B, C sequences ZnT1-siRNA group was34.82±9.35%,42.98±4.85%,65.78±1.71%respectively at mRNA level and32.11±1.70%,39.57±2.55%,63.15±3.82%respectively at protein level. Theinhibition on BDNF of A, B, C sequences in ZnT1-siRNA group was14.91±1.80%,24.46±4.11%,37.10±4.35%respectively at mRNA level and 36.94±2.09%,22.09±1.79%,46.92±1.91%respectively at protein. What wasmore, there were positive correlations between inhibitions of ZnT1and BDNFboth at mRNA level and protein level (Spearman rho=0.787, P<0.01at mRNAlevel, Spearman rho=0.453, P=0.030at protein level). MTT showed therewere no differents in cell vitality.3. At2h after SCI, the vacuolation of mitochondria and swelling ofendoplasmic reticulum could be seen. At24h after injured, the formations ofautophagic vacuoles containing membranous structures and cytoplasmicorganelles, secondary lysosomes, and homogenous membrane structurescould be detected. There were positive correlations between LC3, Bax andCaspase-3(Spearman rho=0.448, P=0.014at protein level and spearman rho=0.787, P <0.01at mRNA level between LC3and Bax; spearman rho=0.397,P=0.027at protein level and spearman rho=0.641, P <0.01at mRNA levelbetween LC3and caspase-3). Bcl-2expression increased after SCI followingCaspase-3and Bax, however while Caspase-3and Bax were at their peak, theexpression of Bcl-2was significantly inhibited.4. Within24h after SCI, ZnT1genes expression and autophagy (LC3Ⅱ/LC3Ⅰ) expression showed the simLar change, there was a positive correlationbetween the ZnT1and LC3Ⅱ/LC3Ⅰ, spearman rho=0.829, P=0.042.Conclusions1. ZnT1-siRNA lentivirus vector has been constructed successfully, MOI=6is the best transfect condition and it can effectively silence the ZnT1expressionin neurons.2. The expression of BDNF could be reduced by lentivirus-mediatedZnT1-RNAi in neurons. These results suggested that zinc might regulate BDNFexpression via ZnT1which could be a potential target for the regulation of BDNFexpression.3. Autophagy is induced after SCI, and it has a positive correlation withapoptosis, suggesting apoptosis and autophagy may cooperate with each other.What’s more, we showed dynamic changes of apoptosis and autophagy after SCI, and this will direct us intervention after SCI including time point andtreatment.4. Autophagy may play a certain effect in the zinc transportion andmetabolism after SCI.