| Background and purpose Newcastle disease virus(NDV)belongs to the poultry virus,which can cause explosive infection of poultry,causing serious economic losses to poultry industry,and is listed as one of the Class A infectious diseases that must be reported.At the same time,NDV is also an oncolytic virus,which is basically non-pathogenic to humans and is a potential candidate for tumor biotherapy.NDV can infect tumor cells,directly induce tumor cell membrane fusion,and cause tumor cell necrosis and apoptosis,which is one of the main anti-tumor mechanisms of the virus.Hemagglutinin-neuraminidase(HN)is the main anti-tumor protein in NDV.Lentivirus(lentivirus)is a diploid RNA retrovirus.The vector is based on the genome of the lentivirus,which removes multiple sequences of genes related to the pathogenicity of the virus,and then inserts the desired target gene fragments into the vector genome.Due to high transfection efficiency,lentivirus vectors can infect many kinds of cells,and the vectors can contain large target gene fragments,so they are widely used in research.Micro RNA is a small RNA composed of 20-24 nucleotides,which plays an important role in the development of tumor.Studies have shown that micro RNA-204 is related to the apoptosis of lung cancer cells.Bcl-2 is an oncogene that inhibits apoptosis and is associated with lung cancer cell apoptosis.It was found that the apoptosis of liver cancer cell lines transfected with NDV-HN gene was related to NDV-HN,suggesting that NDV could play an anticancer role through the apoptosis of liver cancer cells induced by HN.By constructing human lung adenocarcinoma A549 cell line overexpressed HN protein,this study aims to study the apoptosis of HN-induced lung adenocarcinoma A549 cells,the expression of micro RNA-204 in cells and the expression of anti-apoptotic gene Bcl-2,providing theoretical basis for the basic research of NDV7793-HN for anti-tumor biological therapy.Methods1.The plasmid pc DNA3.1-HN with the full-length NDV7793-HN gene was saved by our laboratory and it was sent to the company to construct the sf GFP-HN vector and sequencing NDV7793-HN.2.The sf GFP-HN plasmid and auxiliary plasmids PH1 and PH2 were co-transfected into 293 V cells for lentivirus packaging.The supernatant was collected to concentrate the virus particles.The lentivirus in the experimental group was named Lv-sf GFP-HN.At the same time,the unloaded plasmid group was set as the control group,and lentiviruses in the unloaded group were packaged and concentrated in the same way,and the lentivirus in the unloaded group was named Lv-sf GFP.3.The lentivirus particles were diluted 10 times gradient to infect 293 V cells,the expression of green fluorescent protein was observed under fluorescence microscope.Meanwhile,Puromycin was used to determine the lentivirus titer,and drug screening was conducted to select stable transfected monoclonal cell lines 293V-sf GFP-HN and 293V-sf GFP.4.The obtained lentivirus particles were infected with A549 cells,and the expression of GFP was observed under a fluorescence microscope after 72 h of infection.The screening medium containing puromycin was replaced for drug screening,and stable transfected monoclonal cell lines A549-sf GFP-HN and A549-sf GFP were selected.5.The transfection rates of 293V-sf GFP-HN cells,293V-sf GFP cells,A549-sf GFP-HN cells and A549-sf GFP cells were determined by flow cytometry.6.The expression of NDV7793-HN m RNA in 293V-sf GFP-HN cells,293V-sf GFP cells,293 V cells,A549-sf GFP-HN cells,A549-sf GFP cells and A549 cells were detected by RT-PCR.7.Western blot analysis was used to detect the expression of HN protein in A549-sf GFP-HN cells,A549-sf GFP cells and A549 cells.8.RT-PCR was used to detect the expression of micro RNA-204 in A549-sf GFP-HN cells,A549-sf GFP cells and A549 cells.9.RT-PCR and western blot were used to detect Bcl-2 expression in A549-sf GFP-HN cells,A549-sf GFP cells and A549 cells.10.Western blot was used to detect Bcl-2 protein expression in A549-sf GFP-HN cells,A549-sf GFP cells and A549 cells.11.The apoptosis of A549-sf GFP-HN cells and A549 cells were detected by flow cytometer and their apoptosis after cisplatin addition were detected by flow cytometer.Results1.It was showed by the sequencing analysis that the vector plasmid sf GPF-HN had been successfully constructed,which was consistent with the pc DNA3.1-HN sequence.2.After transfection for 48 h,GFP expression was observed in 293 V cells by fluorescence microscopy,indicating successful plasmid co-transfection,3.After infection for 72 h,GFP expression was observed in 293 V cells under fluorescence microscopy,indicating successful lentivirus packaging,Puromycin titer determination showed that Lv-sf GFP-HN concentrated virus titer was 1×10~6 TU/ml,and Lv-sf GFP concentrated virus titer was 1×10~7 TU/ml.4.After infection for 72 h,GFP expression was observed in A549 cells under fluorescence microscope,indicating that lentivirus successfully infected A549,and Puromycin was successfully screened,indicating the stable transfected monoclonal cell lines with A549-sf GFP-HN and A549-sf GFP.5.The transfection rates of 293V-sf GFP-HN cells,293V-sf GFP cells,A549-sf GFP-HN cells and A549-sf GFP cells were detected by flow cytometry,and the transfection efficiency reached about 90%,indicating successful cell screening.6.RT-PCR showed that HN m RNA expression was appeared in293v-sf GFP-HN cells and A549-sf GFP-HN cells,but was not in other cells.7.Western blot showed that HN protein expression in A549-sf GFP-HN cells,but no HN expression in A549-sf GFP cells and A549 cells.8.RT-PCR showed that micro RNA-204 expression in A549-sf GFP-HN cells was significantly up-regulated compared with that in A549-sf GFP and A549 cells.9.RT-PCR showed that the m RNA expression of Bcl-2 in A549-sf GFP-HN cells was significantly down-regulated compared with that in A549-sf GFP and A549 cells.10.Western blot showed that Bcl-2 protein expression in A549-sf GFP-HN cells was significantly down-regulated compared with A549-sf GFP cells and A549 cells.11.Flow cytometry showed that the apoptosis rate of A549-sf GFP-HN cells increased after the addition of cisplatin,compared with that of A549 cells after the addition of cisplatin.Conclusions1.In order to investigate whether the NDV-HN protein causes lung cancer apoptosis and its mechanism,we successfully packaged lentivirus Lv-sf GFP-HN and Lv-sf GFP,and constructed a stable transfected A549-sf GFP-HN cell line,and confirmed that A549-sf GFP-HN cell line can express HN protein.2.A549-sf GFP-HN can up-regulate the expression of micro RNA-204 related to apoptosis of human lung adenocarcinoma A549 cells,meanwhile,the expression of anti-apoptotic protein Bcl-2 in A549 cells was down-regulated,and the apoptosis rate of A549-sf GFP-HN cells after cisplatin was higher than that of A549 cells.This indicates that HN can down-regulate the expression of Bcl-2 through micro RNA-204,and increase the apoptosis rate of A549-sf GFP-HN cells induced by cisplatin.It is preliminarily proved that HN can play an anticancer role by promoting the apoptosis of lung adenocarcinoma. |
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