Construction Of Recombinant Coxsackievirus MiR-204/375-pMKS1-CVB3 Eukaryotic Expression Vector And Validation Of Its Expression In Gastric Cancer Cells

Research background:Gastric cancer currently is the fifth most common malignancy worldwide,with increasing incidence and mortality rates in China.Surgical resection and postoperative radiotherapy remain the principal treatments,however,it often accompany with adverse effects.Therefore,requirement of new adjuvant treatments for gastric cancer is urgent.Immunotherapy,including oncolytic virus therapy,cellular immunotherapy,immune checkpoint inhibitor therapy,and cancer vaccines,is promising approach to cancer treatment that involves manipulating the human immune system to restart the anti-tumor immune response.Oncolytic viruses(OVs)treatment is at the forefront of immunotherapy and offers new possibilities for adjuvant treatment of gastric cancer.OVs can selectively infect tumor cells and cause their death without harming normal cells.However,modifying these lysing viruses to reduce the risk of pathogenicity during infection is currently the primary focus of research in this area.Coxsackievirus B3(CVB3)is one of the most commonly used oncolytic viruses.However,its potential risk in gastrointestinal(GI)tumors is causing myocardium and pancreas injury.Therefore,modifying oncolytic viruses to address these limitations and shortcomings has become a hot topic of research in this area.Micro RNA(miRNA),approximately 22 nt,non-coding RNA,plays crucial roles in gene regulations.Abnormal expression of miRNAs is associated with various human diseases,including tumors.In this study,we constructed a recombinant oncolytic virus vector encoding Coxsackievirus B3(CVB3)using genetic engineering.The aim was to support subsequent investigations into the mechanism of the anti-tumor effect of recombinant miR-204/375-p MKS1-CVB3 in gastric cancer,which has significant scientific and clinical translation value.Research methods:In this study,we conducted bioinformatics analysis using the db DEMC(database of differentially expressed miRNAs in human cancers)and miRCancer database to screen for miRNAs related to the suppression of gastric cancer.We obtained and identified the significantly differentially expressed miRNA set comparing gastric cancer tissues to paracarcinoma tissues.Screened miRNAs were further validated by literature analysis using the miRCancer database and potential miRNAs down-regulated during gastric cancer pathogenesis were identified.Bioenrichment analysis was also performed to study their preliminary biological functions.Moreover,based on the publication of Markian’s Lab,miR-375-ts,working as a protector to reduce the virulence effect of the virus on the pancreas,was selected to be inserted into the recombinant vector.The sequences with primiR-204 and three times miR-375-ts were planed to insert into the 5’UTR region of the CVB3 genome using the multiple overlapping-PCR method.The recombinant expression plasmid clones were selected and screened by transformation assay and identified by sequencing.The recombinant viral expression vector and original vector were separately transfected into human gastric cancer AGS cell line.The viral replication levels of time course were determined by RT-q PCR.Finally,we obtained alive viral particles by harvesting transfected AGS cells,which would be applied into subsequent studies on the biological traits and functions of the virus.Research results:The down-regulation of miR-204-5p expression in gastric cancer cells was determined which would promote the proliferation of gastric cancer cells.The anti-tumor effect of miR-204-5p and the protecting effect of miR375 on CVB3-induced pancreatic damage were used to modify the p MKS1-CVB3 eukaryotic expression vector,and the recombinant mir-204/375-p MKS1-CVB3 eukaryotic expression vector was successfully constructed.The recombinant mir-204/375-p MKS1-CVB3 eukaryotic expression vector was successfully constructed and validated by sequencing.The plasmid was transfected with human gastric cancer AGS cell line,and the replication level of viral genomic m RNA in the host at different time points was detected by RT-q PCR.The recombinant virus replication levels were reduced compared to the wild type virus vector(p<0.05).At 72 h the virus started to replicate at high levels and produced a cytopathic effect(CPE).Conclusion:(1)Bioinformatics analysis showed that miR-204-5p was significantly differentially expressed in gastric cancer and paracarcinoma tissues.Down-regulation of miR-204-5p in gastric cancer cells could promote the proliferation of gastric cancer cells;(2)A recombinant CVB3 eukaryotic expression vector with pri-miR-204-5p and miR-375-ts was successfully constructed,and recombinant viral particles were harvested.(3)The recombinant viral plasmid was expressed in human gastric cancer AGS cells for 96 h at a lower replication level compared to the original viral expression vector.