Identification And Preliminary Verification Of Host Factors That Restrict Oncolytic HSV-1 (oHSV-1) Replication In Cancer Cells

Cancer is a major public health problem worldwide.The launch of multiple novel agents,companion diagnostics,and new types of treatment,makes a series of breakthrough in cancer treatment,which has contributed to improved treatment outcomes and a reduction in cancer mortality rates for major developed countries over the past decade.Oncolytic virotherapy recognized as an emerging cancer therapeutic modality,has become one of the most potential and promising cancer treatment options.In recent years,more than 60 oncolytic virus-related clinical trails have been conducted,oncolytic virus T-VEC was launched for marketing in 2015,which has become the first FDA approved virotherapy for advanced cancer.Although numerous oncolytic viruses in clinical trails has been proved with good safety and antitumor activity,due to the complexity and heterogeneity of the tumor,the efficacy of oncolytic virotherapy varied in different types of tumors or in different patients of the same type of tumor.This difference in clinical response prompted us to study the mechanism of selective killing of oncolytic viruses in order to identify the host restricting factors that may play a key role in vrial oncolysis.These findings would help guide the future clinical application of oncolytic viruses,or provide a good chance to increase response rate by developing new therapeutic drugs to release the limitations of the host factor so as to further enhance the efficacy of oncolytic viruses.In this study,using a well constructed oncolytic virus oHSV-1,a small number of virus-resistant HeLa cells were found and proved to be able to resist the killing of oHSV-1.The resistant cells were enriched by repeated viral infection and finally established as a cell line,named HeLa-R.To investigate the molecular mechanisms of virus-resistant effect of HeLa-R cells,we first compared the gene expression levels of HeLa and HeLa-R cells using transcriptome sequencing(RNA-Seq),we obtained 448 differentially expressed genes,of which 197 genes were up-regulated and 251 genes were down-regulated in HeLa-R.Then we further performed GO functional annotation and KEGG enrichment analysis on these 448 genes.We found that GO functional annotations of the differential genes were mainly enriched in biological processes and cell components.KEGG enrichment analysis showed that the most significant enrichment was the interaction of extracellular matrix receptors.In addition,multiple biological processes such as cell matrix adhesion and cancer-related signaling pathways were also enriched.Thus,we analyzed and validated the RNA-Seq results from two aspects,significantly up-regulated genes and enriched signaling pathways in HeLa-R.Twenty-two genes with significantly upregulated expression were selected and knocked-down by RNA interference technology.We then explore the impact of the down-regulation of these gene on cell killing ability of oHSV-1 in HeLa-R,using microscopic observation,crystal violet staining and WST-1 cell viability assay.The results showed that the three genes,CREB3L1,CXCL1 and OLR1 may play important roles in preventing HeLa-R cells from virus-mediated cell lysis.Among them,CXCL1 was constantly expressed at a relatively high level in other virus-resistant cancer cells,and the cell killing ability of oHSV-1 was enhanced in CXCL1 knocked-down cancer cells.In addition,we selected corresponding small molecule inhibitors for key signaling molecules in partial KEGG enrichment signaling pathways to verify whether the relevant signaling pathways play a role in HeLa-R cell resistance to oHSV-1 killing.It revealed that the MEK1/2 inhibitor U0126(MAPK signaling pathway)and the Cdk4/6 inhibitor Palbociclib(Cell cycle signaling pathway)can enhance the lytic activity of oHSV-1 in HeLa-R.In this study,RNA-Seq was used to screen the host factors for restricting the cell killing ability and replication of oncolytic viruses.We found that CREB3L1,CXCL1 and OLR1,all play imoportnat roles in virus-resistant cells,the anti-viral mechanism of these genes was also preliminarily explored.The results will provide guidance for the clinical application of oncolytic virus oHSV-1,and provide potential molecular targets for the improvement of viral oncolytic activity.