Oral squamous cell carcinoma (OSCC) is a significant global health concern. Epstein–Barr virus (EBV) infection as well as long non-coding RNA (lncRNAs) associated EBV infection, have been linked to OSCC development and are known to influence cancer progression. LINC00944 is associated with various cancers and immune cells, but its role in oral cancer remains underexplored. This study investigated the role of EBV-induced LINC00944 in OSCC and its impact on the tumor microenvironment. The LINC00944 expression was analyzed from a database of head and neck squamous cell carcinoma (HNSCC) tissues, and its expression in EBV-positive and EBV-negative OSCC cell lines was examined via qRT-PCR. We overexpressed LINC00944 in SCC25 and ORL-48T oral cancer cell lines and evaluated its impact on migration and invasion ability using wound healing and transwell experiments. Additionally, we studied its influence on macrophage differentiation. The results showed that LINC00944 expression was higher in HNSCC than in normal tissues and was linked to EBV-positive OSCC cell lines. LINC00944 overexpressed-OSCC cell lines significantly increased cellular motility and invasiveness. Additionally, LINC00944 was secreted in a cultured medium, delivered to macrophages, and promoted macrophage differentiation into the M1 subtype. Predicted interactions suggested that LINC00944 targets miRNAs that regulate NFKB1 and RELA. In conclusion, EBV-induced LINC00944 contributes to OSCC progression by enhancing tumor cell migration, invasion, and macrophage differentiation, potentially regulating these processes through NFKB1 and RELA. These findings provide valuable directions for LINC00944’s future studies on its mechanisms and suggest that it could be a target of study in EBV-associated OSCC.

Dysregulated long non-coding RNA (lncRNA) expression is linked to various cancers and may be influenced by oncogenic Epstein–Barr virus (EBV) infection, a known and detectable risk factor in oral squamous cell carcinoma (OSCC) patients. However, research on the oncogenic role of EBV-induced lncRNAs in OSCC is limited. To identify lncRNA-associated EBV infection and OSCC carcinogenesis, the differential expression of RNA-seq datasets from paired normal adjacent and OSCC tissues, and microarray data from EBV-negative and EBV-positive SCC25 cells, were identified and selected, respectively, for interaction, functional analysis, and CCK-8 cell proliferation, wound healing, and invasion Transwell assays. In OSCC tissues, 6731 differentially expressed lncRNAs were identified when compared to normal tissues from RNA-seq datasets, with 295 linked to EBV-induced OSCC carcinogenesis from microarray datasets. The EBV-induced lncRNA VWA8-AS1 showed significant upregulation in EBV-positive SCC25 cells and EBV-infected adjacent and OSCC tissue samples. VWA8-AS1 potentially promotes OSCC via the lncRNA–miRNA–mRNA axis or direct protein interactions, affecting various cellular processes. Studies in OSCC cell lines revealed that elevated VWA8-AS1 levels enhanced cell migration and invasion. This study demonstrates VWA8-AS1’s contribution to tumor progression and possible interactions with its targets in OSCC, offering insights for future research on functional mechanisms and therapeutic targets in EBV-associated OSCC.