The HPV-E7/miR-106a/RUNX3/TGF-β1 axis regulates malignant progression in both HPV-positive and negative head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) remains a prevalent malignancy worldwide, posing significant health threats due to its high recurrence and metastatic potential. HPV-positive and negative HNSCC subtypes exhibit distinct prognostic profiles and their underlying pathogenic mechanisms remain poorly characterized.

METHODS: Four HNSCC cell lines were selected: two HPV-positive (UM-SCC-47 and UPCI-SCC-090) and two HPV-negative (FaDu and UM-SCC-4). Basal miR-106a expression was measured in HPV-positive and -negative cells, followed by RT-qPCR validation of miR-106a, HPV-E7, RUNX3 overexpression and knockdown efficiency. Functional assays included CCK-8 for proliferation, wound healing for migration, Transwell for invasion, and flow cytometry for apoptosis. RT-qPCR quantified HPV-E7, miR-106a, RUNX3, and TGF-β1 mRNA levels; RUNX3 and TGF-β1 protein expression was assessed via Western blot. Dual-luciferase reporter assays confirmed the direct targeting of miR-106a to RUNX3. Finally, xenograft nude mouse models assessed miR-106a's effects on tumor growth and downstream molecular regulation in HPV-positive and -negative HNSCC.

RESULTS: Comparative analysis revealed that miR-106a was significantly upregulated in HPV-positive HNSCC cells compared to their HPV-negative counterparts. Functional assays demonstrated that miR-106a overexpression enhanced HNSCC cell proliferation, migration, and invasion while suppressing apoptosis, whereas ectopic expression of RUNX3 exerted opposing effects on these oncogenic phenotypes. Mechanistically, miR-106a overexpression transcriptionally downregulated RUNX3 and concurrently elevated TGF-β1 expression, while RUNX3 overexpression inversely suppressed TGF-β1 levels. Dual-luciferase reporter assays confirmed a direct binding interaction between miR-106a and the 3'UTR of RUNX3. Rescue experiments further established that HPV E7-driven oncogenic effects-enhanced proliferation, migration, invasion, and apoptosis suppression-were abrogated by miR-106a inhibition, concomitant with restored expression of RUNX3 and attenuated TGF-β1 signaling. In vivo studies corroborated these findings, showing that miR-106a overexpression accelerated tumor growth in xenograft models, accompanied by progressive RUNX3 downregulation and TGF-β1 upregulation, consistent with its in vitro regulatory axis.

CONCLUSIONS: Our findings suggest that the E7/miR-106a/RUNX3/TGF-β1 axis modulates proliferation, migration, invasion, and apoptosis in HPV-positive versus negative HNSCC, implicating its pathogenic role in tumor progression.