Co-Transfection With Anti-Fibrotic microRNAs for Treating Oral Submucous Fibrosis

Background: Oral submucous fibrosis is characterized by excessive collagen deposition and is highly associated with a patient's betel nut chewing habit. Arecoline initiates the transforming growth factor-beta (TGF-β)/Smads signaling pathway and activates downstream fibrotic genes. Dysregulation of microRNA (miR) expression is involved in the OSF progression, and miR modulation is a promising treatment. As one miR can target multiple mRNAs, and one mRNA has multiple binding sites to different miRs, we thus propose that simultaneous co-transfection of anti-fibrotic miRs may have a better therapeutic effect than single miR transfection. Methods: Human oral fibroblasts were first subjected to arecoline stimulation and then transfected with 16 miRs individually. Based on the ability to downregulate TGFB1 and actin alpha 2, smooth muscle (ACTA2) mRNA levels, the miR-29a-3p mimic, miR-196a-3p mimic, and miR-509-5p mimic were selected for co-transfection. Results: In addition to downregulation of collagen type I alpha 1 chain (COL1A1), COL3A1, COL5A1, matrix metalloproteinase-1 (MMP1), MMP7, tissue inhibitor of metalloproteinase-1 (TIMP1), and TIMP2 mRNA expressions, co-transfection with the three miRs led to a more significant downregulation of COL1A1 and MMP1 expressions. A Western blot analysis revealed that co-transfection of the miRs efficiently suppressed the TGF-β/Smads pathway and extracellular matrix component productions. Furthermore, co-transfection with miRs more effectively inhibited wound closure and collagen gel contraction compared to single miR transfection. Conclusions: Co-transfection of anti-fibrotic miRs can be a promising treatment for oral submucous fibrosis.