Supplementary Material for: YTHDC1-modified m6A methylation of hsa_circ_0102678 promotes keratinocyte inflammation induced by Cutibacterium acnes biofilm through regulating miR-146a/TRAF6 and IRAK1 axis

Introduction: CircRNAs are closely related to many human diseases, however, their role in acne remains unclear. This study aimed to determine the role of hsa-circ_0102678 in regulating inflammation of acne. Methods: Firstly, microarray analysis was performed to study the expression of circRNAs in acne. Subsequently, RNase R digestion assay and FISH assay were utilized to confirm the characteristics of hsa-circ_0102678. Finally, qRT-PCR, Western blotting analysis, Immunoprecipitation, Luciferase reporter assay, circRNA probe pull-down assay, Biotin-labeled miRNA pull-down assay, RNA immunoprecipitation (RIP) assay and m6A dot blot assay were utilized to reveal the functional roles of hsa-circ_0102678 on inflammation induced by C. acnes biofilm in human primary keratinocytes. Results: Our investigations showed that the expression of hsa-circ_0102678 was significantly decreased in acne tissues and hsa-circ_0102678 was a type of circRNAs, which was mainly localized in the cytoplasm of primary human keratinocytes. Moreover, hsa-circ_0102678 remarkably affected the expression of IL-8, IL-6, and TNF-α, which induced by C. acnes biofilm. Importantly, mechanistic studies indicated that the YTHDC1 could bind directly to hsa-circ_0102678 and promote the export of N6-methyladenosine modified hsa-circ_0102678 to the cytoplasm. Besides, hsa-circ_0102678 could bind to miR-146a and sponge miR-146a to promote the expression of IRAK1 and TRAF6. Conclusion: Our findings revealed a previously unknown process by which hsa_circ_0102678 promoted keratinocyte inflammation induced by C. acnes biofilm via regulating miR-146a/TRAF6 and IRAK1 axis.