m6A reader YTHDC1 modulates autophagy by targeting SQSTM1 in diabetic skin.

ABSTRACT

Dysregulation of macroautophagy/autophagy contributes to the delay of wound healing in diabetic skin. N6-methyladenosine (m6A) RNA modification is known to play a critical role in regulating autophagy. In this study, it was found that SQSTM1/p62 (sequestosome 1), an autophagy receptor, was significantly downregulated in two human keratinocyte cells lines with short-term high-glucose treatment, as well as in the epidermis of diabetic patients and a db/db mouse model with long-term hyperglycemia. Knockdown of SQSTM1 led to the impairment of autophagic flux, which was consistent with the results of high-glucose treatment in keratinocytes. Moreover, the m6A reader protein YTHDC1 (YTH domain containing 1), which interacted with SQSTM1 mRNA, was downregulated in keratinocytes under both the acute and chronic effects of hyperglycemia. Knockdown of YTHDC1 affected biological functions of keratinocytes, which included increased apoptosis rates and impaired wound-healing capacity. In addition, knockdown of endogenous YTHDC1 resulted in a blockade of autophagic flux in keratinocytes, while overexpression of YTHDC1 rescued the blockade of autophagic flux induced by high glucose. In vivo, knockdown of endogenous Ythdc1 or Sqstm1 inhibited autophagy in the epidermis and delayed wound healing. Interestingly, we found that a decrease of YTHDC1 drove SQSTM1 mRNA degradation in the nucleus. Furthermore, the results revealed that YTHDC1 interacted and cooperated with ELAVL1/HuR (ELAV like RNA binding protein 1) in modulating the expression of SQSTM1. Collectively, this study uncovered a previously unrecognized function for YTHDC1 in modulating autophagy via regulating the stability of SQSTM1 nuclear mRNA in diabetic keratinocytes.Abbreviations ACTB actin beta; AGEs glycation end products; AL autolysosome; AP autophagosome; ATG autophagy related; AKT AKT serine/threonine kinase; ANOVA analysis of variance; BECN1 beclin 1; Co-IP co-immunoprecipitation; DEGs differentially expressed genes; DM diabetes mellitus; ELAVL1 ELAV like RNA binding protein 1; FTO FTO alpha-ketoglutarate dependent dioxygenase; G glucose; HaCaT human keratinocyte; GO Gene Ontology; GSEA Gene Set Enrichment Analysis; HE hematoxylin-eosin; IHC immunohistochemical; IRS immunoreactive score; KEAP1 kelch like ECH associated protein 1; KEGG Kyoto Encyclopedia of Genes and Genomes; m6A N6-methyladenosine; M mannitol; MANOVA multivariate analysis of variance; MAP1LC3 microtubule associated protein 1 light chain 3; MAP1LC3B microtubule associated protein 1 light chain 3 beta; MeRIP methylated RNA immunoprecipitation; METTL3 methyltransferase 3, N6-adenosine-methytransferase complex catalytic subunit; MTOR mechanistic target of rapamycin kinase; MTORC1 mechanistic target of rapamycin complex 1; NBR1 NBR1 autophagy cargo receptor; NFE2L2 nuclear factor, erythroid 2 like 2; NG normal glucose; NHEK normal human epithelial keratinocyte; OE overexpressing; p- phospho-; PI propidium iodide; PPIN Protein-Protein Interaction Network; RBPs RNA binding proteins; RIP RNA immunoprecipitation; RNA-seq RNA-sequence; RNU6-1 RNA, U6 small nuclear 1; ROS reactive oxygen species; siRNAs small interfering RNAs; SQSTM1 sequestosome 1; SRSF serine and arginine rich splicing factor; T2DM type 2 diabetes mellitus; TEM transmission electron microscopy; TUBB tubulin beta class I; WT wild-type; YTHDC1 YTH domain containing 1.