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 <i>SQSTM1</i> 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 <i>SQSTM1</i> mRNA, was downregulated in keratinocytes under both the acute and chronic effects of hyperglycemia. Knockdown of <i>YTHDC1</i> affected biological functions of keratinocytes, which included increased apoptosis rates and impaired wound-healing capacity. In addition, knockdown of endogenous <i>YTHDC1</i> resulted in a blockade of autophagic flux in keratinocytes, while overexpression of <i>YTHDC1</i> rescued the blockade of autophagic flux induced by high glucose. <i>In vivo</i>, knockdown of endogenous <i>Ythdc1</i> or <i>Sqstm1</i> inhibited autophagy in the epidermis and delayed wound healing. Interestingly, we found that a decrease of YTHDC1 drove <i>SQSTM1</i> 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 <i>SQSTM1</i>. Collectively, this study uncovered a previously unrecognized function for YTHDC1 in modulating autophagy via regulating the stability of <i>SQSTM1</i> nuclear mRNA in diabetic keratinocytes.<b>Abbreviations:</b> 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; <i>RNU6-1</i>: 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.