N6-methyladenosine modification of lnc-CCKAR-5 regulates autophagy in human umbilical cord mesenchymal stem cells by destabilizing LMNA and inhibits diabetic wound healing

Long noncoding RNAs(lncRNAs) are pivotal contributors to the development of human diseases. However, their significance in the context of diabetic wound healing regulated by human umbilical cord mesenchymal stem cells(hUCMSCs) remains unclear. This study sheds light on the involvement of lncCCKAR5 in this process. We found that hUCMSCs exposed to high glucose conditions exhibited a significant downregulation of lncCCKAR5 expression, and lncCCKAR5 played a critical role in modulating autophagy, thus inhibiting apoptosis in hUCMSCs. Additionally, the reduction of LncCCKAR5 in cells exposed to high glucose effectively thwarted cellular senescence and facilitated filopodium formation. Mechanistically, LncCCKAR5 served as a scaffold that facilitated the interaction between MKRN2 and LMNA, a key regulator of cytoskeletal function and autophagy. The LncCCKAR5/LMNA/MKRN2 complex played a pivotal role in promoting the ubiquitin-mediated degradation of LMNA, with this effect being further augmented by N6-adenosine methylation of LncCCKAR5. Consequently, our findings underscore the critical role of LncCCKAR5 in regulating the autophagic process in hUCMSCs, particularly through protein ubiquitination and degradation. This intricate regulatory network presents a promising avenue for potential therapeutic interventions in the context of diabetic wound healing involving hUCMSCs. Overall design: RNA-seq libraries were constructed by NewCore Biotech (Shanghai, China). Firstly, RNA was isolated with TRIZOL reagent (Invitrogen, Grand Island, NY), total RNA was purified by the Qiagen RNeasy mini kit (Qiagen, Valencia, CA). Purified RNA was analyzed on an ND-8000 spectropho tometer (Nanodrop Technologies, Wilmington, DE). High quality total RNA was used as the starting material. Briefly, total RNA was used for removing the rRNAs with NEBNext rRNA Depletion Kit (New England Biolabs, Inc., Massachusetts, USA) following the manufacturer's instructions. RNA libraries were constructed by using NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs, Inc., Massachusetts, USA) according to the manufacturer's instructions. Libraries were controlled for quality and quantified using the BioAnalyzer 2100 system (Agilent Technologies,Inc., USA). Library sequencing was performed on an Illumina NovaSeq 6000 instrument with 151 bp paired end reads.