Inhibition of piR48444 promotes osteogenesis via METTL7A-mediated BMP2 mRNA m6A 1 methylation

Non-coding RNAs, including piwi-interacting RNAs (piRNAs), regulate the osteogenic 33 differentiation of bone marrow-derived MSCs (BMSCs). However, the role and mechanism of 34 piRNAs in the osteogenic differentiation of stem cells from human exfoliated deciduous teeth 35 (SHED) are unknown. Here, the piRNA/mRNA-seq revealed that piR48444 downregulation is 36 critically related to the upregulated osteogenic genes METTL7A, BMP2, and FOXO1 in SHED. 37 PiR48444 overexpression inhibited, and piR48444 knockdown promoted osteogenesis by targeting 38 METTL7A to regulate BMP2 mRNA m6A methylation, increasing BMP2 mRNA stability and 39 translation. Furthermore, METTL7A-eIF4E complex binds with BMP2 mRNA, further facilitating 40 the translation. BMP2 promoted FOXO1 expression, which induced the expression of ALP and 41 RUNX2. Moreover, piR48444 knockdown increased the osteogenic differentiation of MSCs from 42 various sources via the METTL7A/BMP2 axis. The treatment with piR48444 antagomir protected 43 bone loss in lipopolysaccharide-treated and aging mice. In conclusion, piR48444 inhibits 44 osteogenic differentiation of MSCs via METTL7A-mediated regulation of BMP2 mRNA m6A 45 methylation, suggesting bioengineering of piR48444 inhibitors to promote MSCs-based bone 46 regeneration.

非编码RNA（non-coding RNAs）包括piwi相互作用RNA（piRNAs），可调控骨髓来源间充质干细胞（bone marrow-derived MSCs, BMSCs）的成骨分化。然而，piRNAs在脱落乳牙来源干细胞（human exfoliated deciduous teeth, SHED）成骨分化中的作用与机制尚未阐明。本研究通过piRNA/mRNA测序发现，piR48444的下调与SHED中成骨相关基因METTL7A、骨形态发生蛋白2（BMP2）及叉头框蛋白O1（FOXO1）的上调显著相关。过表达piR48444可抑制成骨过程，而敲低piR48444则通过靶向METTL7A调控BMP2 mRNA的m6A甲基化，增强BMP2 mRNA的稳定性与翻译效率，从而促进成骨。进一步研究显示，METTL7A与真核翻译起始因子4E（eIF4E）形成的复合物可结合BMP2 mRNA，进一步促进其翻译。BMP2可上调FOXO1的表达，而FOXO1又可诱导碱性磷酸酶（ALP）及runt相关转录因子2（RUNX2）的表达。此外，敲低piR48444可通过METTL7A/BMP2轴，促进多种来源间充质干细胞的成骨分化。向经脂多糖（lipopolysaccharide, LPS）处理及衰老的小鼠体内给予piR48444反义寡核苷酸拮抗剂（antagomir），可改善其骨丢失症状。综上，piR48444可通过METTL7A介导的BMP2 mRNA m6A甲基化调控，抑制间充质干细胞的成骨分化，这提示可通过开发piR48444抑制剂以促进基于间充质干细胞的骨再生治疗。