m6A RNA modification regulates GAS5 RNA stability and strengthens contraction ability of myometrium cells. (A) m6A immunohistochemistry staining in not in labor (NIL) and in labor (IL) myometrium. (B) RIP analyze was done to detect the underlying upstream regulating m6A enzymes, including METTL3, METTL16, AKKBH5, FTO, YTHDC1, YTHDF1/2/3, and IGF2BP1/2. (C) METTL3 and IGF2BP1 mRNA were detected higher expressed in IL myometrium than that in NIL myometrium by qRT-PCR. (D) METTL3 and IGF2BP1 immunohistochemistry staining in NIL and IL myometrium. (E) Transfection efficiency in primary myometrium cells with specific METTL3 siRNAs and its overexpression plasmid. (F) 3D Collagen gel matrix was tested in myometrium cells after METTL3 siRNA#3 and plasmid transfected. (G) Transfection efficiency in primary myometrium cells with specific IFG2BP1 siRNAs and its overexpression plasmid. (H) 3D collagen gel matrix applied after IFG2BP1 siRNA#3 and plasmid transfected. (I-J) Actinomycin test was done to find the effect of METTL3 and IGF2BP1 on GAS5 RNA stability. (K-M) MeRIP was done to find the specific conjugated sites of GAS5 RNA. IGF2BP1 enrichment was found to be at the 68-147 bp and 471-557 bp in the 5′ untranslated region (5′UTR) of GAS5. Mettl3 enrichment was found to be at the 68-147 bp in the 5′ untranslated region (5′UTR).