Mir 100 5p mimic

The full length of Circ‐9110 was extended by PCR and then cloned in a pcD2.1‐cir vector (Geneseed Biotech, Guangzhou, China). Given that the CDSs of HOXA1 X1 were not amplified in dairy goats, the CDSs of HOXA1 X2 were cloned and inserted in pcDNA3.1(+) vectors (Promega, Madison, USA).
MiR‐100‐5p mimic, NC mimic, miR‐100‐5p inhibitor, NC inhibitor, Circ‐9110‐siRNA and HOXA1‐siRNA were synthesized with Ribobio (Guangzhou, China). The ESCs were seeded at a density of 7.5 × 10⁵ cells/well in 6‐well plates and then transfected at 60% confluency with MiR‐100‐5p mimic, miR‐100‐5p inhibitor, NC mimic or NC inhibitor, pcDNA3.1, pcDNA3.1‐HOXA1 and pcD2.1‐ciR or pcD2.1‐circ‐9110 at 100 nM final concentrations by using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's specifications.

MiR-221-3p mimic (5′‐AGC UAC AUU GUC UGC UGG GUU UC‐3′), miR-221-3p inhibitor (5′‐GAA ACC CAG ACA AUG UAG CU‐3′) and their corresponding negative controls (NC mimic, NC inhibitor), and overexpression plasmids of fibroblast growth factor 2 (FGF2) (pcDNA-FGF2) and its negative control (vector) were obtained from RiboBio (Guangzhou, China). AMSCs were seeded in 6-well plates at 37°C in 5% CO₂. When the cells grew to 80% confluence, Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA) was used to transfect NC mimic (50 nM), miR-100-5p mimic (50 nM), NC inhibitor (50 nM), miR-100-5p inhibitor (50 nM), vector (30 nM), and pcDNA-FGF2 (30 nM) into AMSCs according to the manufacturer's instructions. Cells were collected after transfection for 48 h for further experiments.