Identification of acvr2a as a target gene of miR-181a in mouse granulosa cells (mGC).

<p>(A) Putative binding sites for human (hsa) miR-181a and mouse (mmu) miR-181a in the 3′-UTR of the acvr2a gene. (B) mGC was infected Ad-miR-181a or Ad-LacZ and cotransfected with the luciferase-acvr2a-3′UTR construct. After 48 h, luciferase assays were performed. *p<0.05, **p<0.01, compared with Ad-LacZ group. (C) HEK293T cells were transfected with plasmid pEGFP-C1 carrying the 3′-UTR of acvr2a at the 3′-terminus of green fluorescent protein (GFP) alone or together with plasmid Ad-miR-181a. After 48 h, GFP expression was monitored by observation of GFP fluorescence and Western blot analysis. Acvr2a mRNA (D) and protein (E) levels in mGC were measured by qRT-PCR and Western blotting after infection of Ad-miR-181a for 48 h. *p<0.05, compared with Ad-LacZ group. (F) Result of a CCK-8 assay examining the proliferation of mGC being infected with Ad-miR-181a and/or Ad-flag-m acvr2a for 48 h. *p<0.05, compared with Ad-LacZ group; ^#p<0.05, compared with Ad-flag-m acvr2a and Ad-miR-181a alone. Relative protein levels were measured by densitometry using Quantity One Software and normalized to β-actin, the control group; the ratios were presented above the Western blot bands.