Bta mir 16a

The second exon sequence of the INSR gene was inserted into the pCD2.1 vector (Geneseed Biotech, Guangzhou, China) and psi-CHECK2 vector (Promega, Fitchburg, WI, USA). Small interfering RNA (siRNA) oligonucleotides were designed to combine with the back-splice region of circINSR (RiboBio, Guangzhou, China). These siRNAs inhibited the expression of circINSR after transfection into the cell and was named si-circINSR. The mimics of bta-miR-15a, bta-miR-15b, bta-miR-16a, and bta-miR-16b were purchased from RiboBio (Guangzhou, China). The 3′-untranslated regions (UTRs) of the cyclin D1 (CCND1) and B-cell lymphoma 2 (Bcl-2) genes containing the miR-15/16 binding sites were amplified using the PCR enzyme mix (Platinum II Taq Hot-Start DNA Polymerase, Invitrogen). The wild-type and mutant 3'-UTR gene sequences were cloned into the psi-CHECK2 vector. The Renilla: Firefly ratio was measured and compared against that for the non-treated control. The mimics (50 nM) or vectors (2 μg/mL) were transfected into cells using a transfection reagent (R0531, Thermo Fisher Scientific, USA). For the overexpression of the miR-15/16 family, the miR-15a, miR-15b, miR-16a, and miR-16b mimics were mixed in equal amounts for transfection.