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. 2021 Sep 23;13(2):425–440. doi: 10.1016/j.jcmgh.2021.09.009

Figure 2.

Figure 2

STAT3 activates microRNA expression in a SIGIRR-dependent manner. (A) Quantitative-ChIP assay was performed using anti-STAT3 antibody to analyze the recruitment of STAT3 on promoters of miR-146a, miR-155, and mir-215 in HIECs. Values were quantified against IgG controls. (B and C) ChIP quantitative PCR values of STAT3 recruitment on microRNA promoters in HIECs with indicated transfection. (D) Promoter reporter activity assay was performed in HEK293T cells transfected with luciferase reporter plasmid pGL4.1 empty vector or pGL4.1 containing human miR-146a promoter, and thymidine kinase promoter–Renilla luciferase reporter plasmid (pRL-TK), serving as a internal transfection control. Luciferase assay was performed with cell lysates using the Dual-Luciferase Reporter Assay System(Promega, Madison, WI). (E) Promoter reporter activity assays were performed in HEK293T cells co-transfected with pGL4.1–miR-146a promoter and indicated STAT3 plasmids. (F) Promoter reporter activity assays were performed in HEK293T cells using PGL4.1–miR-146a promoter with nucleotide mutations at STAT3 binding sites. HEK293T were co-transfected with wild-type miR-146a promoter luciferase reporter or mutant reporter. The relative luciferase activities obtained from 5 to 6 independent experiments are shown as means ± SD. (G) microRNA expression was quantified by real-time PCR in HIECs treated with STAT3 inhibitor, STATTIC, for 24 hours at the indicated concentration. (H) microRNA expression was quantified by real-time PCR in HIECs transduced with control shRNA or Stat3 shRNA. ∗P < .05, ∗∗∗P < .005. ACTB, actin beta; DM, mutations at STAT3 binding site 1 and site 2; EV, pcDNA3–empty vector; M1, mutations at STAT3 binding site 1; SCR, scrambled; SIGIRR, pcDNA3–SIGIRR; 168X, pcDNA3–SIGIRR P168X.