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

Figure 4.

Figure 4

SIGIRR suppresses TLR-induced inflammation partially via anti-inflammatory microRNAs. (A) SIGIRR inhibits flagellin-induced proinflammatory cytokine expression through STAT3. HIECs stably expressing lentiviral Stat3 shRNA or scramble shRNA (SCR) were transfected with pcDNA3–empty vector (EV) or pcDNA3-SIGIRR for 48 hours, then treated with flagellin at 50 ng/mL for 8 hours. Proinflammatory cytokines and microRNA expression were quantified by real-time PCR. (B) miR-146a inhibitor restores IL1b and C-X-C motif chemokine ligand 1 (CXCL1) expression suppressed by SIGIRR. HIECs were treated with flagellin for 8 hours after 48 hours of transfection of pcDNA3-SIGIRR and miR-146a inhibitor or scramble control. IL1b and CXCL1 expression were analyzed by real-time PCR. (C) HIECs were transfected with plasmids and miR-146a mimic or scramble control as indicated for 48 hours, then treated with flagellin for 6 hours at 100 ng/μL. IL1b and CXCL1 expression were analyzed by real-time PCR. (D) HIECs were transfected with pcDNA3–EV, pcDNA3–SIGIRR, and pcDNA3–SIGIRR p.P115R mutant, and cell lysates were analyzed by Western blot using the indicated antibodies. Densitometry quantification is shown graphically. (E) microRNA expression was quantified by real-time PCR in HIECs with indicated transfection. ∗P < .05, ∗∗P < .01, ACTB, actin beta.