Figure 4

Inhibition of miR-21 in INS1 cells blocks the effects of inflammatory cytokines on β-cell identity and overexpression of Tgfb2 ameliorates effects of miR-21 on β-cells. Compared to 24-h treatment with IL1- β alone, pretreatment with a miR-21 inhibitor resulted in (A) reduced Aldh1a3 expression. (B) Cytokine-induced reductions in Tgfb2 and Smad2, Pdx1, and Ins1 were also abrogated by pre treatment with a miR-21 inhibitor, with a trend toward an increase in levels of MafA, Ins2, and Glut2. Wild-type cytokine untreated results are shown for comparison, with statistical comparisons performed between the cytokine-treated control inhibitor and miR-21 inhibitor groups. (C) The qRT-PCR analysis demonstrated that transcripts for Tgfb2 and Smad2 are increased in INS1-miR-21 cells treated with a Tgfb2 overexpression vector as compared to INS1-miR-21 control cells. (D) Overexpression of Tgfb2 resulted in decreased expression of β-cell progenitor markers Ngn3, Nanog, and L-myc in INS1-miR-21 cells. (E) Overexpression of Tgfb2 also resulted in increased expression of Pdx1, Ins1, Ins2, and Neurod1. n = 3–5; ∗p < 0.05.