Loss of miR-29a/b1 promotes inflammation and fibrosis in acute pancreatitis
Shatovisha Dey, Lata M. Udari, Primavera RiveraHernandez, Jason J. Kwon, Brandon Willis, Jeffrey J. Easler, Evan L. Fogel, Stephen Pandol, Janaiah Kota
Shatovisha Dey, Lata M. Udari, Primavera RiveraHernandez, Jason J. Kwon, Brandon Willis, Jeffrey J. Easler, Evan L. Fogel, Stephen Pandol, Janaiah Kota
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Research Article Gastroenterology Genetics

Loss of miR-29a/b1 promotes inflammation and fibrosis in acute pancreatitis

Abstract

MicroRNA-29 (miR-29) is a critical regulator of fibroinflammatory processes in human diseases. In this study, we found a decrease in miR-29a in experimental and human chronic pancreatitis, leading us to investigate the regulatory role of the miR-29a/b1 cluster in acute pancreatitis (AP) utilizing a conditional miR-29a/b1–KO mouse model. miR-29a/b1-sufficient (WT) and -deficient (KO) mice were administered supramaximal caerulein to induce AP and characterized at different time points, utilizing an array of IHC and biochemical analyses for AP parameters. In caerulein-induced WT mice, miR-29a remained dramatically downregulated at injury. Despite high-inflammatory milieu, fibrosis, and parenchymal disarray in the WT mice during early AP, the pancreata fully restored during recovery. miR-29a/b1–KO mice showed significantly greater inflammation, lymphocyte infiltration, macrophage polarization, and ECM deposition, continuing until late recovery with persistent parenchymal disorganization. The increased pancreatic fibrosis was accompanied by enhanced TGFβ1 coupled with persistent αSMA+ PSC activation. Additionally, these mice exhibited higher circulating IL-6 and inflammation in lung parenchyma. Together, this collection of studies indicates that depletion of miR-29a/b1 cluster impacts the fibroinflammatory mechanisms of AP, resulting in (a) aggravated pathogenesis and (b) delayed recovery from the disease, suggesting a protective role of the molecule against AP.

Authors

Shatovisha Dey, Lata M. Udari, Primavera RiveraHernandez, Jason J. Kwon, Brandon Willis, Jeffrey J. Easler, Evan L. Fogel, Stephen Pandol, Janaiah Kota

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Figure 3

Validation of transgenic miR-29a/b1–KO mouse model.

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Validation of transgenic miR-29a/b1–KO mouse model. (A) Schematic for th...
(A) Schematic for the generation of conditional pancreas-specific miR-29a–KO transgenic mice. Representation of miR-29a/b1 WT allele (top), miR-29a/b1–floxed allele with 2 LoxP sites (blue triangles) flaking miR-29a/b1 cluster (middle) and miR-29a/b1–floxed allele after Cre recombination (bottom). miR-29a/b1–floxed mice were crossed with a mouse strain that express Cre recombinase under pancreas-specific promoter (Pdx1-Cre) to generate miR-29a/b1–KO mice. (B) Genotypes of the miR-29a/b1–KO mice were confirmed from tail snips utilizing a PCR strategy to generate fragments with specific sizes for WT allele (miR-29ab1+/+; 140 bp), heterozygous allele (miR-29ab1+/–; 140 and 180 bps), and homozygous miR-29a/b1–KO allele (miR-29ab1–/–; 180 bp). Cre+ was determined by the presence or absence of a 410 bp fragment. (C–E) Expression of miR-29a, -b, and -c respectively in WT (miR-29ab1+/+, Cre-; n = 6), miR-29a/b1 Het (miR-29ab1+/–, Cre+; n = 6), and miR-29a/b1–KO (miR-29ab1–/–, Cre+; n = 6) mice confirmed by qPCR analysis of total RNA obtained from the mouse pancreata (n = 6). (F–H) miR-29a expression in WT and miR-29a/b1–KO mouse acinar cells (n = 4) (F), islets (n = 4) (G), and PSCs (n = 4) (H) isolated from the pancreas and measured by qPCR analysis (n = 4). Graphs represent mean ± SEM; *P < 0.05; **P < 0.01, 1-way ANOVA with Tukey’s post hoc test (C–E), 2-tailed Student’s t test (F–H).