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 2

Generation of miR-29a/b1–floxed allele for development of miR-29a/b1–KO murine model.

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Generation of miR-29a/b1–floxed allele for development of miR-29a/b1–KO ...
(A) Schematic representation for the generation of miR-29a/b1–floxed allele using CRISPR/Cas9 technology. (B) PCR analysis for validation of cleaved fragment consisting of guide RNA generated by cleavage assay. Lane 1: 1 kb ladder with 100 bp fragment at the bottom (labeled), with 200, 300, 400, 500, and 600 bp (labeled) at the top respectively. Lane 2: miR-29b (5′) Control, 563 bp. Lane 3: miR-29b (5′) crRNA, 333/230 bp (80% cleavage). Lane 4: miR-29b (3′) Control, 625 bp. Lane 5: miR-29a (3′) crRNA, 336/289 bp (80% cleavage). (C) TaqMan assays were designed to screen for allelic fragments consisting of both 5′ and 3′ LoxP sites, utilizing a unique LoxP dual-labeled probe for each site. ΔCt for each LoxP site was measured by quantification of the (Fam-labeled) target LoxP Ct value relative to that of an endogenous housekeeping gene Tcrd (VIC labeled) in each sample. Presence of the target locus was screened based on the ΔCt value, where the value closest to zero (least difference between the genomic copies of the housekeeping gene and the target site) represented significant genomic copy number for the site. Each blue dot for the 3′ and 5′ LoxP sites represents the positive controls used in the assay. Orange dots for each site depict some of the screened animals, with the 2 orange dots exactly above the positive control (blue dots) for each 3′ and 5′ sites that were double-positive for the 2 LoxP sites.