MicroRNA-9 modified bone marrow-derived mesenchymal stem cells (BMSCs) repair severe acute pancreatitis (SAP) via inducing angiogenesis in rats

Abstract

Background: Severe acute pancreatitis (SAP) is an acute abdominal disease characterized by pancreatic necrosis and systemic disease. In a previous study, we showed that bone marrow-derived mesenchymal stem cells (BMSCs) can reduce SAP by secreting microRNA (miR)-9; however, the underlying mechanism remains unclear. The present study investigated the mechanism underlying BMSC-induced pancreatic regeneration.

Methods: BMSCs were isolated, and miR-9 modified/antagonized BMSCs (pri-miR-9-BMSCs/TuD-BMSCs) were generated and injected into SAP rats. The levels of inflammatory cytokines and histopathologic changes were examined using ELISA and H&E staining. Angiogenesis was analyzed by qRT-PCR, western blotting, and immunohistochemistry. Cell function tests, dual luciferase reporter assays, cell co-culture, western blotting, and cell tracing were used to explore the mechanisms underlying miR-9 induced angiogenesis.

Results: Pri-miR-9-BMSCs induced angiogenesis in SAP rats (Ang-1↑, TIE-2↑, and CD31↑) and repaired damaged vascular endothelial cells (VECs) in vitro, promoting angiogenesis (Ang-1↑, TIE-2↑, PI3K↑, AKT↑, p-AKT↑, CD31↑, and CD34↑). Pri-miR-9-BMSCs released miR-9 into VECs or injured pancreatic tissue, targeting the VE-cadherin gene and promoting PI3K/AKT signaling to treat SAP (VE-cadherin↓, β-catenin↓, PI3K↑, p-AKT↑), whereas antagonizing miR-9 in BMSCs did not alleviate or aggravated SAP.

Conclusions: Pri-miR-9-BMSCs can repair injured pancreatic tissue by secreting miR-9 and promoting angiogenesis.

Fig. 1

miR-9 modified BMSCs (pri-miR-9-BMSCs) alleviate SAP. a BMSCs infected by pri-miR-9 and empty virus expressing GFP. b The recombinant pri-miR-9-1-PCDH plasmid was identified by dual-enzyme digestion. c pri-miR-9-1 sequence was amplified from pri-miR-9-1 plasmid using special primers. d, e The expression of mature miR-9 in pri-miR-9-BMSCs was higher than in empty virus BMSCs (EV-BMSCs) by gPCR and qRT-PCR. Data are shown as the mean ± SD for at least 3 separate experiments. ^###p < 0.001, compared with EV-BMSCs by paired t test, gPCR, General PCR, qRT-PCR, quantitative real-time PCR. f, g, h, i pri-miR-9-BMSCs significantly reduced pancreatic edema, infiltration, hemorrhage and necrosis, the release of serum amylase, lipase and pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and increased anti-inflammatory cytokines (IL-4, IL-10, and TGF-β) compared with SAP, SAP+PBS, BMSCs, EV-BMSCs and TuD-BMSCs groups. Data are shown as the mean ± SD for at least three separate experiments. ^%p < 0.05,^%%p < 0.01, ^%%%p < 0.001, compared with NC, **p < 0.01 and ***p < 0.001, compared with NC, ^@@p < 0.01 and ^@@@p < 0.001, compared with SAP, ^&&p < 0.01 and ^&&&p < 0.001, compared with PBS treatment, ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001, compared with BMSCs, ^$p < 0.05, ^$$p < 0.01, and ^$$$p < 0.001, compared with EV-BMSCs, ⁺p < 0. 05 and ⁺⁺⁺p < 0.001, compared with TuD-BMSCs by using two-tailed t test

Fig. 2

pri-miR-9-BMSCs promote angiogenesis. a Angiogenesis-related genes (CXCR4, Ang-1, TIE-2, and CD31) were significantly upregulated after pri-miR-9-BMSC transplantation, compared with SAP, SAP+PBS, BMSCs, or TuD-BMSCs groups. Anti-angiogenic genes (VE-cadherin, β-catenin) were significantly decreased compared with SAP, SAP+PBS, BMSCs, EV-BMSCs, or TuD-BMSCs groups. The PI3K/AKT signaling pathway (PI3K, p-AKT, AKT) was activated by pri-miR-9-BMSCs. Data are shown as the mean ± SD for at least 3 separate experiments. *p < 0.05, **p < 0.01 and ***p < 0.001, compared with NC, ⁺⁺p < 0.01 and ⁺⁺⁺p < 0.001, compared with SAP, ^$p < 0.05 and ^$$p < 0.01, compared with SAP+PBS, ^#p < 0.01,^##p < 0.001, compared with BMSCs, ^&p < 0.05,^&&p < 0.01, compared with EV-BMSCs, ^@p < 0.05,^@@p < 0.01, compared with TuD-BMSCs. b The results of IHC showed that the CD31 and CD34 were significantly upregulated by pri-miR-9-BMSCs. Data are shown as the mean ± SD. ***p < 0.001, compared with pancreas by using two-tailed t test

Fig. 3

(a, b) The distributions in vivo of CM-Dil- and SPION-labeled BMSCs were observed by fluorescence microscopy and Prussian Blue staining. The number of cells migrating to the damaged pancreas was smaller than that of cells migrating to the liver, spleen and lung. Data are shown as the mean ± SD. ***p < 0.001, compared with liver, ^###p < 0.001, compared with spleen, ^@@@p < 0.001, compared with lung by using two-tailed t test

Fig. 4

miR-9, released from BMSCs into VECs, can target the VE-cadherin gene and induce the expression of angiogenic genes in VECs. a miR-9 in pancreatic tissues was downregulated in the PBS + SAP group compared with the NC group, whereas it was significantly upregulated by pri-miR-9-BMSCs, compared with SAP, SAP+PBS, BMSCs, EV-BMSCs, and TuD-BMSCs. Data are shown as the mean ± SD for at least three separate experiments. ^%p < 0.05, compared with NC, ^**p < 0.01, compared with NC, ^@@p < 0.01, compared with SAP, ^&&p < 0.01, compared with PBS treatment, ^#p < 0.05, compared with BMSCs, ^$p < 0.05, compared with EV-BMSCs, ⁺p < 0.05, compared with TuD-BMSCs by using two-tailed t test. b GFP-BMSCs could deliver exogenous Cy3-miR-9a-5p to the liver, spleen, lung, and pancreas, and the number was higher in the liver and spleen. c Eight paired base between miR-9 and VE-cadherin and the structure of the wild-type VE-cadherin 3′-UTR (wtUTR) or mutant VE-cadherin 3′-UTR (CAAAG→TCGCT) (mutUTR) were cloned into the psiCHECK-2 plasmid to produce the recombinant vectors, wtUTR psiCHECK-2 and mutUTR psiCHECK-2, harboring the predicted binding sites of miR-9. d The activity of firefly luciferase was significantly decreased by miR-9 and rescued by mutant of VE-cadherin 3′-UTR. e–g Co-culture of VECs and BMSCs transfected with cy3-miR-9 showed that BMSCs secreted cy3-miR-9 into VECs and inhibited the expression of VE-cadherin and β-catenin and upregulated Ang-1, CXCR4, TIE-2 and p-AKT. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001, compared with NC group, ^@p < 0.05, ^@@p < 0.01, and ^@@@p < 0.001, compared with miR-9 con by using paired t test. VECs, vascular endothelial cells; BMSCs, bone marrow-derived mesenchymal stem cells; GFP, green fluorescent protein; gPCR, general PCR; qRT-PCR, quantitative real-time PCR; SAP, severe acute pancreatitis; NC, normal control; miR-9 con, miR-9 mimic control

Fig. 5

miR-9 promoted proliferation, migration and angiogenesis, and inhibited apoptosis in VECs. a–b miR-9 significantly inhibited apoptosis in VECs induced by LPS compared with LPS + miR-9 control. *p < 0.05, compared with LPS + miR-9 control. c miR-9 promoted the proliferation of VECs.**p < 0.001 and ***p < 0.001, compared with miR-9 control group. d–g miR-9 increased migration and induced angiogenesis. *p < 0.05, compared with miR-9 control

Fig. 6

a, b AP was induced by caerulein and 3% NaT, and H&E staining showed that pancreatic damage was more severe in the 3% NaT group than in the caerulein group. c, d miR-9 expression was lower in damaged pancreatic tissues and the serum of AP rats than in the NC or Sham group. Data are shown as the mean ± SD. ^&&&p < 0.001 and ^###p < 0.01, compared with NC, *p < 0.05, compared with NC, ^%%%p < 0.001, compared with Sham, ^$$p < 0.01, compared with Caerulein group by using paired t test. e Western blot analysis showed that angiogenic genes (VEGFA, Ang-1, TIE-2, p-AKT, CXCR4) were downregulated in damaged pancreatic tissues. f The level of miR-9 and the expression of angiogenic genes were negatively correlated with the severity of AP, and miR-9 was positively correlated with angiogenesis-related as determined by Pearson’s correlation analysis (p < 0.05). AP, acute pancreatitis; H&E, hematoxylin eosin; NC, normal control; NaT, sodium taurocholate; SD, standard deviation; miR-9, microRNA-9