Abstract
Background
Acute pancreatitis is a common inflammatory disease. MicroRNAs have been implicated in the pathogenesis of acute pancreatitis.
Aims
The purpose of this study was to investigate the precise roles of miR-193a-5p and miR-320-5p in AP.
Methods
The levels of miR-193a-5p, miR-320-5p and tumor necrosis factor receptor-associated factor 3 were detected by quantitative real-time polymerase chain reaction. Cell apoptosis was determined using flow cytometry. Enzyme-linked immunosorbent assay was performed to measure TNF-α, IL-6, IL-1β and IL-8 production, amylase activity, and malondialdehyde content. Targeted relationship between miR-193a-5p or miR-320-5p and TRAF3 was confirmed by the dual-luciferase reporter and RNA immunoprecipitation assays.
Results
Our data showed that miR-193a-5p and miR-320-5p were down-regulated in acute pancreatitis serum and caerulein-treated AR42J cells. The increased expression of miR-193a-5p or miR-320-5p alleviated caerulein-induced cell injury in AR42J cells. Tumor necrosis factor receptor-associated factor 3 was a direct target of miR-193a-5p and miR-320-5p in AR42J cells. Moreover, miR-193a-5p and miR-320-5p regulated caerulein-induced AR42J cells injury through targeting tumor necrosis factor receptor-associated factor 3.
Conclusion
The present findings demonstrated that miR-193a-5p and miR-320-5p protected AR42J cells against caerulein-induced cell injury by targeting tumor necrosis factor receptor-associated factor 3, highlighting their roles as potential therapeutic targets for acute pancreatitis treatment.
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Abbreviations
- AP:
-
Acute pancreatitis
- TNF:
-
Tumor necrosis factor
- qRT-PCR:
-
Quantitative real-time polymerase chain reaction
- CCK-8:
-
Cell counting kit-8
- ELISA:
-
Enzyme-linked immunosorbent assay
- MDA:
-
Malondialdehyde
- RIP:
-
RNA immunoprecipitation
- KLF2:
-
Kruppel-like factor 2
- FITC:
-
Fluorescein isothiocyanate
- PI:
-
Propidium iodide
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Supplement Figure 1
The effect of caerulein on cell viability, Bcl-2, Bax, C-capsase 3 and t-caspase 3 expression. AR42J cells were treated with 10 nM of caerulein or mock for 4 and 8 h. (A and B) Cell viability by CCK-8 assay. (C) The levels of Bcl-2, Bax, C-capsase 3 and t-caspase 3 by western blot in treated cells. *P < 0.05
Supplement Figure 2
The increased level of miR-193a-5p alleviated caerulein-mediated injury in primary rat pancreatic acinar cells. Primary rat pancreatic acinar cells were transfected with or without NC mimic or miR-193a-5p mimic and then treated with 10 nM of caerulein or mock for 8 h. (A) CCK-8 assay for cell viability. (B) Flow cytometry for cell apoptosis. (C-F) ELISA assay for TNF-α, IL-1β, IL-6 and IL-8 levels. (G and H) Amylase activity and MDA content using the commercial assay kits. *P < 0.05
Supplement Figure 3
The effect of miR-193a-5p on cell viability, Bcl-2, Bax, C-capsase 3 and t-caspase 3 expression in caerulein-treated AR42J cells. AR42J cells were transfected with or without NC mimic or miR-193a-5p mimic and then treated with 10 nM of caerulein or mock for 8 h. (A) qRT-PCR for miR-193a-5p expression. (B) CCK-8 assay for cell viability. (C) Western blot for Bcl-2, Bax, C-capsase 3 and t-caspase 3 levels. *P < 0.05
Supplement Figure 4
The effect of miR-320-5p on cell viability, Bcl-2, Bax, C-capsase 3 and t-caspase 3 expression in caerulein-treated AR42J cells. AR42J cells were transfected with or without NC mimic or miR-320-5p mimic and then treated with 10 nM of caerulein or mock for 8 h, followed by the detection of miR-320-5p level by qRT-PCR (A), cell viability by CCK-8 assay (B), Bcl-2, Bax, C-capsase 3 and t-caspase 3 levels by western blot (C). *P < 0.05
Supplement Figure 5
MiR-193a-5p and miR-320-5p regulated cell viability, Bcl-2, Bax, C-capsase 3 and t-caspase 3 expression by TRAF3 in caerulein-treated AR42J cells. AR42J cells were transfected with or without miR-193a-5p mimic, miR-320-5p mimic, miR-193a-5p mimic + TRAF3 (TRAF3 overexpression plasmid) or miR-320-5p mimic + TRAF3 (TRAF3 overexpression plasmid) and then treated with 10 nM of caerulein for 8 h, followed by the determination of TRAF3 protein level by western blot (A), cell viability by CCK-8 assay (B), the levels of Bcl-2, Bax, C-capsase 3 and t-caspase 3 by western blot (C). *P < 0.05
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Yu, W., Zhang, M., Li, X. et al. Protective Effect of miR-193a-5p and miR-320-5p on Caerulein-Induced Injury in AR42J Cells. Dig Dis Sci 66, 4333–4343 (2021). https://doi.org/10.1007/s10620-020-06800-7
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DOI: https://doi.org/10.1007/s10620-020-06800-7