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Protective Effect of miR-193a-5p and miR-320-5p on Caerulein-Induced Injury in AR42J Cells

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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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Correspondence to Wei Wu.

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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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