Abstract
This study was performed to screen miRNAs and mRNAs that are differentially expressed during trypsinogen activation in acute pancreatitis and to verify their role in the process of trypsinogen activation. The function enrichment analysis showed that the functions of miR-352 and its regulatory targets lysosome-associated membrane protein 2 (LAMP2) and cathepsin L1 (CTSL1) were lysosome related. The results of the verification experiment showed that in the TLC-S-treated AR42J (pancreatic cell line) cells, miR-352 expression increased, expression levels of LAMP2 and CTSL1 were significantly reduced, trypsinogen activation was increased, and the autophagy pathway was blocked. In the miR-352 mimic-transfected cells, miR-352 expression increased, expression levels of LAMP2 and CTSL1 were significantly reduced, trypsinogen activation was increased, intracellular lysosomal pH increased, cathepsins L activity decreased and the amount of autophagolysosomes increased. In the miR-352 inhibitor-transfected cells, miR-352 expression was reduced, expression levels of LAMP2 and CTSL1 were significantly increased, trypsinogen activation was decreased, intracellular lysosomal pH decreased, cathepsins L activity increased and the amount of autophagolysosomes decreased. In the process of taurolithocholic acid 3-sulfate (TLC-S) induced trypsinogen activation, overexpression of miR-352 could down-regulate LAMP2 and CTSL1, resulting in the dysfunction of autophagic lysosome. Thus, the autophagy pathway was blocked, and trypsinogen activation was enhanced.
Highlights
Acute pancreatitis (AP) has high morbidity and mortality rates
The results showed that the lysosomal pH in the taurolithocholic acid 3-sulfate (TLC-S)-treated cells was significantly higher than that of the control group and the group transfected with the rno-miR-352 inhibitor (5.001 ± 0.144 vs. 4.539 ± 0.097, P < 0.05; 5.001 ± 0.144 vs. 4.177 ± 0.03, P < 0.05), the lysosomal pH in the AR42J cells transfected with the rnowww.impactjournals.com/oncotarget miR-352 mimic was significantly higher than that of the control group (5.115 ± 0.35 vs. 4.539 ± 0.097, P < 0.05) (Figure 7B)
The results of cathepsins L activity assay showed that, compared with the control group and the group transfected with the rno-miR-352 inhibitor, cathepsins L activity in the AR42J cells treated with TLC-S was significantly decreased, the cathepsins L activity in the AR42J cells transfected with the rno-miR-352 mimic was significantly decreased compared with the control group after treated with TLC-S for 40 min (9.37 ±1.776 vs.33.447 ± 2.149) (Figure 7C)
Summary
Its pathogenesis is not yet fully elucidated, it is widely accepted that the self-digestion of cells caused by the trypsinogen activation in pancreatic acinar cells is the initiating factor for all types of AP and has become an important indicator in determining the occurrence of AP [1, 2]. Autophagy (mainly macroautophagy) is the lysosome-mediated process for intracellular degradation and for the recycling of organelles, longevity proteins, and lipids. Orlichenko et al [10] indicated that in bombesin-induced AP, ARF1 could promote the activation of trypsinogen by promoting the transportation and processing of the precursor of lysosomal cysteine protease CTSB and the maturation of the autophagosome
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