Study on the mechanism of miR-181a-5p targeting ATG5 to inhibit autophagy in rat pancreatic acinar AR42J cells induced by cerulein
Objective To investigate the mechanism of miR-181a-5p targeting ATG5 to inhibit autophagy in rat pancreatic acinar AR42J cells induced by cerulein.Methods AR42J cells were treated with 100 nmol/L cerulein in vitro to simulate acute pancreatitis(AP).miR-181a-5p mimics and oe-ATG5 were transfected into AR42J cells.AR42J cells were divided into Control group(without any treatment),AP group(model group),AP+mimic NC and AP+mimic miR groups(transfected mimic NC or miR-181a-5p mimic into AR42J cells for 24 h,and then modeled induced by cerulein),and AP+mimic miR+oe-NC and AP+mimic miR+oe-ATG5 groups(transfected miR-181a-5p mimic,oe-NC or oe-ATG5 into AR42J cells for 24 h,and then modeled induced by cerulein).ELISA was used to determine the expression levels of TNF-α and IL-6,and RT-qPCR was performed to determine the expression of miR-181a-5p.MTT assay was used to assess cell viability,and immunofluorescence was conducted to detect the autophagy marker LC3.The miRWalk database predicted potential binding sites between miR-181a-5p and ATG5,and the dual-luciferase reporter assay was used to verify the targeting relationship between miR-181a-5p and ATG5.Western blot analysis was employed to measure the expression levels of autophagy-related proteins(Beclin-1,LC3 Ⅱ/Ⅰ)and ATG5 protein.Results An in vitro AP model was successfully established.Significantly increased levels of TNF-α and IL-6 were noticed in the AP group compared to the Control group.miR-181a-5p expression was down-regulated in cerulein-induced AR42J cells.Compared to the AP+mimic NC group,the AP+mimic miR group showed increased cell viability,down-regulated expression patterns of autophagy-related proteins Beclin-1 and LC3 Ⅱ/Ⅰ,and a reduced number of LC3-positive cells.Dual-luciferase reporter assay revealed that miR-181a-5p negatively regulated ATG5.Further experiments indicated that re-expression of ATG5 could partially reverse the inhibitory effect of miR-181a-5p overexpression on autophagy induced by cerulein in AR42J cells.Conclusion miR-181a-5p inhibits cerulein-induced autophagy in AR42J cells by targeting ATG5.This mechanism may play a crucial role in autophagy regulation in AP.
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