目的

探讨miR-181a-5p 靶向ATG5 抑制雨蛙素诱导的大鼠胰腺腺泡细胞AR42J 自噬的机制。

方法

通过100 nmol/L 雨蛙素处理AR42J 细胞体外模拟急性胰腺炎（AP），并将miR-181a-5p mimics 和oe-ATG5 转染至AR42J 细胞，分为Control组（未经任何处理）、AP组（模型组）、AP+mimic NC 和AP+mimic miR组（转染mimic NC 或miR-181a-5p mimic 至AR42J 细胞24 h，再经雨蛙素诱导建模）、AP+mimic miR+oe-NC 和AP+mimic miR+oe-ATG5组（共转染miR-181a-5p mimic、oe-NC 或oe-ATG5 至AR42J 细胞24 h，再经雨蛙素诱导建模）。使用ELISA检测TNF-α 和IL-6 的表达水平，RT-qPCR 检测miR-181a-5p 表达。采用MTT 测定细胞活力，免疫荧光检测自噬标记物LC3。miRWalk 数据库预测miR-181a-5p 和ATG5 的潜在结合位点，并通过双荧光素酶报告实验验证miR-181a-5p 与ATG5 的靶向关系。Western blot 检测自噬相关蛋白（Beclin-1、LC3 Ⅱ/I）和ATG5 蛋白的表达。

结果

成功构建AP 体外模型，AP组中TNF-α 和IL-6 表达水平较Control组显著升高，且miR-181a-5p 在雨蛙素诱导的AR42J 细胞中显著下调。与AP+mimic NC组相比，AP+mimic miR组细胞活力显著提高，自噬相关蛋白Beclin-1 和LC3 Ⅱ/I的表达显著下调，LC3阳性细胞数量减少。双荧光素酶实验结果显示，miR-181a-5p 可靶向负调控ATG5 的表达。进一步实验表明，过表达ATG5 可部分逆转miR-181a-5p 过表达对雨蛙素诱导自噬的抑制作用。

结论

miR-181a-5p通过靶向负调控ATG5，抑制雨蛙素诱导的AR42J 细胞自噬。这一机制可能在AP 的自噬调控中发挥重要作用。

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.