Study on the mechanism of miR-181a-5p targeting ATG5 to inhibit autophagy in rat pancreatic acinar AR42J cells induced by cerulein

doi:10.3877/cma.j.issn.2095-2015.2024.06.008

Abstract

Abstract:

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.

Key words: Acute pancreatitis, miR-181a-5p, ATG5, Autophagy, AR42J cells

Qingquan Shi, Bin Miao, Shuo Wang, Lin Tao, Chen Shen. Study on the mechanism of miR-181a-5p targeting ATG5 to inhibit autophagy in rat pancreatic acinar AR42J cells induced by cerulein[J]. Chinese Journal of Digestion and Medical Imageology(Electronic Edition), 2024, 14(06): 524-530.

/ / Recommend

Add to citation manager EndNote|Ris|BibTeX

URL: https://zhxhbyyxzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.2095-2015.2024.06.008

https://zhxhbyyxzz.cma-cmc.com.cn/EN/Y2024/V14/I06/524

Figures/Tables 6

References 30

[1]	Zhou J，Zhou P，Zhang Y，et al. Signal Pathways and Markers Involved in Acute Lung Injury Induced by Acute Pancreatitis[J]. Dis Markers，2021，2021： 9947047.
[2]	Földi M，Gede N，Kiss S，et al. The characteristics and prognostic role of acute abdominal on-admission pain in acute pancreatitis： A prospective cohort analysis of 1432 cases[J]. Eur J Pain，2022，26（3）：610-623.
[3]	Gómez-Virgilio L，Silva-Lucero MD，Flores-Morelos DS，et al.Autophagy： A Key Regulator of Homeostasis and Disease： An Overview of Molecular Mechanisms and Modulators[J]. Cells，2022，11（15）： 2262.
[4]	Ding WX，Ma X，Kim S，et al. Recent insights about autophagy in pancreatitis. eGastroenterology，2024，2（2）： e100057.
[5]	Silwal P，Kim YS，Basu J，et al. The roles of microRNAs in regulation of autophagy during bacterial infection[J]. Semin Cell Dev Biol，2020，101： 51-58.
[6]	Akkoc Y，Gozuacik D. MicroRNAs as major regulators of the autophagy pathway[J]. Biochim Biophys Acta Mol Cell Res，2020，1867（5）： 118662.
[7]	Ashrafizadeh M，Zarrabi A，Orouei S，et al. MicroRNA-mediated autophagy regulation in cancer therapy： The role in chemoresistance/chemosensitivity[J]. Eur J Pharmacol，2021，892： 173660.
[8]	Wei JL，Li YC，Ma ZL，et al. MiR-181a-5p promotes anoikis by suppressing autophagy during detachment induction in the mammary epithelial cell line MCF10A[J]. Protein Cell，2016，7（4）： 305-309.
[9]	Benedetti R，Papulino C，Sgueglia G，et al. Regulatory Interplay between miR-181a-5p and Estrogen Receptor Signaling Cascade in Breast Cancer[J]. Cancers（Basel），2021，13（3）： 543.
[10]	Changotra H，Kaur S，Yadav SS，et al. ATG5： A central autophagy regulator implicated in various human diseases[J]. Cell Biochem Funct，2022，40（7）： 650-667.
[11]	Honda S，Arakawa S，Yamaguchi H，et al. Association Between Atg5-independent Alternative Autophagy and Neurodegenerative Diseases[J]. J Mol Biol，2020，432（8）： 2622-2632.
[12]	Park JW，Kim Y，Lee SB，et al. Autophagy inhibits cancer stemness in triple-negative breast cancer via miR-181a-mediated regulation of ATG5 and/or ATG2B[J]. Mol Oncol，2022，16（9）： 1857-1875.
[13]	王伟，朱婷婷，刘家豪. miR-132 靶向调控PTEN 基因对雨蛙素诱导的急性胰腺炎模型胰腺腺泡细胞增殖及凋亡的影响[J]. 国际消化病杂志，2022，42（4）： 261-266.
[14]	Padula D，Mauro A，Maggioni P，et al. Practical approach to acute pancreatitis： from diagnosis to the management of complications[J].Intern Emerg Med，2024，19（8）： 2091-2104.
[15]	Zhou W，Dong S，Chen Z，et al. New challenges for microRNAs in acute pancreatitis： progress and treatment[J]. J Transl Med，2022，20（1）： 192.
[16]	Hidalgo NJ，Pando E，Mata R，et al. Impact of comorbidities on hospital mortality in patients with acute pancreatitis： a populationbased study of 110，021 patients[J]. BMC Gastroenterol，2023，23（1）： 81.
[17]	Tsomidis I，Voumvouraki A，Kouroumalis E. The Pathogenesis of Pancreatitis and the Role of Autophagy[J]. Gastroenterol Insights，2024，15，303-341.
[18]	Zhang T，Gan Y，Zhu S. Association between autophagy and acute pancreatitis[J]. Front Genet，2023，14： 998035.
[19]	Ge T，Ning B，Wu Y，et al. MicroRNA-specific therapeutic targets and biomarkers of apoptosis following myocardial ischemiareperfusion injury[J]. Mol Cell Biochem，2024，479（10）： 2499-2521.
[20]	Galagali H，Kim JK. The multifaceted roles of microRNAs in differentiation[J]. Curr Opin Cell Biol，2020，67： 118-140.
[21]	Fu Y，Xin Z，Ling Z，et al. A CREB1-miR-181a-5p loop regulates the pathophysiologic features of bone marrow stromal cells in fibrous dysplasia of bone[J]. Mol Med，2021，27（1）： 81.
[22]	Zhang Y，Guan Y，Zheng X，et al. Hypoxia-induced miR-181a-5p up-regulation reduces epirubicin sensitivity in breast cancer cells through inhibiting EPDR1/TRPC1 to activate PI3K/AKT signaling pathway[J]. BMC Cancer，2024，24（1）： 167.
[23]	Ye X，Zhou XJ，Zhang H. Exploring the Role of Autophagy-Related Gene 5（ATG5） Yields Important Insights Into Autophagy in Autoimmune/Autoinflammatory Diseases[J]. Front Immunol，2018，9： 2334.
[24]	张瑜，辛前有，冯亚飞，等. 急性胰腺炎患者血清miR-181a-5p 表达及与Th17/Treg 免疫平衡的关系[J]. 河北医学，2023，29（1）： 115-120.
[25]	Li Fan，Hu Zhenfei，Huang Yidan，et al. Dexmedetomidine ameliorates diabetic cardiomyopathy by inhibiting ferroptosis through the Nrf2/GPX4 pathway[J]. J Cardiothorac Surg，2023，18： 223.
[26]	Runwal G，Stamatakou E，Siddiqi FH，et al. LC3-positive structures are prominent in autophagy-deficient cells[J]. Sci Rep，2019，9（1）： 10147.
[27]	潘春风. miR-181a-5p 靶向HMGB1 调控急性胰腺炎腺泡细胞增殖及凋亡的实验研究[D]. 右江民族医学院，2023.
[28]	黄燕，杨艳清，万睿，等. miR-181-5p 靶向PTEN/AKT/mTOR 通路对氧化应激诱导下人皮肤成纤维细胞自噬及老化的影响[J]. 中国老年学杂志，2023，43（18）： 4538-4542.
[29]	陈晨晖，程楠，彭伟，等. 急性胰腺炎患者外周血 ATG5、miR-30-5p 水平与患者病情严重程度及预后的关系[J]. 检验医学与临床，2022，19（14）： 1886-1890.
[30]	Shan C，Chen X，Cai H，et al. The Emerging Roles of Autophagy-Related MicroRNAs in Cancer[J]. Int J Biol Sci，2021，17（1）： 134-150.

Gene	Forward primer（5’-3’）	Reverse primer（5’-3’）
miR-181a-5p	ACATTCAACGCTGTCGGT GAGT	GTGCAGGGTCCGAGGT
U6	GTGCTCGCTTCGGCAGCA CATATA	AATATGGAACGCTTCAC GAATT

Gene	Forward primer（5’-3’）	Reverse primer（5’-3’）
miR-181a-5p	ACATTCAACGCTGTCGGT GAGT	GTGCAGGGTCCGAGGT
U6	GTGCTCGCTTCGGCAGCA CATATA	AATATGGAACGCTTCAC GAATT

Please choose a citation manager

Content to export