微小RNA-141-3p靶向调控含CUE结构域蛋白2基因对雨蛙素诱导的大鼠胰腺腺泡细胞损伤的影响

Effect of microRNA-141-3p on the targeted regulation of the CUE domain-containing protein 2 gene on cerulein-induced injury of rat pancreatic acinar cells

目的 探讨微小RNA-141-3p(miR-141-3p)靶向调控含CUE结构域蛋白2(CUEDC2)对雨蛙素诱导的大鼠胰腺腺泡细胞损伤的影响。方法 2018年7月至2019年12月,从美国ATCC购买大鼠胰腺腺泡细胞AR42J。100 nm/L雨蛙素(CAE)刺激大鼠胰腺腺泡细胞AR42J 6 h建立急性胰腺炎细胞模型。实时荧光定量PCR(qRT-PCR)和蛋白质印迹法(Western blotting)检测雨蛙素干预后AR42J细胞中miR-141-3p和CUEDC2的表达水平。双荧光素酶报告基因实验和蛋白质印迹法验证miR-141-3p和CUEDC2的靶向关系。利用脂质体转染法将miR-141-3p抑制物(anti-miR-141-3p)、miRNA抑制物阴性对照(anti-miR-NC)、CUEDC2过表达载体(pcDNA-CUEDC2)、空载体(pcDNA)分别转染AR42J细胞,经雨蛙素干预处理后,流式细胞术检测细胞凋亡,蛋白质印迹法检测B细胞淋巴瘤/白血病-2(Bcl-2)和Bcl-2相关X蛋白(Bax)的表达水平,酶联免疫吸附试验(Elisa)试剂盒检测细胞培养上清中肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)水平。结果 雨蛙素干预处理后AR42J细胞中miR-141-3p的表达水平显著升高[(2.43±0.24)比(1.00±0.09)],CUEDC2的表达水平显著降低。miR-141-3p靶向负性调控CUEDC2表达。与转anti-miR-NC和雨蛙素协同处理组比较,转染anti-miR-141-3p和雨蛙素协同处理组AR42J细胞凋亡率显著增加[(27.48±2.33)%比(15.64±1.51)%],Bax蛋白的表达显著增加,Bcl-2蛋白的表达显著降低,TNF-α[(136.54±14.58)ng/L比(226.48±20.54)ng/L]和IL-6[(115.89±11.65)ng/L比(193.47±18.63)ng/L]的分泌显著降低;与转染pcDNA和雨蛙素协同处理组比较,转染pcDNA-CUEDC2和雨蛙素协同处理组AR42J细胞凋亡率显著增加[(23.87±2.35)%比(14.23±1.44)%],Bax蛋白的表达显著增加,Bcl-2蛋白的表达显著降低,TNF-α[(158.74±15.32)ng/L比(236.87±18.66)ng/L]和IL-6[(135.77±14.97)ng/L比(189.67±17.32)ng/L]的分泌显著降低;转染si-CUEDC2可逆转转染anti-miR-141-3p对雨蛙素诱导的大鼠胰腺腺泡细胞损伤的影响。结论 抑制miR-141-3p通过靶向CUEDC2可促进急性胰腺炎腺泡细胞的凋亡,抑制TNF-α和IL-6分泌,从而减轻雨蛙素诱导的大鼠胰腺腺泡细胞损伤。

Objective To investigate the effect of micro RNA-141-3p (mi R-141-3p) targeting CUE domain containing 2 protein(CUEDC2) on cerulein-induced pancreatic acinar cell injury in rats.Methods This study was conducted from July 2018 to December2019.Rat AR42J pancreatic acinar cells were purchased from ATCC (USA).A cell model of acute pancreatitis was established by stimulating rat pancreatic acinar AR42J cells with 100 nm/L cerulein (CAE) for 6 h.Real-time quantitative PCR (q RT-PCR) and Western blotting were used to detect the expression levels of mi R-141-3p and CUEDC2 in AR42J cells after cerulein intervention.Dual-luciferase reporter gene assay and Western blotting verified the targeting relationship between mi R-141-3p and CUEDC2.mi R-141-3p inhibitor (anti-mi R-141-3p),mi RNA inhibitor negative control (anti-mi R-NC),CUEDC2 overexpression vector (pc DNA-CUEDC2) and empty vector (pc DNA) were transfected into AR42J cells by lipofection,and after treatment with cerulein,cell apoptosis was detected by flow cytometry,and the expression of B-cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2 related X protein (Bax) were detected by Western blotting.The levels of tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6) in the cell culture supernatant were detected by an enzymelinked immunosorbent assay (ELISA) kit.Results After treatment with cerulein,the expression level of mi R-141-3p in AR42J cells was significantly increased[(2.43±0.24) vs.(1.00±0.09)],and the expression level of CUEDC2 was significantly decreased.mi R-141-3p targets and negatively regulates CUEDC2 expression.Compared with the transfected anti-mi R-NC and cerulein cotreatment group,the AR42J cell apoptosis rate significantly increased in the transfected anti-mi R-141-3p and cerulein cotreatment group[(27.48±2.33)%vs.(15.64±1.51)%],the expression of Bax protein was significantly increased,the expression of Bcl-2 was significantly decreased,and the secretion of TNF-α[(136.54±14.58) ng/L vs.(226.48±20.54) ng/L]and IL-6[(115.89±11.65) ng/L vs.(193.47±18.63) ng/L]was significantly decreased.Compared with the transfected pc DNA and cerulein-treated group,the AR42J cell apoptosis rate in the transfected pc DNA-CUEDC2 and cerulein synergistic treatment group was significantly increased[(23.87±2.35)%vs.(14.23±1.44)%],the expression of Bax protein was significantly increased,the expression of Bcl-2 protein was significantly decreased,and the secretion of TNF-α[(158.74±15.32) ng/L vs.(236.87±18.66) ng/L]and IL-6[(135.77±14.97) ng/L vs.(189.67±17.32) ng/L]was significantly decreased.Transfection of si-CUEDC2 reversed the effect of transfection of anti-mi R-141-3p on cerulein-induced rat pancreatic acinar cell injury.Conclusion Inhibition of mi R-141-3p by targeting CUEDC2 can promote the apoptosis of acinar cell in acute pancreatitis and inhibit the secretion of TNF-αand IL-6,thereby alleviating cerulein-induced rat pancreatic acinar cell injury in rats.