miR-181a-5p靶向HMGB1调节急性胰腺炎腺泡细胞凋亡的实验研究

Experimental study on the effect of miR-181a-5p targeting HMGB1 regulation of on apoptosis in acinar cells of acute pancreatitis

目的探讨miR-181a-5p调控HMGB1的表达在雨蛙素(cerulein)联合脂多糖(lipopolyasccharide,LPS)诱导的严重急性胰腺炎(acute pancreatitis,AP)大鼠胰腺腺泡细胞(AR42J)凋亡相关基因半胱氨酸天冬氨酸蛋白酶3(Caspase-3)的影响。方法以未处理的AR42J细胞作为正常组,1×10-8 mol/L的cerulein联合10μg/mL的LPS诱导AR42J细胞24 h建立体外严重AP组,用脂质体转染法将miR-NC(空载体)和miR-181a-5p mimic转染至严重AP组作为miR-NC对照组和miR-181a-5p过表达组。采用qRT-PCR检测AR42J细胞中miR-181a-5p、HMGB1和Caspase-3 m RNA的表达水平;Western Blot检测HMGB1和Caspase-3蛋白表达;双荧光素酶报告基因实验明确miR-181a-5p和HMGB1的靶向关系;Pearson相关分析确定miR-181a-5p、HMGB1和Caspase-3 mRNA表达的相互关系。结果与正常组相比,严重AP组中miR-181a-5p mRNA表达水平显著下调(P<0.01),HMGB1 mRNA表达水平显著升高(P<0.01),Caspase-3 mRNA表达水平升高(P<0.05),HMGB1、Caspase-3的蛋白表达升高(P<0.05);与miR-NC对照组相比,miR-181a-5p过表达组miR-181a-5p mRNA表达水平显著上调(P<0.01),HMGB1、Caspase-3 mRNA表达水平显著下降(P<0.01),HMGB1、Caspase-3的蛋白表达下降(P<0.05);双荧光素酶报告基因显示miR-181a-5p可抑制野生型HMGB1细胞的荧光活性,miR-181a-5p可靶向调控HMGB1的表达;Pearson相关性分析显示,miR-181a-5p与HMGB1表达水平呈负相关(r=-0.905,P<0.01);miR-181a-5p与Caspase-3表达水平呈负相关(r=-0.809,P<0.01);HMGB1与Caspase-3表达水平呈正相关(r=0.691,P<0.05)。结论miR-181a-5p在cerulein+LPS诱导AR42J细胞中低表达,HMGB1和Caspase-3高表达,过表达miR-181a-5p可抑制凋亡相关基因Caspase-3的表达,其机制可能与靶向HMGB1有关,这将为AP的诊断及治疗提供新的思路。

Objective To investigate the effect of miR-181a-5p regulation of HMGB1 expression on the apoptosis-related gene Caspase-3 in pancreatic acinus cells(AR42J)of rats with severe acute pancreatitis(AP)induced by cerulein combined with lipopolysaccharide(LPS).Methods Untreated AR42J cells were used as the normal group,and AR42J cells were induced by 1×10-8 mol/L cerulein combined with 10μg/mL LPS for 24 h to establish in vitro the severe AP group.By liposome method,miR-NC(empty vector)and miR-181a-5p mimic were transfected into the severe AP group as miR-NC control group and miR-181a-5p over-expression group,respectively.qRT-PCR was used to detect the expression of miR-181a-5p,HMGB1,and Caspase-3 mRNA in AR42J cells.Western Blot was used to detect the protein expression of HMGB1 and Caspase-3.The dual-luciferase reporter assay was conducted to confirm the targeting relationship between miR-181a-5p and HMGB1.Pearson correlation analysis was used to determine the relationship between the expression of miR-181a-5p,HMGB1,and Caspase-3 mRNA.Results Compared with the normal group,the severe AP group had significantly down-regulated mRNA expression of miR-181a-5p(P<0.01),significantly increased mRNA expression of HMGB1(P<0.01),increased mRNA expression of Caspase-3(P<0.05),and increased protein expressions of HMGB1 and Caspase-3(P<0.05).Compared with the miR-NC control group,the miR-181a-5p over-expression group had significantly increased mRNA expression of miR-181a-5p(P<0.01),significantly decreased mRNA expression of HMGB1 and Caspase-3(P<0.01),and decreased protein expression of HMGB1 and Caspase-3(P<0.05).The dual-luciferase reporter gene assage showed that miR-181a-5p could inhibit the fluorescence activity of wild-type HMGB1 cells,and miR-181a-5p could target and regulate the expression of HMGB1.Pearson correlation analysis showed a negative correlation between miR-181a-5p and HMGB1 expression(r=-0.905,P<0.01),while there was a negative correlation between the expression of miR-181a-5p and Caspase-3(r=-0.809,P<0.01).And there was a positive correlation between HMGB1 and Caspase-3 expression(r=0.691,P<0.05).Conclusion miR-181a-5p is low-expressed in cerulein+LPS-induced AR42J cells,while HMGB1 and Caspase-3 are high-expressed.Over-expression of miR-181a-5p can inhibit the expression of apoptosis-related gene Caspase-3,and its mechanism may be related to targeting HMGB1.This study provides a new direction for the diagnosis and treatment of AP.