BACKGROUND The incidence rate of acute pancreatitis(AP), which is a pathophysiological process with complex etiology, is increasing globally. miR-125b-5p, a bidirectional regulatory miRNA, is speculated to exhibit ant...BACKGROUND The incidence rate of acute pancreatitis(AP), which is a pathophysiological process with complex etiology, is increasing globally. miR-125b-5p, a bidirectional regulatory miRNA, is speculated to exhibit anti-tumor activity. However,exosome-derived miR-125b-5p in AP has not been reported.AIM To elucidate the molecular mechanism of exosome-derived miR-125b-5p promoting AP exacerbation from the perspective of the interaction between immune cells and acinar cells.METHODS Exosomes derived from AR42J cells were isolated and extracted in active and inactive states by an exosome extraction kit, and were verified via transmission electron microscopy, nanoparticle tracking analysis, and western blotting. RNA sequencing assay technology was used to screen differentially expressed miRNAs in active and inactive AR42J cell lines, and bioinformatics analysis was used to predict downstream target genes of miR-125b-5p. The expression level of miR-125b-5p and insulin-like growth factor 2(IGF2) in the activated AR42J cell line and AP pancreatic tissue were detected by quantitative real-time polymerase chain reaction and western blots. The changes in the pancreatic inflammatory response in a rat AP model were detected by histopathological methods. Western Blot was used to detect the expression of IGF2, PI3K/AKT signaling pathway proteins, and apoptosis and necrosis related proteins.RESULTS miR-125b-5p expression was upregulated in the activated AR42J cell line and AP pancreatic tissue,while that of IGF2 was downregulated. In vitro experiments confirmed that miR-125b-5p could promote the death of activated AR42J cells by inducing cell cycle arrest and apoptosis. In addition,miR-125b-5p was found to act on macrophages to promote M1 type polarization and inhibit M2type polarization, resulting in a massive release of inflammatory factors and reactive oxygen species accumulation. Further research found that miR-125b-5p could inhibit the expression of IGF2 in the PI3K/AKT signaling pathway. Additionally, in vivo experiments revealed that miR-125b-5p can promote the progression of AP in a rat model.CONCLUSION miR-125b-5p acts on IGF2 in the PI3K/AKT signaling pathway and promotes M1 type polarization and inhibits M2 type polarization of macrophage by inhibiting IGF2 expression, resulting in a large release of pro-inflammatory factors and an inflammatory cascade amplification effect, thus aggravating AP.展开更多
基金Supported by The Beijing Municipal Science and Technology Commission,Capital Research and Demonstration Application of Clinical Diagnosis and Treatment Technology,No. Z191100006619038 and No. Z171100001017077The Capital Health Research and Development of Special,No. 2020-1-2012
文摘BACKGROUND The incidence rate of acute pancreatitis(AP), which is a pathophysiological process with complex etiology, is increasing globally. miR-125b-5p, a bidirectional regulatory miRNA, is speculated to exhibit anti-tumor activity. However,exosome-derived miR-125b-5p in AP has not been reported.AIM To elucidate the molecular mechanism of exosome-derived miR-125b-5p promoting AP exacerbation from the perspective of the interaction between immune cells and acinar cells.METHODS Exosomes derived from AR42J cells were isolated and extracted in active and inactive states by an exosome extraction kit, and were verified via transmission electron microscopy, nanoparticle tracking analysis, and western blotting. RNA sequencing assay technology was used to screen differentially expressed miRNAs in active and inactive AR42J cell lines, and bioinformatics analysis was used to predict downstream target genes of miR-125b-5p. The expression level of miR-125b-5p and insulin-like growth factor 2(IGF2) in the activated AR42J cell line and AP pancreatic tissue were detected by quantitative real-time polymerase chain reaction and western blots. The changes in the pancreatic inflammatory response in a rat AP model were detected by histopathological methods. Western Blot was used to detect the expression of IGF2, PI3K/AKT signaling pathway proteins, and apoptosis and necrosis related proteins.RESULTS miR-125b-5p expression was upregulated in the activated AR42J cell line and AP pancreatic tissue,while that of IGF2 was downregulated. In vitro experiments confirmed that miR-125b-5p could promote the death of activated AR42J cells by inducing cell cycle arrest and apoptosis. In addition,miR-125b-5p was found to act on macrophages to promote M1 type polarization and inhibit M2type polarization, resulting in a massive release of inflammatory factors and reactive oxygen species accumulation. Further research found that miR-125b-5p could inhibit the expression of IGF2 in the PI3K/AKT signaling pathway. Additionally, in vivo experiments revealed that miR-125b-5p can promote the progression of AP in a rat model.CONCLUSION miR-125b-5p acts on IGF2 in the PI3K/AKT signaling pathway and promotes M1 type polarization and inhibits M2 type polarization of macrophage by inhibiting IGF2 expression, resulting in a large release of pro-inflammatory factors and an inflammatory cascade amplification effect, thus aggravating AP.
文摘目的 探讨长链非编码RNA(long non-coding RNA,LncRNA)核富集转录本1(nuclear enriched abundant transcript 1,NEAT1)通过调节微小核糖核酸(micro RNAs,miR)-125b-5p/胰岛素样生长因子结合蛋白5(insulinlike growth factor binding protein 5,IGFBP5)轴对血管瘤内皮细胞增殖、凋亡和迁移的影响。方法 实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)、蛋白免疫印迹(Western blot)分别检测血管瘤组织(2016年3月~2019年3月收集,n=18)、瘤旁组织样本(2016年3月~2019年3月收集,n=18)以及人脐静脉内皮细胞HUVES,人血管瘤内皮细胞HemECs,HDEC中NEAT1,miR-125b-5p及IGFBP5蛋白表达。构建沉默NEAT1,同时沉默NEAT1和miR-125b-5p的HemECs细胞系,通过细胞活力检测试剂盒(cell counting kit-8,CCK-8)、台盼蓝染色、流式细胞术、划痕愈合实验、Western blot分别观察NEAT1和miR-125b-5p对HemECs细胞增殖、凋亡、迁移及IGFBP5,增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、B细胞淋巴瘤/白血病-2(B cell lymphoma/lewkmia-2,Bcl-2)和基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)蛋白表达的影响;双荧光素酶报告基因实验检测NEAT1与miR-125b-5p,mi R-125b-5p与IGFBP5的关系。结果 与瘤旁组织比较,血管瘤组织中NEAT1(2.87±0.22 vs 1.00±0.00),IGFBP5蛋白(1.45±0.14 vs 0.27±0.02)表达水平升高,miR-125b-5p(0.24±0.02 vs 1.00±0.00)表达水平降低,差异具有统计学意义(t=35.400~161.220,均P <0.05);与HUVES细胞比较,HemECs,HDEC细胞中NEAT1(2.76±0.24,1.78±0.13 vs 1.00±0.00),IGFBP5蛋白(1.31±0.15,0.78±0.06 vs 0.24±0.02)表达升高,miR-125b-5p表达(0.19±0.02,0.45±0.04 vs 1.00±0.00)降低,差异具有统计学意义(t=17.320~99.204,14.697~33.680,均P<0.05),且HemECs细胞中NEAT1和IGFBP5蛋白表达量最高,miR-125b-5p表达量最低,因此,选取HemECs细胞为研究对象;与si-NC组比较,si-NEAT1组NEAT1(0.32±0.02 vs 1.01±0.12)表达、A值(0.45±0.04 vs 1.13±0.11)、细胞生长率(32.28%±2.79%vs 99.41%±0.22%)、划痕愈合率(20.33%±1.23%vs 49.24%±2.43%)及IGFBP5(0.41±0.04 vs 1.31±0.20),PCNA(0.36±0.04 vs 1.27±0.14),Bcl-2(0.48±0.04 vs 1.39±0.16)和MMP-9(0.21±0.02 vs 1.09±0.10)蛋白表达降低,mi R-125b-5p(1.87±0.15 vs 1.02±0.10)表达、细胞凋亡率(45.58%±3.34%vs 12.36%±1.07%)升高,差异具有统计学意义(t=10.809~58.755,均P <0.05);下调miR-125b-5p减弱了沉默NEAT1对HemECs细胞增殖、迁移的抑制及对细胞凋亡的促进作用(t=9.218~15.010,均P <0.05);NEAT1与miR-125b-5p,miR-125b-5p与IGFBP5存在靶向调控关系。结论 沉默NEAT1通过上调miR-125b-5p来抑制IGFBP5表达,从而抑制HemECs细胞增殖、迁移,并促进细胞凋亡。
