摘要
背景 本研究旨在探究影响急性胰腺炎进程的潜在分子机制.通过功能缺失实验分析H19敲低对细胞功能的影响.并通过生物信息学预测以及实验验证确定H19与miR-30a-5p的互作关系.我们推测H19可能通过靶向miR-30a-5p来促进急性胰腺炎进程.目的 研究lnc RNA H19靶向miR-30a-5p对雨蛙肽诱导的急性胰腺炎细胞凋亡、炎症因子分泌和自噬的影响.方法 培养人胰腺AR42J细胞,雨蛙肽诱导建立胰腺损伤细胞模型.将实验分为雨蛙肽+si-NC组、雨蛙肽+si-H19组、雨蛙肽+miR-NC组、雨蛙肽+miR-30a-5p组、雨蛙肽+si-H19+anti-miR-NC组、雨蛙肽+si-H19+anti-miR-30a-5p组.实时荧光定量PCR(RTqPCR)检测细胞中H19和m i R-30a-5p表达水平;流式细胞术检测细胞凋亡;酶联免疫吸附实验检测细胞中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白介素-1β(interleukin-1β,IL-1β)和白介素-6(interleukin-6, IL-6)水平;蛋白质印迹(Western blot)法检测细胞自噬相关的微管相关蛋白1轻链3(microtubule-associatedprotein1lightchain3,LC3)-Ⅰ、LC3-Ⅱ蛋白表达.双荧光素酶实验检测细胞中H19和miR-30a-5p靶向结合关系.结果 与正常胰腺细胞比较,雨蛙肽诱导损伤的胰腺炎细胞中H19表达水平显著升高, miR-30a-5p表达水平显著降低(P<0.05).与雨蛙肽+si-NC组比较,雨蛙肽+siH19组H19表达水平显著降低;细胞存活率显著升高;TNF-α、IL-1β和IL-6水平显著降低;LC3-Ⅰ蛋白表达显著降低、LC3-Ⅱ蛋白表达显著升高(P<0.05).与雨蛙肽+miR-NC组比较,雨蛙肽+miR-30a-5p组miR-30a-5p表达水平显著升高;细胞存活率显著升高;TNF-α、IL-1β和IL-6水平显著降低;LC3-Ⅰ蛋白表达显著降低、LC3-Ⅱ蛋白表达显著升高(P<0.05).与雨蛙肽+si-H19+anti-miR-NC比较,雨蛙肽+si-H19+antimiR-30a-5p组miR-30a-5p表达显著降低;细胞存活率显著降低;TNF-α、IL-1β和IL-6水平显著升高;LC3-Ⅰ蛋白表达显著升高、LC3-Ⅱ蛋白表达显著降低(P<0.05).结论 �
BACKGROUND We hypothesized that H19 might promote the progression of acute pancreatitis by targeting miR-30a-5p.AIM To investigate the effect of lnc RNA H19 targeting mi R-30a-5p on cell apoptosis, secretion of inflammatory factors, and autophagy in a caerulein-induced acute pancreatitis cell model.METHODS Human pancreatic AR42J cells were cultured and treated with caerulein to establish a pancreatic injury cell model. The cells were divided into cerulean + si-NC group, cerulean + siH19 group, cerulean + mi R-NC group, cerulean + mi R-30a-5p group, cerulean + si-H19 + anti-mi R-NC group, and cerulean + si-H19 + anti-mi R-30a-5p group. Real-time fluorescence quantitative PCR was used to detect the expression levels of H19 and mi R-30a-5p in cells;flow cytometry was used to detect apoptosis;enzyme-linked immunosorbent assay(ELISA) was used to detect TNF-α, IL-1β, and IL-6 levels;Western blot was used to detect autophagy-related microtubule-associated protein 1 light chain 3-Ⅰ(LC3-Ⅰ) and microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ) protein expression;dual luciferase assay was used to detect the target relationship between H19 and mi R-30a-5p in cells.RESULTS Compared with normal pancreatic cells, H19 expression was increased and mi R-30a-5p expression was decreased in pancreatitis cells induced by caerulein(P < 0.05). Compared with the cerulean + si-NC group, H19 expression was reduced, the cell survival rate was increased, the levels of TNF-α, IL-1β, and IL-6 were reduced, and LC3-Ⅰ protein expression was reduced and LC3-Ⅱ protein expression was increased in the cerulein+si-H19 group(P < 0.05). Compared with the caerulein + mi R-NC group, mi R-30a-5p expression was increased, the cell survival rate was increased, the levels of TNF-α, IL-1β, and IL-6 were decreased, and LC3-Ⅰ protein expression was reduced and LC3-Ⅱ protein expression was increased in the caerulein + mi R-30a-5p group(P < 0.05). Compared with the cerulean + si-H19 + anti-mi R-NC group, the expression of mi R-30a-5p w
作者
王春晖
潘卓
唐忠明
Chun-Hui Wang;Zhuo Pan(The First People’s Hospital Surgery of Huzhou City,Huzhou 313000,Zhejiang Province,China;Gastroenterology Department,The First People’s Hospital of Huzhou City,Huzhou 313000,Zhejiang Province,China)
出处
《世界华人消化杂志》
CAS
2022年第22期997-1003,共7页
World Chinese Journal of Digestology
