microRNA-198通过调控PI3K/Akt信号通路对小鼠急性胰腺炎的影响及作用机制

Effect of microRNA-198 on Acute Pancreatitis in Mice by Regulating PI3 K/Akt Signaling Pathway and Its Mechanism

目的探究microRNA-198 ( miR-198 )通过调控磷脂酰肌醇3-激酶(phosphoinositide 3-kinase,PI3K)/蛋白激酶B (protein kinase B, Akt)信号通路对小鼠急性胰腺炎(acute pancreatitis, AP)的影响及作用机制。方法 RT-PCR检测胰腺炎组织miR-198的表达。将AP小鼠分为Control, AP、AP + mock、AP + inhibitor组,RT-PCR检测miR-198表达,HE染色检测心肌病理变化。试剂盒检测血清淀粉酶(serumamylase, AMS)、脂肪酶(lipase)、髓过氧化物酶(myeloperoxidase, MPO)、炎性因子白介素 6 (interleukin6, IL-6)肿瘤坏死因子a (tumor necrosis factor-α, TNF-α)的含量。Western印迹检测第10号染色体缺失的磷酸酶及张力蛋白同源蛋白(phosphatase and tensin homolog deleted on chromosome ten, PTEN)、PI3K, p-PI3K、Akt、p-Akt 的表达。将细胞分为 Control、cerulein、cerulein + mock、cerulein + inhibitor 组,RT-PCR检测miR-198表达。流式细胞术检测细胞凋亡。试剂盒检测AMS、lipase浓度。Western印迹检测PTEN, PI3K、p-PI3K、Akt、p-Akt的表达。预测miR-198的靶向结合位点并检测miR-198与PTEN靶向关系。结果miR-198表达随病情严重程度增加而升高;在动物实验中,与Control组比较,AP组miR-198表达,AMS、lipase、MPO, IL-6, TNF-α浓度,PTEN 表达升高,p-PI3K、p-Akt 表达降低;与 AP 组比较,AP + inhibitor 组 miR-198 表达,AMS、lipase、MPO、IL-6, TNF-α浓度,PTEN 表达降低,p-PI3K、p-Akt 表达升高;在细胞实验中,与Control组比较,cerulein组miR-198表达,细胞凋亡率,AMS , lipase, PTEN表达升高,p-PI3K、p-Akt表达降低;与cerulein组比较,cerulein + inhibitor组miR-198表达,细胞凋亡率,AMS、lipase, PTEN 蛋白表达降低,p-PI3K, p-Akt 表达升高;此外,与 PTEN wt 比较,miR-198 mimic +PTENwt荧光素酶活性降低,结论 miR-198抑制PTEN表达.激活PI3K/Akt通路加重小鼠急性胰腺炎。

Objective To investigate the effects of microRNA-198 on mice acute pancreatitis (AP) by regulating the PI3 K/Akt signaling pathway and the mechanism. Methods The expres sion of miR-198 was detected by RT-PCR. The AP mice model was established. The AP mice were divided into control, AP, AP + mock and AP + inhibitor group. RT-PCR was used to detect miR- 198 expression. The pathological changes of pancreatic tissue were observed by HE staining. The kits were used to detect the concentrations of serum amylase ( AMS), lipase, monocyte myeloperoxidase (MPO ), inflammatory factor IL-6, tumor necrosis factor alpha ( TNF-α) in each group. Western blotting was used to detect the expression of PTEN, PI3 K, p-PI3 K, Akt and p- Akt. The cells were divided into control, cerulein, cerulein + mock, cerulein + inhibitor group. The expression of miR-198 was detected by RT-PCR. Flow cytometry was used to detect apoptosis. The kits were used to measure the concentration of AMS and lipase. Western blotting was used to detect the expression of PTEN, PI3 K, p-PI3 K, Akt and p-AKT. The targeted binding site of miR-198 was predicted and the relationship between miR-198 and PTEN was detected. Results The expression of miR-198 increased with the aggravation of the disease. In animal experiments, compared with control group, the expression of miR-198 in AP group was increased, the concentration of AMS, lipase, MPO, IL-6 and TNF-α were increased, and the expression of PTEN was increased, while the expression levels of p-PI3 K and p-Akt were decreased. Compared with the AP group, the expression of miR-198 in AP + inhibitor group was decreased, the concentration of AMS, lipase, MPO, IL-6 and TNF-α was decreased, the expression of PTEN was decreased, and expression of p-PI3 K and p- Akt was increased. In cytologic al experiments, compared with the control group, the expression of miR-198 in cerulein group was increased, the apoptosis rate of cells was increased, the concentration of AMS, lipase, MPO, IL-6 and TNF-α was increased, and the expression level of PTEN was increased, while the expression levels of p-PI3 K and p-Akt were decreased. Compared with the cerulein group, the expression level of miR-198 in cerulein + inhibitor group was decreased ,the apoptosis rate of cells was decreased, the concentration of AMS, lipase, MPO, IL-6 and TNF-α was decreased, the expression of PTEN was decreased, expression of p-PI3 K and p- Akt was increased, furthermore, the luciferase activity of PTEN wild-type treated with miR-198 mimic was reduced compared to the PTEN wild-type. Conclusion miR-198 inhibits the expression of PTEN, thereby activating PI3 K/Akt pathway and aggravating acute pancreatitis in mice.