Zhang Xiaoliang, Ye Jinshan, Liang Xing, Yang Lixia
Department of Postgraduate, Third Military Medical University, Chongqing 400038, China.
Department of Cardiology, Kunming General Hospital of Chengdu Military Area Command, Kunming 650032, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2017 Aug;33(8):1079-1086.
Objective To investigate the effect of microRNA-155 on inflammatory response and lipid uptake of macrophages after the cells are stimulated by ox-LDL and its potential mechanism. Methods Macrophage RAW264.7 cells were treated with 0, 25, 50 and 100 μg/mL ox-LDL for 24 hours or with 50 μg/mL ox-LDL for 0, 6, 12, 24 hours. The level of miR-155 was evaluated in all above samples through real-time quantitative PCR. In our research, RAW264.7 cells were divided into six groups: control group, ox-LDL group, ox-LDL/negative control group, ox-LDL/anti-miR-155 group, ox-LDL/shRNA negative control group and ox-LDL/PPARγ-shRNA group. Oil red O staining was used to observe lipid uptake in the cells. Filipin staining was used to evaluate the cellular uptake of ox-LDL. Cholesterol testing was performed to examine the levels of total cholesterol (TC) and free cholesterol (FC). Real-time quantitative PCR was done to detect the expressions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 mRNAs. According to study purpose, we explored the potential mechanisms of miR-155 inhibitor (including control group, ox-LDL group, ox-LDL/negative control group and ox-LDL/miR-155 inhibitor group), miR-155 mimic (including negative control group and miR-155 mimic group), and PPARγ shRNA (including control group, ox-LDL group, ox-LDL/shRNA negative control group and ox-LDL/PPARγ shRNA group) in ox-LDL-treated RAW264.7 cells through evaluating the expressions of p-STAT3, PPARγ, CD36 and NF-κBp65 using Western blotting. Results Ox-LDL stimulation increased the relative expression of miR-155 in a dose- and time-dependent manner. Through oil red O staining, Filipin staining, cholesterol testing and real-time PCR experiment, we found the relative absorbance, levels of TC and FC, filipin fluorescence intensity, and levels of TNF-α, IL-1β and IL-6 mRNAs were significantly lower in ox-LDL/anti-miR-155 group than in ox-LDL and ox-LDL/negative control group. Similarly, the relative absorbance, levels of TC and FC, filipin fluorescence intensity and levels of TNF-α, IL-1β and IL-6 mRNAs were significantly lower in ox-LDL/ PPARγ shRNA group than in ox-LDL group and ox-LDL/shRNA negative control group. The expressions of p-STAT3, PPARγ, CD36 and NF-κBp65 proteins were suppressed in ox-LDL/anti-miR-155 group as compared with ox-LDL group and ox-LDL/negative control group. Similarly, p-STAT3, PPARγ, CD36 and NF-κBp65 protein levels decreased in ox-LDL/PPARγ shRNA as compared with ox-LDL/vector group. Moreover, p-STAT3, PPARγ, CD36 and NF-κBp65 protein levels were higher in miR-155 mimic group than in negative control group. Conclusion Mediated by PPARγ, miR-155 induced inflammation response and lipid uptake of macrophages via STAT3/NF-κB signal pathway and CD36.
目的 探讨微小RNA-155对氧化型低密度脂蛋白（ox-LDL）刺激后巨噬细胞炎症反应和脂质摄取的影响及其潜在机制。方法 用0、25、50和100μg/mL的ox-LDL处理巨噬细胞RAW264.7细胞24小时，或用50μg/mL的ox-LDL处理0、6、12、24小时。通过实时定量PCR评估上述所有样本中miR-155的水平。在本研究中，RAW264.7细胞分为六组：对照组、ox-LDL组、ox-LDL/阴性对照组、ox-LDL/抗miR-155组、ox-LDL/shRNA阴性对照组和ox-LDL/PPARγ-shRNA组。采用油红O染色观察细胞内脂质摄取情况。采用 Filipin 染色评估细胞对ox-LDL的摄取。进行胆固醇检测以检测总胆固醇（TC）和游离胆固醇（FC）水平。采用实时定量PCR检测肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）和IL-6 mRNA的表达。根据研究目的，通过蛋白质印迹法评估p-STAT3、PPARγ、CD36和NF-κBp65的表达，探讨miR-155抑制剂（包括对照组、ox-LDL组、ox-LDL/阴性对照组和ox-LDL/miR-155抑制剂组）、miR-155模拟物（包括阴性对照组和miR-155模拟物组）以及PPARγ shRNA（包括对照组、ox-LDL组、ox-LDL/shRNA阴性对照组和ox-LDL/PPARγ shRNA组）在ox-LDL处理的RAW264.7细胞中的潜在机制。结果 ox-LDL刺激以剂量和时间依赖性方式增加miR-155的相对表达。通过油红O染色、Filipin染色、胆固醇检测和实时PCR实验，我们发现ox-LDL/抗miR-155组的相对吸光度、TC和FC水平、Filipin荧光强度以及TNF-α、IL-1β和IL-6 mRNA水平均显著低于ox-LDL组和ox-LDL/阴性对照组。同样，ox-LDL/PPARγ shRNA组的相对吸光度、TC和FC水平、Filipin荧光强度以及TNF-α、IL-1β和IL-6 mRNA水平均显著低于ox-LDL组和ox-LDL/shRNA阴性对照组。与ox-LDL组和ox-LDL/阴性对照组相比，ox-LDL/抗miR-155组中p-STAT3、PPARγ、CD36和NF-κBp65蛋白的表达受到抑制。同样，与ox-LDL/载体组相比，ox-LDL/PPARγ shRNA组中p-STAT3、PPARγ、CD36和NF-κBp65蛋白水平降低。此外，miR-155模拟物组中p-STAT3、PPARγ、CD36和NF-κBp65蛋白水平高于阴性对照组。结论 miR-155通过PPARγ介导，经由STAT3/NF-κB信号通路和CD36诱导巨噬细胞的炎症反应和脂质摄取。
