Long non-coding RNA MEG3 promotes tumor necrosis factor-alpha induced oxidative stress and apoptosis in interstitial cells of cajal via targeting the microRNA-21 /I-kappa-B-kinase beta axis

Interstitial Cells of Cajal (ICC) plays a critical role in the peristaltic contractions of the gastrointestinal and urinary tract. The dysfunction and loss of ICC contributes to hypokinetic disease, such as gallstoneand ureteropelvic junction obstruction . In the present study, we identified the underlying driving molecular signals of oxidative stress and apoptosis in ICC. ICC was isolated from small intestine of Balb/c mice, and stimulated with tumor necrosis factor-alpha (TNF-α). MTT and flow cytometry were performed to assess cell viability, apoptosis, and the level of reactive oxygen species in ICC, respectively. The level of malondialdehyde, superoxide dismutase, and glutathione peroxidase in cells were measured to assess oxidative stress. The expression of inflammatory factors (interleukin, IL-1 and IL-6) and apoptosis-related proteins were detected by western blot. We observed that TNF-αinduced inflammation, oxidative stress and cell apoptosis in ICC. By using quantitative real-time PCR , we verified that the expression of long non-coding RNAMEG3 was elevated by TNF-α in ICC. Silencing MEG3 reversed inflammation, oxidative stress, and cell apoptosisin TNF-α-treated ICC. Subsequently, we confirmed that MEG3 sponged cytoprotective miR-21 to upregulate the expression of I-kappa-B-kinase beta (IKKB) and activate the nuclear factor kappa-B (NF-κB) pathway. Both miR-21 overexpression and IKKB knockdown reduced TNF-α-induced above symptoms in ICC. Taken together, we can conclude that MEG3 mediates inflammation, oxidative stress and apoptosis in TNF-α-treated ICC via the miR-21/IKKB-NF-κB axis. The study improves our understanding of the molecular mechanism of ICC reduction related diseases.卡哈尔间质细胞（Interstitial Cells of Cajal，ICC）在胃肠道与泌尿道的蠕动收缩中发挥关键作用。ICC的功能异常与缺失会引发运动减退性疾病，例如胆结石（gallstone）与肾盂输尿管连接部梗阻（ureteropelvic junction obstruction）。本研究明确了ICC中氧化应激与细胞凋亡的潜在分子驱动信号。研究人员从Balb/c小鼠的小肠中分离得到ICC，并采用肿瘤坏死因子-α（tumor necrosis factor-alpha，TNF-α）对其进行刺激。分别通过MTT法与流式细胞术（flow cytometry）检测ICC的细胞活力、凋亡情况以及活性氧（reactive oxygen species）水平；通过检测细胞内丙二醛（malondialdehyde）、超氧化物歧化酶（superoxide dismutase）与谷胱甘肽过氧化物酶（glutathione peroxidase）的表达水平评估氧化应激状态；采用蛋白质印迹法（western blot）检测炎症因子（白细胞介素-1（interleukin-1，IL-1）与白细胞介素-6（interleukin-6，IL-6））以及凋亡相关蛋白的表达情况。研究结果显示，TNF-α可诱导ICC产生炎症反应、氧化应激与细胞凋亡。通过实时定量聚合酶链式反应（quantitative real-time PCR）验证发现，TNF-α处理可上调ICC中长链非编码RNA MEG3（long non-coding RNA MEG3）的表达水平。沉默MEG3可逆转TNF-α处理的ICC中的炎症反应、氧化应激与细胞凋亡现象。进一步研究证实，MEG3可通过海绵吸附作用靶向结合具有细胞保护作用的微小RNA-21（miR-21），进而上调IκB激酶β（I-kappa-B-kinase beta，IKKB）的表达并激活核因子κB（nuclear factor kappa-B，NF-κB）信号通路。过表达miR-21以及敲低IKKB均可减轻TNF-α诱导的ICC上述异常表型。综上，本研究表明MEG3可通过miR-21/IKKB-NF-κB信号轴介导TNF-α处理的ICC的炎症反应、氧化应激与细胞凋亡，本研究加深了我们对ICC相关疾病分子机制的理解。