miR-146a通过靶向TRIB3调节癫痫大鼠海马神经元凋亡及炎症反应

miR-146a regulates apoptosis and inflammatory response in hippocampal neurons of epileptic rats by targeting TRIB3

目的探讨miR-146a靶向调控Tribble同源蛋白3(TRIB3)对癫痫大鼠海马神经元凋亡的影响,以及对炎症反应的调控作用。方法体外培养新生Sprague-Dawley(SD)大鼠的海马神经元,制备癫痫海马神经元模型,采用免疫荧光双染法和膜片钳技术检测癫痫海马神经元模型。将海马神经元随机分为四组:空白对照组(control组):正常海马神经元;癫痫组(EP组):癫痫海马神经元;阴性对照组(miR-146a NC组):转染100 nM miR-146a NC至癫痫海马神经元;miR-146a mimic组:转染100 nM miR-146a mimic至癫痫海马神经元,采用脂质体介导法进行转染;流式细胞术检测各组海马神经元细胞的凋亡情况;Western blot检测各组海马神经元中半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)以及肿瘤抑制基因p53的蛋白表达;ELISA法检测各组海马神经元细胞培养液中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)含量;实时荧光定量PCR(qRT-PCR)检测TNF-α、IL-1β、IL-6 mRNA表达水平;双荧光素酶报告基因实验确定miR-146a与TRIB3的靶向作用情况。结果癫痫组大鼠海马神经元与正常组大鼠海马神经元相比较形态未见明显异常;癫痫大鼠海马神经元自发性放电频率明显较正常大鼠海马神经元明显增加(P<0.05);与空白对照组比较,癫痫组miR-146a表达水平升高,海马神经元凋亡数目显著增加,海马神经元中caspase-3、Bax及p53的蛋白表达显著增加,Bcl-2蛋白表达减少,TNF-α、IL-1β和IL-6 mRNA表达水平与细胞培养液中的含量均升高,差异均有统计学意义(P<0.01)。与癫痫组比较,miR-146a mimic组海马神经元凋亡数目下降,caspase-3、Bax及p53的蛋白表达显著减少,Bcl-2蛋白表达增加,TNF-α、IL-1β和IL-6 mRNA表达水平与含量均下降,差异均有统计学意义(P<0.01)。TRIB3与miR-146a存在靶向关系,在野生型TRIB3-WT中,miR-146a mimic组荧光素酶活性较miR-146a NC组显著降低(P<0.01)。结论高表达miR-146a通过靶向调节TRIB3 mRNA表达来抑制海马神经元凋亡的发生,并减少TNF-α、IL-1β和IL-6等炎症因子的释放。

Objective To investigate the effects of miR-146 a targeting tribble homolog 3(TRIB3)on hippocampal neuron apoptosis in epilepsy rats,and to regulate the inflammatory response.Methods The hippocampal neurons of newborn Sprague-Dawley(SD)rats were cultured in vitro to prepare the hippocampal neuron model of epilepsy.The immunofluorescence double staining method and patch clamp technique were used to detect the hippocampal neuron model of epilepsy.Hippocampal neurons were randomly divided into four groups:blank control group(control group):normal hippocampal neurons;epilepsy group(EP group):epilepsy hippocampal neurons;negative control group(miR-146 a NC group):transfect 100 nM miR-146 a NC to epilepsy hippocampal neurons;miR-146 a mimic group:transfect 100 nM miR-146 a mimic to epilepsy hippocampal neurons,using liposome-mediated transfection;Flow cytometry to detect the apoptosis of hippocampal neurons in each group;Western blot detection of caspase-3(Caspase-3),B-lymphoma-2(Bcl-2),Bcl-2 related X protein(Bax),and tumor suppression in hippocampal neurons in each group protein expression of gene p53;ELISA was used to detect tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-6(IL-6)in hippocampal neuronal cell culture fluid of each group content;real-time fluorescent quantitative PCR(qRT-PCR)to detect TNF-α,IL-1β,and IL-6 mRNA expression levels;experimental determination of dual luciferase reporter genes targeting of miR-146 a and TRIB3.Results The hippocampal neurons in the epilepsy group had no obvious abnormalities compared with those in the normal group.The frequency of spontaneous firing of hippocampal neurons in epilepsy rats was significantly higher than that in normal rats(P<0.05).Compared with the blank control group,the expression level of miR-146 a in the epilepsy group increased,and the number of hippocampal neuron apoptosis increased significantly.The protein expression of caspase-3,Bax and p53 in the hippocampal neurons increased significantly,the expression of Bcl-2 protein decreased,and the cells the levels of TNF-α,IL-1βand IL-6 in the culture medium and mRNA expression level were increased,and the differences were statistically significant(P<0.01).Compared with the epilepsy group,the number of apoptosis in hippocampal neurons in the miR-146 a mimic group decreased,protein expression of caspase-3,Bax,and p53 decreased significantly,Bcl-2 protein expression increased,and TNF-α,IL-1βand The content of IL-6 decreased,and the mRNA expression level also decreased.The differences were statistically significant(P<0.01).There is a targeting relationship between TRIB3 and miR-146 a.In wild-type TRIB3-WT,the luciferase activity of miR-146 a mimic group was significantly lower than that of miR-146 a NC group(P<0.01).Conclusion Highly expressed miR-146 a can inhibit the occurrence of hippocampal neuron apoptosis through targeted regulation of TRIB3 mRNA expression and reduce the release of inflammatory factors such as TNF-α,IL-1βand IL-6.