基于生信学数据挖掘和实验验证阿魏酸介导间充质干细胞外泌体调控炎症miRNA表达情况

Verification of Inflammatory miRNA Expression Regulated by Ferulic Acid-mediated hUCBMSC-Exos Based on Bioinformatics Data Mining and Experiment

关键词:  阿魏酸  间充质干细胞  外泌体  炎症  miRNA  医学信息学

Key words:  ferulic acid  mesenchymal stem cells  exosomes  inflammation  miRNA  medical informatics

摘要:

目的 基于生信分析筛选出人脐血间充质干细胞来源的外泌体(human umbilical cord blood mesenchymal stem cells-derived exosomes，hUCBMSC-Exos)中可用于调控炎症过程的微小RNA (microRNA，miRNA)。在此基础上，研究阿魏酸(ferulic acid，FA)对hUCBMSC-Exos中与炎症调控相关miRNA的影响。方法 从现有数据库中分别筛选hUCBMSC-Exos中的miRNAs和与调控炎症相关的miRNAs，取2组miRNAs的交集，从而获得hUCBMSC-Exos中与调控炎症相关的候选miRNA分子组。取足月健康新生儿脐带血，将分离所得的单个核细胞置于DMEM/F12培养基培养至P4代细胞，进行成骨、成软骨和成脂诱导分化鉴定。实验分为2个部分来验证FA对hUCBMSC-Exos及hUCBMSCs分泌相关炎症因子的作用：①将鉴定成功的hUCBMSC-Exos随机分为实验组及对照组，实验组用2 mg·L^-1 FA干预，对照组用等体积PBS液处理，培养24 h。采用超速离心法结合超滤法提取hUCBMSC-Exos，通过透射电子显微镜观察其形态，蛋白印迹法鉴定其特征表面标志物CD9、CD63和TSG101，采用BCA法测定hUCBMSC-Exos浓度，RT-qPCR法检测各组外泌体中炎症调控相关候选miRNAs的表达。②另将鉴定成功的人hUCBMSCs按随机数字表法随机分为实验组、模型组和对照组，实验组与模型组用含30 mmo1·L^-1的葡萄糖DMEM/F12培养基培养120 h，建立高糖诱导损伤hUCBMSCs细胞模型后，实验组用2 mg·L^-1 FA干预，模型组和对照组用等容量的PBS液处理。每组均培养24 h后用ELISA试剂盒检测3组上清液中炎性因子含量。结果 通过对GEO测序数据分析和对文献的筛选最终获得7个在hUCBMSC-Exos中可能高表达且与炎症调控相关的miRNAs：miR-125b、miR-126、miR-145、miR-146a、miR-21、miR-221、miR-31-5p。成功培养、分离和鉴定得到hUCBMSCs，并进一步收集到hUCBMSC-Exos，培养24 h后，实验组hUCBMSC-Exos的浓度为(1.179±0.03)μg·mL^-1，明显高于对照组(0.881±0.03)μg·mL^-1(P<0.01)。与对照组相比，实验组中部分炎症调控相关miRNA变化显著，miR-126-3p和miR-146a-5p显著上升，miR-145及miR-31-5p显著下降，差异有统计学意义(P<0.05)。与模型组相比，在2 mg·L^-1 FA干预下，实验组中hUCBMSCs分泌IL-1β、MCP-1水平明显降低，分泌IL-10、M-CSF明显水平升高，差异具有统计学意义(P<0.05)，2组间IL-6分泌差异无统计学意义。与对照组相比，模型组hUCBMSCs分泌IL-1β、IL-6、MCP-1水平明显增高，差异具有统计学意义(P<0.05)，2组间IL-10、M-CSF分泌差异无统计学意义。结论 FA可能通过介导hUCBMSC-Exos中调控炎症的miRNA (miR-126-3p、miR-146a-5p、miR-145及miR-31-5p)分泌，进一步调控hUCBMSCs分泌相关炎症因子从而调控机体炎症反应，本研究进一步从外泌体水平证实FA具有调控炎症的潜能。为进一步研究FA通过hUCBMSC-Exos调控机体炎症反应奠定坚实基础。

Abstract:

OBJECTIVE To screen microRNAs(miRNAs) from human umbilical cord blood mesenchymal stem cells-derived exosomes(hUCBMSC-Exos) that can be used to regulate inflammatory processes, and study the effect of ferulic acid(FA) on miRNAs related to inflammation regulation in hUCBMSC-Exos on this basis. METHODS miRNAs in hUCBMSC-Exos and miRNAs related to inflammation regulation were screened from the existing database, and their intersection was taken to obtain candidate miRNA molecular groups related to inflammation regulation in hUCBMSC-Exos. The umbilical cord blood of full-term healthy newborns was taken, and the isolated mononuclear cells were cultured in DMEM/F12 medium to P4 generation cells, and the differentiation of osteogenesis, chondrogenesis and adipogenesis was identified. The experiment included two parts to verify the effect of FA on hUCBMSC-Exos and inflammatory cytokines secreted by hUCBMSCs: ①The identified hUCBMSC-Exos were randomly divided into experimental group and control group. The experimental group was intervened with 2 mg·L^-1FA, and the control group was treated with equal volume PBS solution for 24 h. hUCBMSC-Exos was extracted by ultracentrifugation combined with ultrafiltration, and its morphology was observed by transmission electron microscope. Its characteristic surface markers CD9, CD63 and TSG101 were identified by Western blotting. The concentration of hUCBMSC-Exos was determined by BCA method, and the expression of candidate miRNAs related to inflammation in exosomes of each group was detected by RT-qPCR. ②The hUCBMSCs were randomly divided into experimental group, model group and control group according to the random number table method. The experimental group and model group were cultured in DMEM/F12 medium containing 30 mmo1·L^-1 glucose for 120h to establish the high glucose damaged hUCBMSCs model. The experimental group was treated with 2 mg·L^-1FA. Model group and control group were treated with PBS solution of equal volume. The content of inflammatory factors in supernatant of each group was detected by ELISA kit after 24h culture. RESULTS The 7 miRNAs such as miR-125b, miR-126, miR-145, miR-146a, miR-21, miR-221 and miR-31-5p, which might be highly expressed in hUCBMSC-Exos and were related to the regulation of inflammation, were obtained through the analysis of GEO sequencing data and the screening of literatures. In the experiment, hUCBMSCs were successfully cultured, isolated and identified, and hUCBMSC-Exos was further collected. After culture for 24 h, the concentration of hUCBMSC-Exos in the experimental group was (1.179±0.03)mg·mL^-1, which was significantly higher than that in the control group of (0.881±0.03)mg·mL^-1 (P<0.01). Compared with the control group, some inflammatory regulation-related miRNAs in the experimental group changed significantly, miR-126-3p and miR-146a-5p were significantly increased, while miR-145 and miR-31-5p were significantly decreased, with statistically significant differences(P<0.05). Compared with the model group, hUCBMSCs secretion of IL-1β and MCP-1 were significantly decreased, while IL-10 and M-CSF were significantly increased in the experimental group after 2 mg·L^-1 FA intervention, with statistically significant differences(P<0.05), while there was no statistically significant difference in IL-6 secretion between the two groups. Compared with the control group, the secretion of IL-1β, IL-6 and MCP-1 of hUCBMSCs in model group was significantly increased, and the difference was statistically significant(P<0.05). There was no significant difference in IL-10 and M-CSF secretion between the two groups. CONCLUSION FA may regulate inflammation by mediating inflammation related miRNA: miR-126-3p, miR-146a-5p, miR-145 and miR-31-5p in hUCBMSC-Exos, to further regulates the inflammatory factors secreted by hUCBMSCs and regulates the body’s inflammatory response. This study further confirmed that FA has the potential to regulate inflammation at the level of exosomes. It laid a solid foundation for further research on the regulation of inflammatory response by FA through hUCBMSC-Exos level.