miR-16-5p Promotes Inflammation and Apoptosis in Oxygen-Glucose Deprivation Microglia Model by Mediating GPR30 Expression
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摘要:
目的 探究在缺血性中风小胶质细胞（BV-2）体外模型中，miR-16-5p通过靶向GPR30表达促进细胞凋亡和炎症反应的分子机制。 方法 通过氧-葡萄糖剥夺（oxygen glucose deprivation，OGD）处理BV-2细胞构建缺血性中风的细胞模型。qRT-PCR评估OGD细胞中miR-16-5p和GPR30 mRNA水平。在OGD细胞中转染miR-16-5p inhibitor沉默miR-16-5p表达，并通过ELISA试剂盒和Annexin V-FITC/PI染色检测miR-16-5p对OGD细胞模型的炎症反应和凋亡的变化。通过Starbase预测和双荧光素酶报告基因实验验证miR-16-5p靶向GPR30的3’-非翻译区（UTR）序列。在OGD细胞中敲低miR-16-5p或/和GPR30检测细胞炎症反应和凋亡的变化。 结果 miR-16-5p在OGD诱导的细胞中表达升高（P < 0. 01），而GPR30表达降低（P < 0. 01）。敲低miR-16-5p抑制了OGD细胞的炎症因子表达水平以及细胞凋亡的比例（P < 0. 01）。抑制miR-16-5p表达能够上调GPR30 mRNA和蛋白质水平（P < 0. 01）。同时沉默miR-16-5p和GPR30，能够部分促进单独转染miR-16-5p inhibitor细胞的炎症因子表达水平和细胞凋亡的比例（P < 0. 05）。 结论 在小胶质细胞OGD模型中，miR-16-5p通过抑制GPR30的表达，激活小胶质细胞的炎症反应，促进其凋亡。 Abstract:Objective To explore the molecular mechanism of miR-16-5p promoting apoptosis and inflammatory response by targeting GPR30 expression in an in vitro ischemic stroke microglia model （BV-2）. Methods An ischemic stroke cell model was established by subjecting BV-2 cells to oxygen-glucose deprivation （OGD）. qRT-PCR was utilized to assess the levels of miR-16-5p and GPR30 mRNA in OGD cells. A miR-16-5p inhibitor was transfected into OGD cells to silence miR-16-5p expression, and alterations in inflammatory response and apoptosis were measured using ELISA kits and Annexin V-FITC/PI staining. Starbase was employed to predict interactions, and dual-luciferase reporter gene assays were conducted to confirm that miR-16-5p targets the 3'-untranslated region （UTR） sequence of GPR30. Changes in cellular inflammatory response and apoptosis were evaluated by knocking down miR-16-5p and/or GPR30 in OGD cells. Results miR-16-5p expression was significantly elevated （P < 0.01）, while GPR30 expression was notably decreased （P < 0.01） in OGD-induced cells. Knockdown of miR-16-5p reduced the expression levels of inflammatory factors and the cell apoptosis ratio （P < 0.01）. Inhibition of miR-16-5p expression led to an upregulation of GPR30 mRNA and protein levels （P < 0.01）. Simultaneous silencing of both miR-16-5p and GPR30 partially enhanced inflammatory factor expression levels and the cell apoptosis ratio compared to cells transfected solely with the miR-16-5p inhibitor （P < 0.05）. Conclusion In the microglia OGD model, miR-16-5p triggers inflammatory responses and enhances apoptosis by inhibiting GPR30 expression. -
Key words:
- Ischemic stroke /
- miR-16-5p /
- GPR30 /
- Inflammatory response /
- Apoptosis
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图 1 miR-16-5p和GPR30在OGD模型BV-2细胞中的表达水平
*P < 0.01；***P < 0.001。
Figure 1. Expression levels of miR-16-5p and GPR30 in OGD model BV-2 cells
图 2 敲低miR-16-5p后各组细胞的炎症因子含量
*P < 0.05；**P < 0.01；***P < 0.001。
Figure 2. Levels of inflammatory factors in each group after miR-16-5p knockdown
图 3 敲低miR-16-5p后各组细胞的凋亡比例
A: 敲低miR-16-5p后对各组细胞凋亡的影响；B：敲低miR-16-5p后各组细胞凋亡率柱状图；***P < 0.001。
Figure 3. Apoptosis ratio of cells in each group after knocking down miR-16-5p
图 4 敲低miR-16-5p后各组细胞的凋亡和炎症蛋白的表达水平变化
A：凋亡和炎症蛋白在各组细胞中的免疫印迹图；B：凋亡和炎症蛋白相对表达量柱状图；*P < 0.05；**P < 0.01。
Figure 4. Changes in apoptosis and expression levels of inflammatory proteins in various groups of cells after knocking down miR-16-5p
图 5 生物信息学预测miR-16-5p和GPR30的靶向调控位点
Figure 5. Bioinformatics prediction of targeted regulatory sites for miR-16-5p and GPR30
图 6 miR-16-5p靶向负调控GPR30的表达
***P < 0.001。
Figure 6. miR-16-5p targeting negative regulation of GPR30 expression
图 7 抑制miR-16-5p对GPR30 mRNA在细胞中的表达水平的影响
*P < 0.05；**P < 0.01。
Figure 7. The effect of inhibiting miR-16-5p on the expression level of GPR30 mRNA in cells
图 8 抑制miR-16-5p对GPR30蛋白质在细胞中的表达水平的影响
A：GPR30蛋白在各组细胞中的免疫印迹图；B：GPR30蛋白相对表达量柱状图；**P < 0.01。
Figure 8. Effect of miR-16-5p inhibition on the expression level of GPR30 protein in cells
图 9 miR-16-5p调控GPR30 mRNA的表达
**P < 0.01；***P < 0.001。
Figure 9. miR-16-5p regulates the expression of GPR30 mRNA
图 12 miR-16-5p调控GPR30蛋白介导细胞凋亡和炎症蛋白的表达水平
A：GRP30，凋亡和炎症蛋白在各组细胞中的免疫印迹图；B：GRP30和凋亡蛋白相对表达量柱状图；C：炎症蛋白相对表达量柱状图；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 12. miR-16-5p regulates GPR30 protein mediated apoptosis and expression levels of inflammatory proteins
图 10 miR-16-5p调控GPR30介导炎症反应
*P < 0.05；**P < 0.01；***P < 0.001。
Figure 10. miR-16-5p regulates GPR30 mediated inflammatory response
图 11 miR-16-5p调控GPR30介导细胞凋亡
A: 敲低miR-16-5p或/和GPR30后对各组细胞凋亡的影响；B：敲低miR-16-5p或/和GPR30后各组细胞凋亡率柱状图；*P < 0.05；**P < 0.01；***P < 0.001。
Figure 11. miR-16-5p regulates GPR30 mediated cell apoptosis
表 1 qRT-PCR引物序列
Table 1. qRT-PCR primer sequences
正向引物序列（5'-3'） 反向引物序列（5'-3'） miR-16-5p GCAGCACGTAAATATTGGC GAACATGTCTGCGTATCTC GPR30 GCCACATAGTCAACCTTGCAGC CGTCTTCTGCTCCACATAGAGC U6 GAACGCCTCATGATTTGCAGG AGAAGACTGAAACAGCACAGAGA GAPDH CATCACTGCCACCCAGAAGACTG ATGCCAGTGAGCTTCCCGTTCAG 
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