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赛培产品文章：小鼠TNF-α、IL-1β、IL- 6、IL- 4、IL- 10、TGF-β
发布时间：2022-07-25 11:18:54
lncRNA LINC01089对氧糖剥夺/复氧损伤后 小胶质细胞极化的影响陈思思 朱馨艺 邓 钢 王 龙 doi:10.13798/j.issn.1009-153X.2022.03.012 作者单位：430060 武汉，武汉大学人民医院神经外科（陈思思、朱馨艺、邓 钢、王 龙） 通讯作者：王 龙，E-mail：361140072@qq.com
【摘要】 目的 探讨长链非编码RNA（lncRNA）LINC01089对小胶质细胞氧糖剥夺/复氧（OGD/R）损伤后表型和炎症反应的 影响及其机制。方法 体外培养小鼠小胶质细胞B-V2细胞，OGD/R损伤后，转染lncRNA LINC01089过表达质粒及其空载质粒、 沉默质粒siRNA及阴性对照，以及miR-449c-5p模拟物、抑制剂及其阴性对照寡核苷酸。采用qRT-PCR检测M1型小胶质细胞 标记物iNOS mRNA和CD86 mRNA、M2型小胶质细胞标记物Arg1 mRNA和CD206 MRNA、lncRNA LINC01089、miR-449c-5p的 表达水平；采用ELISA检测细胞上清液炎性因子的含量；采用免疫印迹法检测细胞STAT6蛋白表达水平。荧光素酶报告基因实 验验证lncRNA LINC01089与miR-449c-5p以及miR-449c-5p与STAT6的靶向关系。结果 OGD/R 损伤后，B-V2细胞lncRNA LINC01089表达水平显著下调，miR-449c-5p表达水平显著上调。荧光素酶报告基因实验结果显示，lncRNA LINC01089靶向负 调控miR-449c-5p表达，miR-449c-5p靶向负调控STAT6表达。过表达lncRNA LINC01089，显著降低M1型标记物iNOS mRNA 和 CD86 mRNA 表达水平及细胞上清液促炎因子 IL-1β、IL-6 和 TNF-α的含量，明显增高 M2 型标记物 Arg-1 mRNA 和 CD206 mRNA 表达水平及细胞上清液抗炎因子 IL-4、IL-10 和 TGF-β的含量。过表达 lncRNA LINC01089 可靶向负调控 B-V2 细胞 miR-449c-5p表达，促进STAT6蛋白表达。结论 小胶质细胞OGD/R损伤后，lncRNA LINC01089表达下调，促使小胶质细胞向 M1型转化，诱导炎症反应，其机制可能与靶向负调控miR-449c-5p/STAT6信号轴有关。 【关键词】 小胶质细胞；长链非编码RNA；lncRNA LINC01089；miR-449c-5p；小胶质细胞极化 【文章编号】 1009-153X（2022）03-0186-07 【文献标志码】 A 【中国图书资料分类号】 R 743 Effect of lncRNA LINC01089 on microglia polarization after oxygen-glucose deprivation/reoxygenation injury CHEN Si- si, ZHU Xin-yi, DENG Gang, WANG Long. Department of Neurosurgery, Renmin Hospital of Wuhan University, Wuhan 430060, China 【Abstract】 Objective To investigate the effect of lncRNA- LINC01089 on the phenotypic polarization of mouse microglia after oxygen- glucose deprivation/reoxygenation (OGD/R) injury. Methods The mouse microglia B- V2 cells were cultured in vitro. After OGD/R injury, lncRNA LINC01089 overexpression plasmid and its empty vector, silencing plasmid siRNA and negative control, as well as miR-449c-5p mimics, inhibitors and its negative control oligonucleotides were transfected into the cells. The expression levels of M1- type microglia markers iNOS and CD86 mRNAs, M2-type microglia markers Arg1 and CD206 mRNAs, lncRNA LINC01089, and miR- 449c-5p were detected by qRT-PCR. The contents of inflammatory factors in cell supernatant were detected by ELISA. Western blotting was used to detect the expression level of STAT6 protein in B-V2 cells. The luciferase reporter gene experiment was used to verify the targeting relationship between lncRNA LINC01089 and miR-449c-5p, and miR-449c-5p and STAT6. Results After OGD/R injury, the expression level of lncRNA LINC01089 in B-V2 cells was significantly down-regulated, and the expression level of miR-449c-5p was significantly up- regulated. The results of the luciferase reporter gene assay showed that lncRNA LINC01089 targeted and negatively regulated the expression of miR- 449c- 5p, and miR- 449c- 5p targeted and negatively regulated the expression of STAT6. Overexpression of lncRNA LINC01089 significantly decreased the expression levels of M1-type markers iNOS and CD86 mRNAs, and significantly decreased the contents of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in the cell supernatant, and significantly increased the M2- type marker Arg- 1 and CD206 mRNAs expression levels, and significantly increased the contents of antiinflammatory factors IL-4, IL-10 and TGF-β in cell supernatants. Overexpression of lncRNA LINC01089 can target and negatively regulate the expression of miR-449c-5p in B-V2 cells and promote the expression of STAT6 protein. Conclusions After OGD/R injury, the expression of lncRNA LINC01089 is down- regulated in microglia, which promotes the transformation of microglia to M1 type and induces inflammatory response. The mechanism may be related to the negative regulation of miR-449c-5p/STAT6 signaling axis. 【Key words】 Microglia; Long non-coding RNA; lnvRNA LINC01089; miR-449c-5p; Microglia polarization
lncRNA_LINC01...损伤后小胶质细胞极化的影响_陈思思.pdf下载
