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Effects of miR-532-5p on human brain microvascular endothelial cells damage induced by ox-LDL via down-regulating
1School of Nursing, Jiangsu Vocational College of Medicine, Yancheng, China.
Abstract:
The effect of miR-532-5p on human brain microvascular endothelial cells damage induced by ox-LDL is studied. HBMEC-3 were cultured and treated with ox-LDL for 24 h to establish a model of cell oxidative damage. The expression of miR-532-5p was detected by qRT-PCR and that of CLIC4 was detected by Western blot. miR-NC, miR-532-5p mimics, si-NC, si-CLIC4, miR-532-5p mimics and pcDNA, miR-532-5p mimics and pcDNA-CLIC4 were transfected into HBMEC-3 cells, respectively, using ox -LDL processing for 24 h. Flow cytometry was used to detect the apoptotic rate. The dual luciferase reporting experiment verified the relationship between miR-532-5p and CLIC4. The ox-LDL treatment led to lower expression of miR-532-5p (p<0.05), higher expression of CLIC4 (P <0.05), enhanced content of MDA (p<0.05), decreased activities of SOD and CAT (p<0.05), increased apoptosis rate (p<0.05), higher protein level of Bax (p<0.05), and lower protein level of Bcl-2 (p<0.05). Compared with ox-LDL + miR-NC group and ox-LDL + si-NC group, ox-LDL + miR-532-5p group and ox-LDL + si-CLIC4 group had decreased content of MDA (P<0.05), increased activities of SOD and CAT (p<0.05), decreased apoptosis rate (p<0.05), lower level of Bax (p<0.05), and higher level of Bcl-2 (p<0.05). The miR-532-5p mitigates human brain microvascular endothelial cells damage induced by ox-LDL via down-regulating CLIC4 expression.
Insights
MicroRNA-532-5p protects human brain microvascular endothelial cells from oxidative damage caused by oxidized low-density lipoprotein (ox-LDL). It achieves this by down-regulating Chloride Intracellular Channel 4 (CLIC4) expression, reducing cell apoptosis and improving antioxidant capacity.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Oxidized low-density lipoprotein (ox-LDL) is implicated in cerebrovascular damage.
- MicroRNAs play crucial roles in regulating cellular processes and disease pathogenesis.
- Understanding the molecular mechanisms underlying ox-LDL-induced endothelial cell injury is vital for developing therapeutic strategies.
Purpose of the Study:
- To investigate the role of microRNA-532-5p (miR-532-5p) in protecting human brain microvascular endothelial cells (HBMEC-3) against ox-LDL-induced damage.
- To elucidate the molecular mechanism by which miR-532-5p exerts its protective effects, focusing on its interaction with Chloride Intracellular Channel 4 (CLIC4).
Main Methods:
- Establishment of an ox-LDL-induced oxidative damage model in HBMEC-3 cells.
- Quantitative real-time PCR (qRT-PCR) and Western blot to assess miR-532-5p and CLIC4 expression.
- Transfection with miR-532-5p mimics, si-CLIC4, and relevant controls.
- Flow cytometry to evaluate apoptosis rates.
- Dual-luciferase reporter assay to confirm the targeting relationship between miR-532-5p and CLIC4.
Main Results:
- Ox-LDL treatment significantly decreased miR-532-5p expression while increasing CLIC4 expression, MDA content, and apoptosis rate, accompanied by reduced SOD and CAT activities and altered Bax/Bcl-2 protein levels.
- Overexpression of miR-532-5p or knockdown of CLIC4 reversed the detrimental effects of ox-LDL, reducing MDA, increasing antioxidant enzyme activities, decreasing apoptosis, and modulating Bax/Bcl-2.
- Dual-luciferase assay confirmed that miR-532-5p directly targets CLIC4.
Conclusions:
- miR-532-5p acts as a protective factor against ox-LDL-induced damage in human brain microvascular endothelial cells.
- The protective mechanism involves the down-regulation of CLIC4 expression by miR-532-5p.
- These findings suggest miR-532-5p as a potential therapeutic target for cerebrovascular diseases associated with ox-LDL.
