Youjiang Medical University for Nationalities, Baise, Guangxi, China.
Department of Medical Laboratory, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, Guangxi, China.
Ann Clin Lab Sci. 2024 Jan;54(1):47-55.
To investigate the clinical significance of miR-499a expression in the serum of ischemic stroke patients and its potential mechanism in regulating astrocytes to promote ischemic stroke.
Serum samples from 99 ischemic stroke patients and 99 healthy individuals were collected and analyzed for miR-499a expression through RT-PCR. Statistical analysis was performed to compare the expression differences between the two groups, and correlation between miR-499a expression and clinical pathological indices in stroke patients was analyzed. MiR-499a mimic, inhibitor, and negative control vectors were constructed and transfected into astrocyte SVGp12 cells. Afterward, miR-499a expression was validated by RT-PCR, cell viability was assessed by CCK8 assay, and apoptosis was detected using flow cytometry. The binding sites of miR-499a and Beclin1 were predicted by the Target-scan database and confirmed by dual luciferase assay. After overexpressing Beclin1, co-transfection with miR-499a mimic or negative control was conducted to observe the reverse effect of miR-499a mimic on Beclin1 overexpression.
MiR-499a was significantly upregulated in the stroke group (<0.001), it was positively correlated with TC (Total Cholesterol), LDL-C (Low-density lipoprotein cholesterol), and APO-A1 (Apolipoprotein A1) (R>0.3, <0.001). MiR-499a mimics promoted cell viability while inhibiting apoptosis of astrocytes. MiR-499a targeted Beclin 1 and inhibited its mRNA and protein expression, as well as the expression of autophagy-related proteins LC-3 and p62. MiR-499a could reverse the impact of Beclin1 overexpression on SVGp12 astrocyte proliferation and apoptosis.
Serum miR-499a in stroke patients may serve as a potential diagnostic indicator. MiR-499a-mediated inhibition of Beclin 1, subsequently leading to suppression of astrocytic autophagy and viability, may represent a pivotal mechanism underlying its promotion of IS.
探讨缺血性脑卒中患者血清 miR-499a 表达的临床意义及其在调节星形胶质细胞促进缺血性脑卒中中的潜在机制。
收集 99 例缺血性脑卒中患者和 99 例健康对照者的血清样本，通过 RT-PCR 分析 miR-499a 的表达。采用统计学方法比较两组间表达差异，分析脑卒中患者 miR-499a 表达与临床病理指标的相关性。构建 miR-499a 模拟物、抑制剂和阴性对照载体，并转染星形胶质细胞 SVGp12 细胞。通过 RT-PCR 验证 miR-499a 表达，CCK8 法评估细胞活力，流式细胞术检测细胞凋亡。Target-scan 数据库预测 miR-499a 与 Beclin1 的结合位点，并通过双荧光素酶报告实验进行验证。过表达 Beclin1 后，与 miR-499a 模拟物或阴性对照共转染，观察 miR-499a 模拟物对 Beclin1 过表达的逆转作用。
miR-499a 在脑卒中组中显著上调（<0.001），与 TC（总胆固醇）、LDL-C（低密度脂蛋白胆固醇）和 APO-A1（载脂蛋白 A1）呈正相关（R>0.3，<0.001）。miR-499a 模拟物促进星形胶质细胞活力，抑制细胞凋亡。miR-499a 靶向 Beclin1 并抑制其 mRNA 和蛋白表达，以及自噬相关蛋白 LC-3 和 p62 的表达。miR-499a 可以逆转 Beclin1 过表达对 SVGp12 星形胶质细胞增殖和凋亡的影响。
脑卒中患者血清 miR-499a 可能作为一种潜在的诊断标志物。miR-499a 介导的 Beclin1 抑制，进而抑制星形胶质细胞自噬和活力，可能是其促进 IS 的关键机制。
