分析 miR-124 和 miR-16 在非酒精性脂肪性肝病中的治疗潜力。
Analysis of the therapeutic potential of miR-124 and miR-16 in non-alcoholic fatty liver disease.
机构信息
Department of Medical Biotechnology and Nanotechnology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Department of Medical Biotechnology and Nanotechnology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
出版信息
J Diabetes Complications. 2024 Apr;38(4):108722. doi: 10.1016/j.jdiacomp.2024.108722. Epub 2024 Mar 12.
BACKGROUNDS
Non-alcoholic fatty liver disease (NAFLD) is a common condition affecting >25 % of the population worldwide. This disorder ranges in severity from simple steatosis (fat accumulation) to severe steatohepatitis (inflammation), fibrosis and, at its end-stage, liver cancer. A number of studies have identified overexpression of several key genes that are critical in the initiation and progression of NAFLD. MiRNAs are potential therapeutic agents that can regulate several genes simultaneously. Therefore, we transfected cell lines with two key miRNAs involved in targeting NAFLD-related genes.
METHODS
The suppression effects of the investigated miRNAs (miR-124 and miR-16) and genes (TNF, TLR4, SCD, FASN, SREBF2, and TGFβ-1) from our previous study were investigated by real-time PCR in Huh7 and HepG2 cells treated with oleic acid. Oil red O staining and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay were utilized to assess cell lipid accumulation and cytotoxic effects of the miRNAs, respectively. The pro-oxidant-antioxidant balance (PAB) assay was undertaken for miR-16 and miR-124 after cell transfection.
RESULTS
Following transfection of miRNAs into HepG2, oil red O staining showed miR-124 and miR-16 reduced oleic acid-induced lipid accumulation by 35.2 % and 28.6 % respectively (p < 0.05). In Huh7, miR-124 and miR-16 reduced accumulation by 23.5 % and 31.3 % respectively (p < 0.05) but without impacting anti-oxidant activity. Real-time PCR in HepG2 revealed miR-124 decreased expression of TNF by 0.13-fold, TLR4 by 0.12-fold and SREBF2 by 0.127-fold (p < 0.05). miR-16 decreased TLR4 by 0.66-fold and FASN by 0.3-fold (p < 0.05). In Huh7, miR-124 decreased TNF by 0.12-fold and FASN by 0.09-fold (p < 0.05). miR-16 decreased SCD by 0.28-fold and FASN by 0.64-fold (p < 0.05). MTT assays showed, in HepG2, viability was decreased 24.7 % by miR-124 and decreased 33 % by miR-16 at 72 h (p < 0.05). In Huh7, miR-124 decreased viability 42 % at 48 h and 29.33 % at 72 h (p < 0.05), while miR-16 decreased viability by 32.3 % (p < 0.05).
CONCLUSION
These results demonstrate the ability of miR-124 and miR-16 to significantly reduce lipid accumulation and expression of key pathogenic genes associated with NAFLD through direct targeting. Though this requires further in vivo investigation.
背景
非酒精性脂肪性肝病（NAFLD）是一种常见疾病，影响全球超过 25%的人口。这种疾病的严重程度从单纯的脂肪堆积（脂肪积聚）到严重的脂肪性肝炎（炎症）、纤维化，最终发展为肝癌。许多研究已经确定了几种关键基因的过度表达，这些基因在 NAFLD 的发生和发展中至关重要。miRNA 是一种潜在的治疗剂，可以同时调节多个基因。因此，我们用两种关键 miRNA（miR-124 和 miR-16）转染细胞系，这两种 miRNA 参与靶向与 NAFLD 相关的基因。
方法
用油酸处理 Huh7 和 HepG2 细胞后，通过实时 PCR 研究了我们之前研究中 TNF、TLR4、SCD、FASN、SREBF2 和 TGFβ-1 基因的抑制作用。油红 O 染色和 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基-2H-四唑溴盐（MTT）试验分别用于评估 miRNA 处理后细胞内脂质积累和细胞毒性作用。miR-16 和 miR-124 转染后进行了促氧化剂-抗氧化剂平衡（PAB）试验。
结果
miRNA 转染 HepG2 后，油红 O 染色显示 miR-124 和 miR-16 分别减少了 35.2%和 28.6%的油酸诱导的脂质积累（p<0.05）。在 Huh7 中，miR-124 和 miR-16 分别减少了 23.5%和 31.3%的积累（p<0.05），但不影响抗氧化活性。HepG2 中的实时 PCR 显示 miR-124 使 TNF 的表达降低了 0.13 倍，TLR4 降低了 0.12 倍，SREBF2 降低了 0.127 倍（p<0.05）。miR-16 使 TLR4 降低了 0.66 倍，FASN 降低了 0.3 倍（p<0.05）。在 Huh7 中，miR-124 使 TNF 降低了 0.12 倍，FASN 降低了 0.09 倍（p<0.05）。miR-16 使 SCD 降低了 0.28 倍，FASN 降低了 0.64 倍（p<0.05）。MTT 试验显示，miR-124 在 HepG2 中使细胞活力在 72 小时时降低了 24.7%，miR-16 降低了 33%（p<0.05）。在 Huh7 中，miR-124 在 48 小时时使细胞活力降低了 42%，在 72 小时时降低了 29.33%（p<0.05），而 miR-16 使细胞活力降低了 32.3%（p<0.05）。
结论
这些结果表明，miR-124 和 miR-16 能够通过直接靶向显著减少与 NAFLD 相关的关键致病基因的脂质积累和表达。尽管这需要进一步的体内研究。
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