Iron overload can promote the development of osteoporosis by inducing apoptosis in osteoblasts. However, the mechanism by which miRNAs regulate apoptosis in osteoblasts under iron overload has not been elucidated. The miRNA expression profile in MC3T3-E1 cells under iron overload was detected by next generation sequencing. qRT-PCR was used to determine the expression of miR-3074-5p in MC3T3-E1 cells under iron overload. The proliferation of MC3T3-E1 cells was tested using CCK-8 assays, and apoptosis was measured using flow cytometry. The miRanda and TargetScan databases were used to predict the target genes of miR-3074-5p. Interaction between miR-3074-5p and the potential target gene was validated by qRT-PCR, luciferase reporter assay and western blotting. We found that iron overload decreased the cell viability and induced apoptosis of MC3T3-E1 cells. The results of next generation sequencing analysis showed that miR-3074-5p expression was significantly increased in MC3T3-E1 cells under iron overload conditions, which was confirmed by further experiments. The inhibition of miR-3074-5p attenuated the apoptosis of iron-overloaded MC3T3-E1 cells. Furthermore, the expression of Smad4 was decreased and was inversely correlated with miR-3074-5p expression, and overexpression of Smad4 partially reversed the viability inhibition of iron-overloaded MC3T3-E1 cells by relieving the suppression of ERK, AKT, and Stat3 phosphorylation, suggesting its regulatory role in the viability inhibition of iron-overloaded MC3T3-E1 cells. The luciferase reporter assay results showed that Smad4 was the target gene of miR-3074-5p. miR-3074-5p functions as an apoptosis promoter in iron-overloaded MC3T3-E1 cells by directly targeting Smad4.

中文翻译：

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铁过载条件下 miR-3074-5p 在 MC3T3-E1 细胞中的凋亡促进特性

铁过载可通过诱导成骨细胞凋亡来促进骨质疏松症的发展。然而，miRNAs 调节铁过载下成骨细胞凋亡的机制尚未阐明。通过二代测序检测铁过载下MC3T3-E1细胞中的miRNA表达谱。qRT-PCR用于确定铁过载下MC3T3-E1细胞中miR-3074-5p的表达。使用CCK-8测定法测试MC3T3-E1细胞的增殖，并使用流式细胞术测量细胞凋亡。miRanda 和 TargetScan 数据库用于预测 miR-3074-5p 的靶基因。miR-3074-5p 与潜在靶基因之间的相互作用通过 qRT-PCR、荧光素酶报告基因测定和蛋白质印迹进行验证。我们发现铁过载降低了细胞活力并诱导了 MC3T3-E1 细胞的凋亡。二代测序分析结果表明，在铁过载条件下，MC3T3-E1细胞中miR-3074-5p的表达显着增加，进一步的实验证实了这一点。miR-3074-5p的抑制减弱了铁过载的MC3T3-E1细胞的凋亡。此外，Smad4 的表达降低并与 miR-3074-5p 的表达呈负相关，Smad4 的过表达通过减轻对 ERK、AKT 和 Stat3 磷酸化的抑制，部分逆转了铁过载的 MC3T3-E1 细胞的活力抑制，表明其在铁过载的 MC3T3-E1 细胞的活力抑制中的调节作用。荧光素酶报告基因检测结果显示Smad4是miR-3074-5p的靶基因。miR-3074-5p 通过直接靶向 Smad4 在铁过载的 MC3T3-E1 细胞中充当凋亡启动子。