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Abstract：
Sepsis is a systemic inflammatory response syndrome resulting from the invasion of the human body by bacteria and other pathogenic microorganisms. One of its most prevalent complications is acute lung injury, which places a significant medical burden on numerous countries and regions due to its high morbidity and mortality rates. MicroRNA (miRNA) plays a critical role in the body\'s inflammatory response and immune regulation. Recent studies have focused on miR-21-5p in the context of acute lung injury, but its role appears to vary in different models of this condition. In the LPS-induced acute injury model of A549 cells, there is differential expression, but the specific mechanism remains unclear. Therefore, our aim is to investigate the changes in the expression of miR-21-5p and SLC16A10 in a type II alveolar epithelial cell injury model induced by LPS and explore the therapeutic effects of their targeted regulation. A549 cells were directly stimulated with 10 µg/ml of LPS to construct a model of LPS-induced cell injury. Cells were collected at different time points and the expression of interleukin 1 beta (IL-1β), tumor necrosis factor-α (TNF-α) and miR-21-5p were measured by RT-qPCR and western blot. Then miR-21-5p mimic transfection was used to up-regulate the expression of miR-21-5p in A549 cells and the expression of IL-1β and TNF-α in each group of cells was measured by RT-qPCR and western blot. The miRDB, TargetScan, miRWalk, Starbase, Tarbase and miR Tarbase databases were used to predict the miR-21-5p target genes and simultaneously, the DisGeNet database was used to search the sepsis-related gene groups. The intersection of the two groups was taken as the core gene. Luciferase reporter assay further verified SLC16A10 as the core gene with miR-21-5p. The expression of miR-21-5p and SLC16A10 were regulated by transfection or inhibitors in A549 cells with or without LPS stimulation. And then the expression of IL-1β and TNF-α in A549 cells was tested by RT-qPCR and western blot in different groups, clarifying the role of miR-21-5p-SLC16A10 axis in LPS-induced inflammatory injury in A549 cells. (1) IL-1β and TNF-α mRNA and protein expression significantly increased at 6, 12, and 24 h after LPS stimulation as well as the miR-21-5p expression compared with the control group (P < 0.05). (2) After overexpression of miR-21-5p in A549 cells, the expression of IL-1β and TNF-α was significantly reduced after LPS stimulation, suggesting that miR-21-5p has a protection against LPS-induced injury. (3) The core gene set, comprising 51 target genes of miR-21-5p intersecting with the 1448 sepsis-related genes, was identified. This set includes SLC16A10, TNPO1, STAT3, PIK3R1, and FASLG. Following a literature review, SLC16A10 was selected as the ultimate target gene. Dual luciferase assay results confirmed that SLC16A10 is indeed a target gene of miR-21-5p. (4) Knocking down SLC16A10 expression by siRNA significantly reduced the expression of IL-1β and TNF-α in A549 cells after LPS treatment (P < 0.05). (5) miR-21-5p inhibitor increased the expression levels of IL-1β and TNF-α in A549 cells after LPS stimulation (P < 0.05). In comparison to cells solely transfected with miR-21-5p inhibitor, co-transfection of miR-21-5p inhibitor and si-SLC6A10 significantly reduced the expression of IL-1β and TNF-α (P < 0.05). MiR-21-5p plays a protective role in LPS-induced acute inflammatory injury of A549 cells. By targeting SLC16A10, it effectively mitigates the inflammatory response in A549 cells induced by LPS. Furthermore, SLC16A10 holds promise as a potential target for the treatment of acute lung injury.
摘要：
脓毒症是细菌等病原微生物侵入人体后产生的一种全身性炎症反应综合征。其最常见的并发症之一是急性肺损伤，由于其高发病率和死亡率，给许多国家和地区带来了巨大的医疗负担。微小RNA(miRNA)在机体炎症反应和免疫调节中起着重要作用。最近的研究集中在miR-21-5p在急性肺损伤的背景下，但是它的作用在这种情况的不同模型中似乎有所不同。在LPS诱导的A549细胞急性损伤模型中,有差异表达，但具体机制尚不清楚。因此,我们的目的是研究miR-21-5p和SLC16A10在LPS诱导的II型肺泡上皮细胞损伤模型中的表达变化,并探讨其靶向调控的治疗作用.用10μg/mlLPS直接刺激A549细胞,构建LPS诱导的细胞损伤模型。收集不同时间点的细胞和白细胞介素1β(IL-1β)的表达,通过RT-qPCR和Westernblot检测肿瘤坏死因子-α（TNF-α）和miR-21-5p。然后使用miR-21-5p模拟转染上调A549细胞中miR-21-5p的表达，并通过RT-qPCR和Westernblot检测各组细胞中IL-1β和TNF-α的表达。miRDB,TargetScan,miRWalk,星基,使用Tarbase和miRTarbase数据库预测miR-21-5p靶基因,使用DisGeNet数据库搜索脓毒症相关基因组.以两组的交集为核心基因。荧光素酶报告基因测定进一步证实SLC16A10是miR-21-5p的核心基因。在有或没有LPS刺激的情况下，A549细胞中miR-21-5p和SLC16A10的表达受到转染或抑制剂的调节。然后通过RT-qPCR和Westernblot检测不同组A549细胞中IL-1β和TNF-α的表达,阐明miR-21-5p-SLC16A10轴在LPS诱导的A549细胞炎症损伤中的作用。(1)LPS刺激后6、12、24hIL-1β和TNF-αmRNA和蛋白表达及miR-21-5p表达均明显高于对照组(P<0.05)。(2)miR-21-5p在A549细胞中过表达后,LPS刺激后IL-1β和TNF-α的表达显著降低,提示miR-21-5p对LPS诱导的损伤具有保护作用。(3)核心基因集,包含与1448个败血症相关基因相交的miR-21-5p的51个靶基因，已确定。此集合包括SLC16A10、TNPO1、STAT3、PIK3R1和FASLG。在文献综述之后，选择SLC16A10作为最终的靶基因。双荧光素酶检测成果证实SLC16A10确实是miR-21-5p的靶基因。(4)siRNA抑制SLC16A10的表达可显著降低LPS处理后A549细胞中IL-1β和TNF-α的表达(P<0.05)。(5)miR-21-5p抑制剂可显著提高LPS刺激后A549细胞中IL-1β和TNF-α的表达水平(P<0.05)。与单独转染miR-21-5p抑制剂的细胞相比，miR-21-5p抑制剂和si-SLC6A10共转染可显著降低IL-1β和TNF-α的表达(P<0.05)。MiR-21-5p在LPS诱导的A549细胞急性炎症损伤中起保护作用。通过靶向SLC16A10，它有效地减轻了LPS诱导的A549细胞的炎症反应。此外，SLC16A10有望成为治疗急性肺损伤的潜在靶标。
