Abstract：
Long noncoding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) alleviates the progression of diabetic nephropathy by inhibiting inflammation and fibrosis. This study investigated how CASC2 impacts renal interstitial fibrosis (RIF) through regulating M1 macrophage (M1) polarization.
Nine-week-old mice underwent unilateral ureteral obstruction (UUO) establishment. Macrophages were induced toward M1 polarization using lipopolysaccharide (LPS) in vitro and cocultured with fibroblasts to examine how M1 polarization influences RIF. LnCeCell predicted that CASC2 interacted with myocyte enhancer factor 2 C (MEF2C), which was validated by dual-luciferase reporter assay. CASC2/MEF2C overexpression was achieved by lentivirus-expressing lncRNA CASC2 injection in vivo or CASC2 and MEF2C transfection in vitro. Renal injury was evaluated through biochemical analysis and hematoxylin-eosin/Masson staining. Macrophage infiltration and M1 polarization in the kidney and/or macrophages were detected by immunofluorescence, flow cytometry, and/or quantitative reverse transcription polymerase chain reaction (qRT-PCR). Expressions of CASC2, MEF2C, and markers related to inflammation/M1/fibrosis in the kidney/macrophages/fibroblasts were analyzed by qRT-PCR, fluorescence in situ hybridization, enzyme-linked immunosorbent assay, and/or Western blot.
In the kidneys of mice, CASC2 was downregulated and macrophage infiltration was promoted time-dependently from days 3 to 14 post-UUO induction; CASC2 overexpression alleviated renal histological abnormalities, hindered macrophage infiltration and M1 polarization, downregulated renal function markers serum creatinine and blood urea nitrogen and inflammation/M1/fibrosis-related makers, and offset UUO-induced MEF2C upregulation. LncRNA CASC2 overexpression inhibited fibroblast fibrosis and M1 polarization in cocultured fibroblasts with LPS-activated macrophages. Also, CASC2 bound to MEF2C and inhibited its expression in LPS-activated macrophages. Furthermore, MEF2C reversed the inhibitory effects of lncRNA CASC2 overexpression.
CASC2 alleviates RIF by inhibiting M1 polarization through directly downregulating MEF2C expression. CASC2 might represent a promising value of future investigations on treatment for RIF.
Nine-week-old mice underwent unilateral ureteral obstruction (UUO) establishment. Macrophages were induced toward M1 polarization using lipopolysaccharide (LPS) in vitro and cocultured with fibroblasts to examine how M1 polarization influences RIF. LnCeCell predicted that CASC2 interacted with myocyte enhancer factor 2 C (MEF2C), which was validated by dual-luciferase reporter assay. CASC2/MEF2C overexpression was achieved by lentivirus-expressing lncRNA CASC2 injection in vivo or CASC2 and MEF2C transfection in vitro. Renal injury was evaluated through biochemical analysis and hematoxylin-eosin/Masson staining. Macrophage infiltration and M1 polarization in the kidney and/or macrophages were detected by immunofluorescence, flow cytometry, and/or quantitative reverse transcription polymerase chain reaction (qRT-PCR). Expressions of CASC2, MEF2C, and markers related to inflammation/M1/fibrosis in the kidney/macrophages/fibroblasts were analyzed by qRT-PCR, fluorescence in situ hybridization, enzyme-linked immunosorbent assay, and/or Western blot.
In the kidneys of mice, CASC2 was downregulated and macrophage infiltration was promoted time-dependently from days 3 to 14 post-UUO induction; CASC2 overexpression alleviated renal histological abnormalities, hindered macrophage infiltration and M1 polarization, downregulated renal function markers serum creatinine and blood urea nitrogen and inflammation/M1/fibrosis-related makers, and offset UUO-induced MEF2C upregulation. LncRNA CASC2 overexpression inhibited fibroblast fibrosis and M1 polarization in cocultured fibroblasts with LPS-activated macrophages. Also, CASC2 bound to MEF2C and inhibited its expression in LPS-activated macrophages. Furthermore, MEF2C reversed the inhibitory effects of lncRNA CASC2 overexpression.
CASC2 alleviates RIF by inhibiting M1 polarization through directly downregulating MEF2C expression. CASC2 might represent a promising value of future investigations on treatment for RIF.
摘要：
背景：LncRNACASC2通过抑制炎症和纤维化减轻糖尿病肾病的进展。这项研究调查了CASC2如何通过调节M1-巨噬细胞（M1）极化来影响肾间质纤维化（RIF）。
方法：9周龄小鼠进行单侧输尿管梗阻(UUO)建立。使用脂多糖（LPS）在体外诱导巨噬细胞向M1极化，并与成纤维细胞共培养以检查M1极化如何影响RIF。lncecell预测CASC2与肌细胞增强因子2C（MEF2C）相互作用，通过双荧光素酶报告基因试验验证。通过体内注射LV-CASC2实现CASC2/MEF2C过表达，或体外CASC2和MEF2C转染。通过生化分析和苏木精-伊红/马森染色评估肾损伤。免疫荧光检测肾和/或巨噬细胞中的巨噬细胞浸润和M1极化，流式细胞术和/或qRT-PCR。qRT-PCR检测肾/巨噬细胞/成纤维细胞中CASC2、MEF2C及炎症/M1/纤维化相关标志物的表达,FISH,ELISA和/或蛋白质印迹。
结果：在小鼠的肾脏中，在UUO诱导后第3天至第14天，CASC2下调，巨噬细胞浸润被时间依赖性地促进；CASC2过表达减轻了肾脏组织学异常，受阻巨噬细胞浸润和M1极化，下调肾功能标志物Scr和Bun，和炎症/M1/纤维化相关的标志物和抵消UUO相关的MEF2C上调。LncRNACASC2过表达抑制共培养的成纤维细胞与LPS激活的巨噬细胞中的成纤维细胞纤维化和M1极化。此外，CASC2与MEF2C结合并抑制其在LPS激活的巨噬细胞中的表达。此外，MEF2C逆转了lncRNACASC2过表达的抑制作用。
结论：CASC2通过直接下调MEF2C表达来抑制M1极化，从而减轻RIF。CASC2可能代表RIF治疗未来研究的有希望的价值。
方法：9周龄小鼠进行单侧输尿管梗阻(UUO)建立。使用脂多糖（LPS）在体外诱导巨噬细胞向M1极化，并与成纤维细胞共培养以检查M1极化如何影响RIF。lncecell预测CASC2与肌细胞增强因子2C（MEF2C）相互作用，通过双荧光素酶报告基因试验验证。通过体内注射LV-CASC2实现CASC2/MEF2C过表达，或体外CASC2和MEF2C转染。通过生化分析和苏木精-伊红/马森染色评估肾损伤。免疫荧光检测肾和/或巨噬细胞中的巨噬细胞浸润和M1极化，流式细胞术和/或qRT-PCR。qRT-PCR检测肾/巨噬细胞/成纤维细胞中CASC2、MEF2C及炎症/M1/纤维化相关标志物的表达,FISH,ELISA和/或蛋白质印迹。
结果：在小鼠的肾脏中，在UUO诱导后第3天至第14天，CASC2下调，巨噬细胞浸润被时间依赖性地促进；CASC2过表达减轻了肾脏组织学异常，受阻巨噬细胞浸润和M1极化，下调肾功能标志物Scr和Bun，和炎症/M1/纤维化相关的标志物和抵消UUO相关的MEF2C上调。LncRNACASC2过表达抑制共培养的成纤维细胞与LPS激活的巨噬细胞中的成纤维细胞纤维化和M1极化。此外，CASC2与MEF2C结合并抑制其在LPS激活的巨噬细胞中的表达。此外，MEF2C逆转了lncRNACASC2过表达的抑制作用。
结论：CASC2通过直接下调MEF2C表达来抑制M1极化，从而减轻RIF。CASC2可能代表RIF治疗未来研究的有希望的价值。
