Department of Clinical Laboratory, The First Affiliated Hospital of Kunming Medical University, Kunming, 650032, Yunnan, China.
Yunnan Institute of Experimental Diagnosis, Kunming, 650032, Yunnan, China.
Mol Cell Biochem. 2022 Aug;477(8):2133-2147. doi: 10.1007/s11010-022-04426-z. Epub 2022 May 24.
To explore the role of the miRNA-1297/phospholipase Cβ1 (PLCβ1) axis in intestinal barrier injury. Abnormally expressed miR-1297 and its target gene PLCβ1 as well as their transcriptome sequencing were confirmed by bioinformatics analysis. Next, the intestinal barrier injury was induced by lipopolysaccharide (LPS) in the CCCHIE-2 cells. Subsequently, the impacts of miR-1297 and PLCβ1 on the transcriptome were estimated. QRT-PCR and Western blotting were conducted to detect the relative mRNA and protein expressions, respectively. The cell viability and permeability were analyzed by MTT assay and fluorescent yellow detection. miR-1297 was significantly upregulated in patients with human immunodeficiency virus/acquired immunodeficiency syndrome and targeted PLCβ1. Moreover, overexpressed PLCβ1 was mainly enriched in the transforming growth factor-beta signaling pathway, while the knockdown of miR-1297 was focused on the arginine biosynthesis pathway. The overexpression of miR-1297 could reduce the PLCβ1 expression and inhibit the viability of CCCHIE-2 cells injured by LPS, while the effect of the downregulation of miR-1297 was on the opposite. Western blotting and cell fluorescence localization experiments revealed that the inhibition of miR-1297 increased the expressions of PLCβ1 and ZO-1. In addition, the upregulation of miR-1297 strengthened the permeability in cells injured by LPS, as did the knockdown of PLCβ1. miR-1297 could restrain the repair of intestinal barrier injury via negatively regulating PLCβ1 and its tight junction downstream protein ZO-1 in CCC-HIE-2 cells injured by LPS, which indicated that PLCβ1 and miR-1297 might be important targets for the repair of intestinal barrier injury.
探讨 miRNA-1297/磷脂酶 Cβ1 (PLCβ1) 轴在肠道屏障损伤中的作用。通过生物信息学分析，证实了异常表达的 miR-1297 及其靶基因 PLCβ1 及其转录组测序。接下来，用脂多糖 (LPS) 在 CCCHIE-2 细胞中诱导肠道屏障损伤。随后，评估了 miR-1297 和 PLCβ1 对转录组的影响。通过 QRT-PCR 和 Western blot 分别检测相对 mRNA 和蛋白表达。通过 MTT 测定和荧光黄检测分析细胞活力和通透性。miR-1297 在人类免疫缺陷病毒/获得性免疫缺陷综合征患者中显著上调，并靶向 PLCβ1。此外，过表达的 PLCβ1 主要富集在转化生长因子-β信号通路中，而 miR-1297 的下调则集中在精氨酸生物合成途径中。过表达 miR-1297 可降低 LPS 损伤的 CCCHIE-2 细胞中 PLCβ1 的表达并抑制其活力，而 miR-1297 下调的作用则相反。Western blot 和细胞荧光定位实验表明，miR-1297 的抑制增加了 PLCβ1 和 ZO-1 的表达。此外，miR-1297 的上调增强了 LPS 损伤细胞的通透性，PLCβ1 的下调也是如此。miR-1297 通过负调控 LPS 损伤的 CCC-HIE-2 细胞中 PLCβ1 及其紧密连接下游蛋白 ZO-1 来抑制肠道屏障损伤的修复，这表明 PLCβ1 和 miR-1297 可能是肠道屏障损伤修复的重要靶点。
