Luo Xin, Luo Sheng-Zheng, Xu Zi-Xin, Zhou Cui, Li Zheng-Hong, Zhou Xiao-Yan, Xu Ming-Yi
Department of Gastroenterology, Shanghai General Hospital, Shanghai 200080, China.
World J Gastroenterol. 2021 Apr 14;27(14):1419-1434. doi: 10.3748/wjg.v27.i14.1419.
Exosomes play an important role in metabolic-associated fatty liver disease (MAFLD), but the mechanism by which exosomes participate in MAFLD still remain unclear.
To figure out the function of lipotoxic exosomal miR-1297 in MAFLD.
MicroRNA sequencing was used to detect differentially expressed miRNAs (DE-miR) in lipotoxic exosomes derived from primary hepatocytes. Bioinformatic tools were applied to analyze the target genes and pathways regulated by the DE-miRs. Quantitative real-time PCR (qPCR) was conducted for the verification of DE-miRs. qPCR, western blot, immunofluorescence staining and ethynyl-20-deoxyuridine assay were used to evaluate the function of lipotoxic exosomal miR-1297 on hepatic stellate cells (LX2 cells). A luciferase reporter experiment was performed to confirm the relationship of miR-1297 and its target gene .
MicroRNA sequencing revealed that there were 61 exosomal DE-miRs ( < 0.05) with a fold-change > 2 from palmitic acid treated primary hepatocytes compared with the vehicle control group. miR-1297 was the most highly upregulated according to the microRNA sequencing. Bioinformatic tools showed a variety of target genes and pathways regulated by these DE-miRs were related to liver fibrosis. miR-1297 was overexpressed in exosomes derived from lipotoxic hepatocytes by qPCR. Fibrosis promoting genes (, ) were altered in LX2 cells after miR-1297 overexpression or miR-1297-rich lipotoxic exosome incubation qPCR and western blot analysis. Immunofluorescence staining and ethynyl-20-deoxyuridine staining demonstrated that the activation and proliferation of LX2 cells were also promoted after the above treatment. was found to be the target gene of miR-1297 and knocking down contributed to the activation and proliferation of LX2 cells modulating the PI3K/AKT signaling pathway.
miR-1297 was overexpressed in exosomes derived from lipotoxic hepatocytes. The lipotoxic hepatocyte-derived exosomal miR-1297 could promote the activation and proliferation of hepatic stellate cells through the PTEN/PI3K/AKT signaling pathway, accelerating the progression of MAFLD.
外泌体在代谢相关脂肪性肝病（MAFLD）中起重要作用，但外泌体参与MAFLD的机制仍不清楚。
明确脂毒性外泌体miR-1297在MAFLD中的作用。
采用微小RNA测序检测原代肝细胞来源的脂毒性外泌体中差异表达的微小RNA（DE-miR）。应用生物信息学工具分析DE-miR调控的靶基因和信号通路。采用定量实时聚合酶链反应（qPCR）验证DE-miR。运用qPCR、蛋白质免疫印迹法、免疫荧光染色和乙炔基-2'-脱氧尿苷检测评估脂毒性外泌体miR-1297对肝星状细胞（LX2细胞）的作用。进行荧光素酶报告基因实验以确认miR-1297与其靶基因的关系。
微小RNA测序显示，与溶剂对照组相比，棕榈酸处理的原代肝细胞来源的外泌体中有61个DE-miR（<0.05），变化倍数>2。根据微小RNA测序，miR-1297上调最为显著。生物信息学工具显示，这些DE-miR调控的多种靶基因和信号通路与肝纤维化相关。通过qPCR检测发现，miR-1297在脂毒性肝细胞来源的外泌体中过表达。在miR-1297过表达或用富含miR-1297的脂毒性外泌体孵育后，LX2细胞中的纤维化促进基因（ ）发生改变（qPCR和蛋白质免疫印迹分析）。免疫荧光染色和乙炔基-2'-脱氧尿苷染色表明，上述处理后LX2细胞的激活和增殖也得到促进。发现 是miR-1297的靶基因，敲低 通过调节PI3K/AKT信号通路促进LX2细胞的激活和增殖。
miR-1297在脂毒性肝细胞来源的外泌体中过表达。脂毒性肝细胞来源的外泌体miR-1297可通过PTEN/PI3K/AKT信号通路促进肝星状细胞的激活和增殖，加速MAFLD的进展。
