Hua Zhijuan, Yang Wenchang, Li Dongli, Cui Yixin, Shen Lu, Rao Lingna, Zheng Yuxiang, Zhang Qiying, Zeng Wenyi, Gong Yi, Yuan Ling
Department of Ophthalmology, The First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, P.R. China.
Department of Pediatric Ophthalmology, The Affiliated Hospital of Yunnan University, Kunming, Yunnan 650021, P.R. China.
Exp Ther Med. 2023 Sep 27;26(5):528. doi: 10.3892/etm.2023.12227. eCollection 2023 Nov.
Subretinal fibrosis (SF) is an important cause of submacular neovascularization that leads to permanent vision loss, but has no effective clinical treatment. The present study examined the influence of metformin on SF, and investigated whether the mechanism involves the microRNA (miR)-140-3p/LIN28B/JNK/STAT3-mediated regulation of oxidative stress, angiogenesis and fibrosis-associated indicators. A mouse model of laser-induced SF was established. In addition, an ARPE-19 fibrotic cell model was established using TGF-β1. A Cell Counting Kit-8 assay was used to examine cell viability. Flow cytometry was used to measure reactive oxygen species levels, and western blotting was used to detect the levels of proteins associated with epithelial-mesenchymal transition (EMT), signaling and fibrosis. The levels of superoxide dismutase, malondialdehyde, glutathione-peroxidase and catalase were measured using kits. Scratch assays and Transwell assays were used to assess cell migration and invasion, respectively, and reverse transcription-quantitative PCR was used to determine the levels of miR-140-3p and LIN28B. Dual-luciferase assays were used to verify the targeting relationship between miR-140-3p and LIN28B, and coimmunoprecipitation was used to confirm the interaction between LIN28B and JNK. Masson staining and hematoxylin and eosin staining were used to examine collagenous fibers and the histopathology of eye tissue. In ARPE-19 cells induced by TGF-β1, metformin promoted miR-140-3p expression and inhibited LIN28B expression and JNK/STAT3 pathway activation, thereby inhibiting oxidative stress, EMT and fibrosis in ARPE-19 cells. The overexpression of LIN28B or treatment with the JNK/STAT3 agonist anisomycin partially reversed the inhibitory effect of metformin on oxidative stress and fibrosis in ARPE-19 cells. The dual-luciferase reporter assay and coimmunoprecipitation assay showed that miR-140-3p targeted the 3' untranslated region of LIN28B mRNA and inhibited LIN28B expression. LIN28B targeted and bound to JNK and regulated the JNK/STAT3 pathway. Therefore, it may be concluded that metformin can promote miR-140-3p expression, inhibit LIN28B and then inhibit the JNK/STAT3 pathway to alleviate SF.
视网膜下纤维化（SF）是导致永久性视力丧失的黄斑下新生血管形成的重要原因，但目前尚无有效的临床治疗方法。本研究探讨了二甲双胍对SF的影响，并研究其作用机制是否涉及微小RNA（miR）-140-3p/LIN28B/JNK/STAT3介导的氧化应激、血管生成及纤维化相关指标的调控。建立了激光诱导的SF小鼠模型。此外，利用转化生长因子-β1（TGF-β1）建立了ARPE-19纤维化细胞模型。采用细胞计数试剂盒-8法检测细胞活力。运用流式细胞术检测活性氧水平，采用蛋白质印迹法检测与上皮-间质转化（EMT）、信号传导及纤维化相关的蛋白质水平。使用试剂盒检测超氧化物歧化酶、丙二醛、谷胱甘肽过氧化物酶和过氧化氢酶的水平。划痕试验和Transwell试验分别用于评估细胞迁移和侵袭能力，采用逆转录-定量聚合酶链反应法测定miR-140-3p和LIN28B的水平。双荧光素酶报告基因检测用于验证miR-140-3p与LIN28B之间的靶向关系，免疫共沉淀法用于证实LIN28B与JNK之间的相互作用。采用Masson染色及苏木精-伊红染色检测眼组织中的胶原纤维及组织病理学变化。在TGF-β1诱导的ARPE-19细胞中，二甲双胍可促进miR-140-3p表达，抑制LIN28B表达及JNK/STAT3信号通路激活，从而抑制ARPE-19细胞中的氧化应激、EMT及纤维化。过表达LIN28B或用JNK/STAT3激动剂茴香霉素处理可部分逆转二甲双胍对ARPE-19细胞氧化应激及纤维化的抑制作用。双荧光素酶报告基因检测及免疫共沉淀试验表明，miR-140-3p靶向LIN28B mRNA的3'非翻译区并抑制LIN28B表达。LIN28B靶向并结合JNK，调控JNK/STAT3信号通路。因此，可以得出结论，二甲双胍可促进miR-140-3p表达，抑制LIN28B，进而抑制JNK/STAT3信号通路以减轻SF。
