circMKLN1海绵吸附调控miR-26a/b在百草枯致肺纤维化中的作用及机制研究

Progressively exacerbated pulmonary fibrosis is the leading cause of death in patients with paraquat (PQ) poisoning. Epithelial-mesenchymal transition (EMT) is an important factor in the formation of pulmonary fibrosis induced by PQ, but the specific mechanism remains unclear. We screened and verified that circMKLN1 was significantly elevated in PQ-induced pulmonary fibrosis with the high-throughput sequencing technology. And PQ-induced EMT was significantly attenuated after inhibition of circMKLN1 in vitro. Bioinformatics analysis suggested that circMKLN1 could bind to miR-26a/b as a “miRNA sponges” and promote the expression of miR-26a/b target genes Smad4 and CTGF. Therefore, we speculate that the high expression of circMKLN1 after PQ poisoning competitively binds to miR-26a/b, relieves the inhibition of target genes by miR-26a/b, induces EMT in alveolar epithelial cells, and promotes the progression of pulmonary fibrosis. In this project, we intend to use the techniques of lentivirus transfection and RNA interference to explore the key role of circMKLN1 in pulmonary fibrosis in the PQ poisoning in vitro and in vivo model, and to clarify the regulation and specific mechanism of circMKLN1 on EMT. This might provide a theoretical basis for finding new therapeutic targets for PQ-induced pulmonary fibrosis.

进行性加重的肺纤维化是百草枯（PQ）中毒患者死亡的主要原因，上皮-间质转化（EMT）是PQ致肺纤维化形成的重要因素，但具体机制仍未明确。我们利用高通量测序技术筛选并验证发现circMKLN1在PQ致肺纤维化中显著升高，体外抑制circMKLN1后PQ诱导的EMT明显减轻。生物信息学分析提示，circMKLN1可作为“miRNA海绵”结合miR-26a/b，促进miR-26a/b靶基因Smad4和CTGF的表达。因此，我们推测：PQ中毒后高表达的circMKLN1可能通过竞争性结合miR-26a/b，解除miR-26a/b对靶基因的抑制，诱导肺泡上皮细胞发生EMT，促进肺纤维化进展。本项目拟在PQ中毒体内外模型中，利用慢病毒转染及RNA干扰等技术，探讨circMKLN1在肺纤维化中发挥的关键作用，明确circMKLN1对EMT的调控及具体机制，为PQ致肺纤维化寻找新的治疗靶点提供理论依据。

肺纤维化特征是肺实质破坏、细胞外基质沉积及成纤维细胞和肺泡上皮细胞表型显著变化，但目前尚缺乏治疗肺纤维有效手段，深入探讨其机制有助于发现新的治疗靶点。研究发现，环状RNA是一类内源性非编码RNA分子，环状RNA在多种疾病的发生发展中具有重要作用。我们前期通过高通量测序技术发现circMKLN1在百草枯（PQ）致肺纤维化中显著升高。本项目通过建立PQ染毒小鼠和肺泡上皮细胞模型，应用腺病毒-shRNA和siRNA技术等在体内外模型中调节circMKLN1表达变化，并应用原位杂交、免疫荧光、Masson染色、天狼猩红染色、Western blot、定量PCR等方法检测小鼠肺组织和肺泡上皮细胞内EMT及纤维化相关指标变化。研究结果显示，PQ染毒小鼠肺组织和肺泡上皮细胞模型中circMKLN1表达均明显增加，且与肺纤维化严重程度呈正相关趋势。体内外模型中抑制circMKLN1表达后，可明显改善PQ中毒诱导的小鼠肺组织胶原蛋白沉积，并抑制肺泡上皮细胞的EMT进程。在体外肺泡上皮细胞内过表达circMKLN1后，可进一步加重PQ诱导的EMT。通过生物信息学分析并应用荧光素酶报告基因实验证实，miR-26a/b是circMKLN1下游靶点，体外抑制circMKLN1后可促进miR-26a/b表达升高。在体内外模型中抑制circMKLN1后，均检测到调控肺纤维化关键因子CTGF的表达明显下降，而CTGF是miR-26a/b靶点之一。体外肺泡上皮细胞中抑制circMKLN1的同时，分别共转染miR-26a/b inhibitor，结果显示抑制circMKLN1后减轻的EMT再次出现加重趋势，且CTGF表达也明显升高。以上提示，circMKLN1通过海绵吸附miR-26a/b促进CTGF表达，进而参与PQ中毒诱导的EMT和纤维化过程。本研究将为寻找治疗PQ致肺纤维化的新靶点提供理论依据。