LRRC15影响A549细胞自噬的作用研究

Effect of LRRC15 on autophagy in A549 cells

特发性肺纤维化(idiopathic pulmonary fibrosis,IPF)是一种致病原因不明、进行性、慢性不可逆的间质性肺疾病。为探讨富含亮氨酸重复蛋白15(leucine-rich repeat-containing protein 15,LRRC15)在IPF的作用及调节机制,本研究构建了博来霉素(bleomycin,BLM)诱导的小鼠肺纤维化和A549细胞损伤模型,检测了LRRC15的表达变化。转染siLRRC15后分别采用MTT、GFP-RFP-LC3双荧光标记系统和免疫印迹等方法检测了细胞活性和自噬的变化。结果表明:BLM处理后,小鼠肺组织和A549细胞中LRRC15表达显著上调;将设计和合成的siLRRC15转入A549细胞,LRRC15的表达极显著降低,可以部分恢复BLM引起的细胞损伤;BLM处理A549细胞后,LC3-Ⅱ和P62蛋白呈现上升的趋势;GFP-RFP-LC3双荧光标记检测发现,BLM处理后自噬体数量明显增多;进一步用siLRRC15处理A549细胞后发现,自噬关键蛋白LC3-Ⅱ、ATG5、ATG7的表达增加,P62蛋白表达下调,自噬流的强度增加。以上研究结果说明LRRC15是IPF中上皮细胞损伤的指示剂,可能通过调节自噬参与纤维化过程中的调节,本研究为进一步阐明IPF的机制提供了必要的理论依据。

Idiopathic pulmonary fibrosis(IPF)is a progressive,chronic,and irreversible interstitial lung disease with unknown cause.To explore the role and regulatory mechanism of leucine-rich repeat-containing protein 15(LRRC15)in IPF,bleomycin(BLM)-induced pulmonary fibrosis in mouse and A549 cells were constructed,and the expression of LRRC15 were detected.Then,MTT,GFP-RFP-LC3 dual fluorescent labeling system and Western blotting were used to investigate the effects of LRRC15 on cell activity and autophagy after transfection of siLRRC15,respectively.The results indicated that the expression of LRRC15 was significantly increased after the BLM treatment in mouse lung tissue and A549 cells.The designed and synthesized siLRRC15 followed by transfection into A549 cells resulted in a dramatic reduction in LRRC15 expression and partially restored the cell damage induced by BLM.Moreover,the expression of LC3-Ⅱ and P62 were up-regulated,the amount of autophagosome were increased by GFP-RFP-LC3 dual fluorescent labeling assay after BLM treatment.Meanwhile,this study also showed that the key autophagy proteins LC3-Ⅱ,ATG5 and ATG7 were up-regulated,P62 was down-regulated and autophagic flux were enhanced after further treatment of A549 cells with siLRRC15.The above findings suggest that LRRC15 is an indicator of epithelial cell damage and may participate in the regulation of fibrosis through autophagy mechanism in IPF.This study provides necessary theoretical basis for further elucidating the mechanism of IPF.