Objective: To study the effect of lysosomal membrane protein Sidt2 deletion on autophagy in human hepatocytes.
Methods: Crispr-Cas9 technology was used to construct a human hepatocyte (HL7702) model of Sidt2 knockout (Sidt2-/-), and the expression levels of the key autophagy proteins LC3II/I, P62 and autophagy-related proteins Atg5, Atg7, and Atg12 were detected.The co-localization of LC3B and P62 in the cells were analyzed with immunofluorescence assay to assess the identification and storage of P62 cargo proteins by the autophagosomes and the degradation of the autophagolysosomes.The co-localization of LC3B and LAMP1 was also determined with immunofluorescence assay to detect the fusion of the autophagosomes with the lysosomes, and LysoTracker was used to trace the acidic lysosomes.
Results: We successfully constructed a HL7702 cell model of Sidt2+/+ and Sidt2-/-, and compared with Sidt2+/+ cells, the Sidt2-/- cell model showed significantly increased expressions of LC3-II/I and P62 (P < 0.01).Immunofluorescence assay showed a significant increase of LC3B and P62 expressions (P < 0.001) and obviously lowered expressions of Atg5, Atg7, and Atg12 in Sidt2-/- cells (P < 0.05).The co-localization of LC3B and P62 and that of LC3B and LAMP1 were both reduced and the number of acidic lysosomes was significantly lowered in Sidt2-/- cells (P < 0.05).
Conclusion: Sidt2 gene deletion disturbs the recognition and sequestration of P62 cargo protein by autophagosomes in human hepatocytes.At the same time, the decreased number of acidic lysosomes and the dysfunction of autophagosome and lysosome fusion cause the block of the autophagy-lysosome pathway, leading eventually to LC3B and P62 accumulation and impaired autophagy in the hepatocytes.
目的: 研究溶酶体膜蛋白Sidt2缺失后对肝脏自噬的影响。
方法: 利用Crispr-Cas9技术构建Sidt2敲除的人肝脏（HL7702）细胞模型，对自噬关键蛋白LC3Ⅱ/Ⅰ、P62及自噬相关蛋白Atg5、Atg7、Atg12进行蛋白水平检测，同时使用免疫荧光方法对LC3B及P62进行共定位，检测自噬小体对P62货物蛋白的识别与封存以及自噬溶酶体的降解。对LC3B及LAMP1进行免疫荧光共定位，检测自噬小体与溶酶体融合过程。使用LysoTracker对酸性溶酶体进行示踪。
结果: 成功构建HL7702细胞Sidt2+/+组及Sidt2-/-组，HL7702细胞Sidt2-/-组较Sidt2+/+组相比，自噬关键蛋白LC3-Ⅱ/Ⅰ及P62表达均增多（P < 0.01），免疫荧光结果显示LC3B及P62亦明显增多（P < 0.001），同时自噬相关蛋白Atg5、Atg7、Atg12表达减少（P < 0.05）。LC3B与P62共定位降低，LC3B与LAMP1共定位降低，酸性溶酶体数量减少（P < 0.05）。
结论: Sidt2基因的缺失导致人肝脏细胞自噬小体对P62货物蛋白的识别和封存障碍，同时酸性溶酶体数量减少、自噬小体与溶酶体融合障碍导致自噬溶酶体途径受阻，最终导致LC3B与P62发生堆积，肝脏细胞自噬受损。
Keywords: Sidt2; autophagosomes; cargo proteins; fusion; lysosomes.