M Geng1,2,3, L Wang4,5, Y Zhang4,5, W Pei4, M Qi3, M Yang3, J Xu3, Y Liang3, K Lü6,7, C He1, J Gao1,2,4. 1. Department of Endocrinology and Genetic Metabolism, Wannan Medical College, Wuhu 241002, China. 2. Institute of Endocrine and Metabolic Diseases, Yijishan Hospital of Wannan Medical College, Wuhu 241002, China. 3. School of Clinical Medicine, Wannan Medical College, Wuhu 241002, China. 4. Anhui Provincial Key Laboratory of Biological Macro-molecules Research, Wannan Medical College, Wuhu 241002, China. 5. Department of Biochemistry and Molecular Biology, Wannan Medical College, Wuhu 241002, China. 6. Central Laboratory, Wannan Medical College, Wuhu 241002, China. 7. Anhui Provincial College Key Laboratory of Non-coding RNA Transformation Research on Critical Diseases, Wannan Medical College, Wuhu 241002, China.
Abstract
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.
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.