Objective: To explore the regulatory mechanism of human hepatocyte apoptosis induced by lysosomal membrane protein Sidt2 knockout.
Methods: The Sidt2 knockout (Sidt2-/-) cell model was constructed in human hepatocyte HL7702 cells using Crispr-Cas9 technology.The protein levels of Sidt2 and key autophagy proteins LC3-II/I and P62 in the cell model were detected using Western blotting, and the formation of autophagosomes was observed with MDC staining.EdU incorporation assay and flow cytometry were performed to observe the effect of Sidt2 knockout on cell proliferation and apoptosis.The effect of chloroquine at the saturating concentration on autophagic flux, proliferation and apoptosis of Sidt2 knockout cells were observed.
Results: Sidt2-/- HL7702 cells were successfully constructed.Sidt2 knockout significantly inhibited the proliferation and increased apoptosis of the cells, causing also increased protein expressions of LC3-II/I and P62(P < 0.05) and increased number of autophagosomes.Autophagy of the cells reached a saturated state following treatment with 50 μmol/L chloroquine, and at this concentration, chloroquine significantly increased the expressions of LC3B and P62 in Sidt2-/- HL7702 cells.
Conclusion: Sidt2 gene knockout causes dysregulation of the autophagy pathway and induces apoptosis of HL7702 cells, and the latter effect is not mediated by inhibiting the autophagy-lysosomal pathway.
目的: 研究溶酶体膜蛋白Sidt2敲除后在肝脏细胞中调控凋亡的途径。
方法: 利用Crispr-Cas9技术构建Sidt2敲除的人肝脏(HL7702)细胞模型, 检测Sidt2和自噬关键蛋白LC3-Ⅱ/Ⅰ、P62蛋白水平, MDC染色观察自噬体形成情况, 通过EdU和流式细胞实验观察Sidt2对肝脏细胞增殖活性和凋亡的影响, 并且使用0、10、25、50、100、200 μmol/L的氯喹(CQ)摸索肝脏细胞CQ饱和浓度, 检测对其自噬流的影响, 以及使用CQ进一步探索影响肝脏细胞的增殖活性其凋亡水平的机制。
结果: 成功构建HL7702细胞Sidt2+/+组及Sidt2-/-组。Sidt2基因缺失可导致肝脏细胞增殖被抑制和凋亡水平的增加, 自噬关键蛋白LC3-Ⅱ/Ⅰ和P62在蛋白水平上的表达均增加且自噬体含量增加(P < 0.05)。结果显示50 μmol/L CQ作用下肝脏细胞自噬达到饱和状态, 50 μmol/L CQ使Sidt2基因缺失时LC3B和P62的表达均增加(P < 0.05), 且CQ进一步降低肝脏细胞活性, 并促进其凋亡水平(P < 0.05)。
结论: 在离体水平上, Sidt2基因剔除后, HL7702细胞可表现自噬通路的异常, 并且不通过抑制自噬溶酶体途径介导和调控凋亡。
Keywords: Sidt2; apoptosis; autophagy; human liver cells; proliferation.