[Lysosomal membrane protein knockout induces apoptosis of human hepatocytes in vitro independent of the autophagy-lysosomal pathway].

OBJECTIVE

To explore the regulatory mechanism of human hepatocyte apoptosis induced by lysosomal membrane protein knockout.

METHODS

The knockout () cell model was constructed in human hepatocyte HL7702 cells using Crispr-Cas9 technology.The protein levels of 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 knockout on cell proliferation and apoptosis.The effect of chloroquine at the saturating concentration on autophagic flux, proliferation and apoptosis of knockout cells were observed.

RESULTS

HL7702 cells were successfully constructed. knockout significantly inhibited the proliferation and increased apoptosis of the cells, causing also increased protein expressions of LC3-II/I and P62( < 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 HL7702 cells.

CONCLUSION

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.