| Golgin-84 belongs to the golgin family and plays an important role in maintaining the structure of Golgi apparatus and regulating vesicle transport.However,the role of golgin-84 in regulating Golgi apparatus and lysosomes function and the physiological function of individuals is not clear.In this paper,golgin-84 knockout HeLa cell line and golgin-84 knockout mice were constructed,and the effects of golgin-84 knockout on cell and mouse growth,Golgi apparatus structure,lysosome function and autophagy were studied by molecular biology,cytobiology and biochemistry.The results obtained in this paper include:(1)Golgin-84 knockout HeLa cell line was successfully constructed by CRISPR-Cas9 technique.The effects of golgin-84 on cell viability,proliferation and migration were detected by MTT test,colony-forming experiment and wound healing.The results showed that after golgin-84 knockout,the cell viability decreased,the number of cell clones formed decreased and the migration rate of cells slowed down.These results suggest that knockout of golgin-84 can significantly inhibit cell viability,proliferation and migration.(2)The expression level of Golgi related proteins and the glycosylation level of lysosome membrane proteins were studied by western bolt experiment,the results showed that knockout of golgin-84 increased the expression of TMF and decreased the expression of CASP in cell,and knockout of golgin-84 significantly decreased the glycosylation level of LAMP1 and LAMP2,which was more obvious after knocking out golgin-84 and TMF at the same time because of the functional complementarity between golgin-84 and TMF.(3)The level of autophagy was studied by western bolt assay,the results showed that after knockout of golgin-84,the ratio of LC3II/LC3-I decreased and the level of p62 remained unchanged,indicating that autophagy was suppressed.And then it was proved by treatment of Torinl and Baf-A1 in cells and mouse starvation experiments that knockout golgin-84 mainly inhibit the initial stage of autophagy.(4)The morphology of the Golgi apparatus and the localization of autophagy-related organelles were detected by immunofluorescence experiment.The results showed that knockout of golgin-84 could lead to Golgi fragmentation,and knockout of golgin-84 significantly increased the co-localization of lysosomes and autophagosomes,but did not affect the co-localization of Golgi apparatus with lysosomes and autophagosomes.It suggests that knockout of golgin-84 can inhibit the function of autolysosome.(5)The study of golgin-84 knockout mice showed that knockout of golgin-84 affected the growth and organ development of mice.To sum up,this study shows that golgin-84 regulates the growth of cells and mice,golgin-84 regulates lysosome function by regulating the glycosylation of lysosomal membrane proteins,and golgin-84 function is associated with autophagy. |
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