The Role And Mechanism Of LASS2 In Inducing Mitophagy In Human Lung Cancer Cells

Objective To investigate the function and mechanism of Lag1 long-lived assurance LAG1 longevity assurance homolog2(LASS2)-induced mitophagy in human lung cancer cells,we aim to establish a theoretical and experimental foundation for future studies on novel anti-tumor target genes.Methods(1)Bioinformatics analysis was performed to investigate the expression profile of the LASS2 gene in lung cancer,its correlation with survival prognosis,its function and pathway enrichment,as well as its correlation with autophagy,mitophagy-related pathways,and pathway proteins.(2)Western blot was used to verify the construction of the cell model for the LASS2 gene silencing and overexpression groups.(3)The changes in mitochondria and autophagosomes in human lung cancer cells were observed through transmission electron microscopy after gene silencing and overexpression of LASS2.(4)The JC-1 fluorescent probe was utilized to detect alterations in mitochondrial membrane potential in human lung cancer cells following gene silencing and overexpression of LASS2.(5)ELISA was utilized to analyze the expression of ATP6V0C in human lung cancer cells following gene silencing and overexpression of LASS2.(6)Western blot analysis was utilized to investigate the impact of LASS2 gene silencing and overexpression on the expression of mitophagy-related proteins,including p62,PINK1,Parkin,and BNIP3L,in human lung cancer cells.Results(1)Bioinformatics analysis results show that the expression of LASS2 is significantly downregulated in lung cancer tissues compared to adjacent tissues(P=0.0028).Additionally,its T4 phase expression was significantly upregulated compared to T1 phase expression in pathological T staging(P=0.0024).However,when compared within the group using age,gender,clinical stage,pathological M stage,and pathological N stage as grouping indicators,there was no statistically significant difference in its expression level.Functional enrichment analysis revealed that the LASS2 is significantly enriched in functional phenotypes such as megaautophagy and lysosomes.The pathway score revealed a significant positive correlation between six autophagy pathways and two mitophagy pathways with LASS2.In the correlation analysis with autophagy and mitochondrial autophagy-related proteins(p62,PINK1,Parkin,BNIP3L,LC3B,and Bcl2),it was found that the LASS2 gene has a significant positive correlation with p62 and PINK1.(2)The results of in vitro experiments on human lung cancer cells show that:(1)Western blot analysis showed that,compared to the blank control group and negative control group,the expression level of LASS2 protein in the LASS2 silencing group significantly decreased(F=27.49,P=0.001),while the expression level of LASS2 protein in the overexpression group significantly increased(F=61.32,P=0.000).This indicates that the previously constructed cell model is effective and usable.(2)Transmission electron microscopy analysis showed that,compared to the blank control group and negative control group,the number of autophagosomes decreased in the LASS2 gene silencing group,while it increased in the overexpression group.(3)JC-1staining showed that,after induction with the same concentration of CCCP,the degree of depolarization of mitochondrial membrane potential was weaker in the LASS2 silencing group cells compared to the blank control group and the negative control group.Conversely,the degree of depolarization of mitochondrial membrane potential was stronger in the overexpression group cells.(4)ELISA detection found that,compared to the blank control group and negative control group,the LASS2 silencing group had a significant increase in intracellular ATP6V0C expression(F=138.1,P=0.000),while the overexpression group had a significant decrease in intracellular ATP6V0C expression(F=8.11,P=0.02).(5)Western blot analysis showed that,compared to the blank control group and negative control group,the expression levels of p62(F=12.39,P=0.007),PINK1(F=79.04,P=0.000),Parkin(F=14.5,P=0.005),and BNIP3L(F=17.68,P=0.003)proteins in the LASS2 silencing group cells were significantly reduced,while the expression levels of p62(F=24.42,P=0.001),PINK1(F=14.91,P=0.028),Parkin(F=22.97,P=0.002),and BNIP3L(F=28.31,P=0.000)proteins in the overexpression group cells were significantly increased.Conclusion The LASS2 gene plays a role in positively regulating mitophagy in lung cancer.The underlying mechanism may result in an increase in intracellular H~+concentration by downregulating intracellular expression of atp6v0c and inhibiting proton pump action.This leads to a decrease in mitochondrial membrane potential,activation of mitophagy PINK1,parkin,and BNIP3L signaling pathways,and ultimately induces mitophagy in human lung cancer cells.