Abstract:

To investigate the effect of zymogen granule protein 16 （ZG16） on epithelial mesenchymal transition （EMT） in hepatocellular carcinoma cells and its potential association with the immune checkpoint molecule programmed death-ligand 1 （PD-L1）. The expression differences of ZG16 between hepatocellular carcinoma （HCC） and normal liver tissues， as well as its relationship with patient prognosis， were analyzed based on the GEO database and the KM-Plot website. Huh7 and HepG2 cells were transfected with the pcDNATM3.1-ZG16 overexpression plasmid to overexpress ZG16. Protein expression levels of ZG16， CTNNA3， E-cadherin， N-cadherin， Vimentin and PD-L1 were detected by Western blot. The localization and protein expression levels of ZG16 and CTNNA3 were examined using immunofluorescence. The interaction between ZG16 and CTNNA3 was detected by co-immunoprecipitation. Cell migration ability was assessed through scratch wound healing assays， and cell colony formation ability was evaluated using colony formation assays.Bioinformatic analysis revealed that ZG16 expression was significantly downregulated in HCC tissues， and higher ZG16 expression was associated with significantly improved patient survival （P<0.05）. In vitro experiments showed that compared to the control group， ZG16 overexpression significantly increased E-cadherin expression and decreased N-cadherin and Vimentin expression （P<0.05）， along with significantly reduced cell migration and colony formation abilities in Huh7 and HepG2 cells （P<0.05）. In HCC patients， ZG16 expression levels were negatively correlated with PD-L1， and ZG16 overexpression significantly reduced PD-L1 protein levels. Mechanistically， ZG16 overexpression significantly increased CTNNA3 protein levels （P<0.05）. Co-immunoprecipitation and co-localization immunofluorescence confirmed that ZG16 and CTNNA3 interact and co-localize in the cytoplasm. Functional rescue experiments demonstrated that， compared to Huh7 cells overexpressing ZG16 alone， simultaneous overexpression of ZG16 and knockdown of CTNNA3 promoted the epithelial-mesenchymal transition process and enhanced cell migration and colony formation abilities （P<0.05）. ZG16 upregulates CTNNA3 to inhibit the EMT process in hepatocellular carcinoma cells. Additionally， ZG16 negatively regulates PD-L1 protein expression. The research results can provide new molecular targets and theoretical basis for the clinical treatment of liver cancer.

Key words: hepatocellular carcinoma, zymogen granule protein 16, epithelial-mesenchymal transformation, CTNNA3, PD-L1