Research On The Mechanism By Which Kallistatin Suppresses Immune Evasion Of Gastric Cancer Cells By Downregulating CD24 Expression

Gastric cancer(GC)is a highly heterogeneous malignancy and the fifth most common cancer and fourth leading cause of cancer-related death worldwide.The incidence and mortality of gastric cancer in East Asia is much higher than in North America and may be related to dietary habits.Kallikrein-binding protein(Kallistatin,KS)belongs to one of the serine protease inhibitor superfamily and has received increasing attention in human oncology research due to its diverse biological functions.Numerous studies have shown that KS is involved in the occurrence and development of tumors and is expected to become a novel biomarker and therapeutic target for tumors.However,although KS is known to have anti-angiogenesis,anti-inflammatory,anti-lymphangiogenesis and other effects,the specific role of KS in gastric cancer needs to be further explored.In this study,we first constructed a gastric cancer cell line that overexpressed KS,and then co-cultured it with macrophages using overexpressed gastric cancer cell lines,and the results showed that KS could enhance the phagocytosis of gastric cancer cells by macrophages.Next,we performed immunohistochemistry and immunotissue fluorescence on tissue chips,and found that the expression of CD24 was increased in gastric cancer compared to next cancer.In addition,we performed Sanger sequencing on cell lines that overexpressed KS and used volcano maps to profile downstream gene expression.We found that gastric cancer cell lines that overexpressed KS had decreased CD24 expression compared to the downstream genes of the control group,and further cell experiments confirmed that KS could indeed downregulate CD24 expression by Western Blot(WB)and Immunofluorescence(IF).GO(Gene Ontology),KEGG(Kyoto encyclopedia of genes and genomes)and GSEA(Gene Set Enrichment Analysis)tools mapped sequenced gene aggregation into certain important signaling pathways,and it was found that the HIF-1α signaling pathway may be involved in the process of KS downregulating CD24 expression.Next,we used WB and IF to detect the content of HIF-1α,and the results showed that the overexpression KS group downregulated the expression of HIF-1α.These conclusions suggest that KS may regulate CD24 expression through the HIF-1α signaling pathway.Next,we went on to investigate the specific molecular mechanism by which KS regulates CD24.Through literature reading and mining,we selected a general signaling pathway: the ligand-AKT-HIF-1α-CD24 signaling pathway.First,we used the activator SC79 of AKT to detect the expression of HIF-1α,AKT,p-AKT,and CD24 by WB.The results confirmed that the addition of SC79 reversed the ability of KS to inhibit HIF-1α,CD24.This was explained that KS regulated CD24 expression through HIF-1α.Next,we added the HIF-1α inhibitor LW6 to the control group,and then measured the expression of HIF-1α and CD24,and the results showed that the inhibitor LW6 performed a similar function to KS.In addition,we added Deferoxamine mesylate(DFO),an agonist of HIF-1α,and then measured the expression of HIF-1α and CD24.The results showed that DFO could reverse the ability of KS to inhibit HIF-1α,thereby increasing the expression of CD24.In addition,the distribution of HIF-1α in the nucleus and cytoplasm was further investigated.The results showed that the overexpression of KS group inhibited the nucleation of HIF-1α,which in turn affected the expression of the downstream gene CD24.In conclusion,we can infer that KS regulates CD24 expression by regulating the HIF-1α signaling pathway.CD24 is a recognized antiphagic protein,so we used a control cell line and phagocyte co-culture,adding anti-CD24 antibody and Ig G to measure phagocytosis respectively.The results showed that with the addition of anti-CD24 antibody group,the phagocytosis was enhanced.Finally,we performed a prognostic analysis of the results of the immune tissue chip,which showed that the high-expression CD24 group had shorter overall survival,higher tumor stage,and easier metastasis than the low-expression CD24 group.Our research results indicate that KS can enhance the phagocytic ability of macrophages towards gastric cancer cells,and its expression level is significantly upregulated in gastric cancer tissues.These findings suggest that KS has important biological functions in gastric cancer,not only as a potential biomarker for early diagnosis and prognosis assessment of gastric cancer,but also as a potential therapeutic target.Based on these results,we believe that KS will become an emerging therapeutic strategy in future clinical practice.However,further research is needed to explore the mechanism of action of KS in gastric cancer and its relationship with other biological processes.We employed a multidisciplinary approach that combines bioinformatics,cellular biology techniques,and statistics,to investigate the role of the key molecule KS in gastric cancer.Our research revealed that KS can regulate the expression of CD24 through the HIF-1α signaling pathway.CD24 plays an important role in the occurrence and progression of gastric cancer,therefore,the mechanism of action of KS further confirms its regulatory role in gastric cancer and provides a theoretical basis for it to be a therapeutic target.This study has the potential to provide new treatment strategies and plans for the treatment of gastric cancer.