Expression Of Chemokine CXCL1 In Macrophage-hepatocellular Carcinoma Microenvironment And Its Influence On The Progression Of Hepatocellular Carcinoma

Object Hepatocellular carcinoma（HCC）is the leading cause of cancer-related death worldwide.HCC morbidity and mortality continue to increase in many countries,and most HCC patients are in advanced stages after diagnosis.During the development of HCC,the liver constructs a special tumor microenvironment rich in fibroblasts,blood vessels,stellate cells,extracellular matrix components,cytokines and enzymes secreted by cancer cells and non-cancer cells.Among them,macrophages are the key cellular components of the liver and have become important players in the maintenance of liver homeostasis and the process of injury and repair in acute and chronic liver diseases.In the HCC microenvironment,macrophages are induced to become tumor-associated macrophages（TAMs）,which have an important impact on cancer progression.Related studies have confirmed that CXCL1 promotes HCC progression in the HCC autocrine regulatory pathway.However,there is no clear report on the expression and function of CXCL1 in the macrophage-hepatocellular carcinoma（M-HCC）microenvironment.The purpose of this experiment was to investigate the expression of CXCL1 in M-HCC and the effect of CXCL1 on HCC.Method The paraffin specimens of 51 HCC patients in our hospital from 2010 to 2022 were retrospectively analyzed,and CXCL1 histochemical staining was performed.The patients were divided into two groups according to the level of CXCL1 expression using semi-quantitative score.The differences in clinical characteristics of the two groups of patients were statistically analyzed.Construction of the M-HCC microenvironment:Phorbol 12-myristate 13-acetate（PMA）was used to induced human monocytes THP-1to obtain macrophages,and the macrophages were co-cultured with Huh7 to construct the M-HCC microenvironment.The effect of TAMs on Huh7 in the co-culture environment was verified by migration and invasion experiments.Validation of the M-HCC microenvironment: RT-PCR validated the expression of CD14 and CD68 in macrophages,and the expression of CD163 and CD206 in TAMs.To study the expression of CXCL1 in M-HCC: The GEPIA tumor infiltration database website was used to analyze the expression of CXCL1 among different types of macrophages.RT-PCR verified the expression of CXCL1 in macrophages,TAMs and Huh7 cells before and after co-culture.To investigate the effect of recombinant protein CXCL1 on HCC: Statistical analysis of the relationship between CXCL1 and HCC prognosis using the TISIDB website.Different concentrations of recombinant protein CXCL1 were co-cultured with Huh7 cells,and the effects of recombinant protein CXCL1 on HCC were studied through cell proliferation,migration and invasion experiments.Exploring CXCL1 and HCC downstream related factors: Statistical analysis of CXCL1 related genes in HCC using TISIDB website.The total RNA of Huh7 cells co-cultured with CXCL1 was extracted for RT-PCR to verify the expression of the above-mentioned related genes.Result The expression level of CXCL1 was significantly correlated with liver cirrhosis and microvascular invasion in HCC patients.Immunofluorescence showed that patients with high CXCL1 expression had higher TAMs-related gene expression（CD68,CD206）.Construction and verification of M-HCC microenvironment: THP-1 was transformed into macrophages after induction by PMA,and RT-PCR verified that the expression of CD14 was down-regulated and the expression of CD68 was up-regulated;macrophages were co-cultured with Huh7 cells and transformed into TAMs,RT-PCR verified that the expression levels of CD163 and CD206 were significantly up-regulated.CXCL1 expression in M-HCC: GEPIA tumor infiltration database analysis showed that CXCL1 expression was different among different types of macrophages.The same results were obtained by RT-PCR validation of CXCL1 between TAMs and macrophages.In addition,the expression of CXCL1 was also significantly up-regulated in Huh7 cells after co-culture.The effect of recombinant protein CXCL1 on HCC: TISIDB website analysis showed that HCC patients with high CXCL1 expression had a worse prognosis.Cell proliferation experiments showed that recombinant protein CXCL1 promoted the proliferation of Huh7 cells,and the difference in proliferation was the most obvious at48 h.In addition,recombinant protein CXCL1 also promoted Huh7 cell migration and invasion.CXCL1 and HCC downstream related factors: TISIDB website analysis showed that NFκB1,IL6 and STAT3 were correlated with CXCL1 in HCC,and the same results were also verified in RT-PCR.Conclusion CXCL1 is associated with the inflammatory microenvironment of HCC.M-HCC co-culture system promotes CXCL1 expression.CXCL1 promotes HCC proliferation,migration and invasion.CXCL1 may act on HCC through NFκB1,IL6 and STAT3.