| Obejective: This research aims to explore the molecular mechanisms of Sorafenibresistant in HCC via vasculogenic mimicry induced by ITGA5/ITGB1 with the purpose of finding a target which can reverse the resistant of Sorafenib,and providing a theoretical basis of combination drugs for subsequent treatment of HCC.Methods: We analyzed the clinical data of Sorafenib-resistant and non-resistant HCC patients in GSE109211 from GEO database,and using bioinformatics analysis software to analyze the differential gene expression,hypoxia score,tumor stroma score and immune score between the Sorafenib-resistant group and non-resistant group.Kaplan Meier-Plotter was used to analyze the survival of HCC patients with the high and low expression of ITGA5/ITGB1 respectively.CCK-8 was conducted to detect the proliferation of Hep G2/Huh7,and with 5 to 10 times of 50% Inhibition concentration of Sorafenib to introduce the Sorafenib-resistant Hep G2/Huh7;and by synthesizing ITGA5/ITGB1 plasmid,constructing a lentiviral vector and infecting Hep G2/Huh7 cells to construct the liver cells with high expression of ITGA5 and ITGB1,respectively.WB was conducted to detect the expression of ITGA5/ITGB1/RUNX1,PAS staining was carried out to detect the level of glycogen.And flow cytometry was ued to dectect the degree of hypoxia and cell cycle via hypoxia probe and PI staining respectively.Results:(1)Bioinformatics results: The analysis of omics difference revealed that there was a significant difference in gene expression between the Sorafenib resistance group and the non-resistant group(P<0.05),and further results showed that cell adhesion and extracellular matrix were closly related to the Sorafenib-resistant through KEGG pathway enrichment.The expression of ITGA5/ITGB1 was higher in Sorafenib resistance group(P<0.01),and the tumor stroma score(P<0.05)and immune score(P<0.05),which indirectly reflcted the proportion of vascular components in tumor tissues,were higher in Sorafenib resistance group too.The survival of HCC patients with low expression of ITGA5/ITGB1 was significantly higher than that of the high expression group.Otherwise,the results of hypoxia analysis showed that the Sorafenib-resistant group with a higher degree of hypoxia(P<0.01).(2)We screened and induced the Sorafenib-resistant Hep G2/Huh7 cells successfully,and detected the up-expression of ITGA5/ITGB1 and the upstream promoter RUNX1 in Sorafenib-resistant Hep G2/Huh7 cells.The Sorafenibresistant Hep G2/Huh7 cells were also performing a high degree of Hypoxia and the level of glycogen,and the cell cycle was arrestd in G1/S phase.(3)The result of WB was confirmed that we had contrcted the liver cells with high expression of ITGA5/ITGB1 successfully,and the high expression of ITGA5/ITGB1 with a reduced sensitivity of Sorafenib,indicating that upregulation of ITGA5/ITGB1 can induce Sorafenib resistance.In addition,the up-regulation of ITGA5/ITGB1 could increase the level of glycogen in liver the degree of Hypoxia in those cells,and could also arrest the cell cycle of liver cancer cells in G1/S phase.(4)We had constructed the subcutaneous liver nude mouse model successfully,and observed the intrahepatic metastasis of the high expression ITGA5/ITGB1 groups.Conclusion: Sorafenib inhibit the tumor angiogenesis which depend on endothelial cells to inhibit HCC,which can further aggravate the degree of Hpoxia in HCC microenvironment.The hypoxia-sensitive transcription factor RUNX1 will be upregulated under the circumstances of Hypoxia,which can regulate the overexpression of the downstream vasculogenic mimicry-related integrin protein ITGA5/ITGB1 to mediate the formation of vascular mimicry in HCC,and ultimately induce the formation of Sorafenib resistance. |
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