Study On The Role And Mechanism Of EPS8L3 In The Development Of Hepatocellular Carcinoma

Background:Hepatocellular carcinoma(HCC)is one of the most common malignancies in the world,and its morbidity and mortality rank in the forefront of all malignant tumors.Although a comprehensive treatment system based on surgical resection and liver transplantation has been established,the current prognosis of HCC patient is still unsatisfactory.Epidermal growth factor receptor(EGFR)has been reported to play an important role in the development of malignant tumors.The EGFR targeted drugs,which were represented by sorafenib,are the most widely used anti-tumor drugs in clinical practice.However,the effect of EGFR targeted drugs in the treatment of HCC is not satisfactory,and with the increase of drug resistance rate,it is urgent to find novel effective therapeutic targets.The epidermal growth factor receptor pathway substrate 8(EPS8)family is expected to provide a novel and potential target for the development of EGFR targeted drugs,providing a new theoretical basis for the clinical treatment of HCC.Therefore,the study focused on EPS8 family has important research significance and clinical value.Objectives:Based on public databases and clinical samples,the study will explore the expression and function of EPS8L3 in HCC,and clarified the relationship between its expression level and clinical features.Meanwhile,the study will explore the effect and underlying molecular mechanisms of EPS8L3 on HCC cell proliferation,migration and invasion through in vitro and in vivo studies,providing potential therapeutic targets for the treatment of HCC.Methods:1.Based on TCGA,GTEx,GEPIA and COSMIC databases,we explored the expression,correlation and gene mutation of EPS8 family in malignant tumors.2.Based on clinical specimens,we studied the expression of EPS8L3 in liver cancer by q RT-PCR,western blot and immunohistochemistry,and analyzed the correlations between the expression level and clinical information.3.Lentivirus and si RNA were used to modulate the m RNA expression of EPS8L3.CCK8 assay was used to detect proliferative ability,colony formation assay to detect colony forming ability,flow cytometry to detect cell cycle changes,transwell and wound healing assay to detect cell migration and invasion.4.Subcutaneous xenograft model and orthotopic transplantation tumor model were used to detect the proliferative ability in vivo.Pulmonary colonization assay was performed to detect the pulmonary colonized ability in vivo.5.The effects of EPS8L3 on cell cycle,epithelial-mesenchymal transition,cell stemness and hepatocyte differentiation were detected by q RT-PCR and western blot.6.The relationships between EPS8L3 and EGFR,SOS1 and ABI1 were detected by western blot,flow cytometry and immunofluorescence.The signal pathway involved was also identified.Results:1.In the EPS8 family,only the m RNA expression of EPS8L3 in HCC tissues was significantly higher than that in adjacent or normal liver tissues,and there was no significant correlations with other family members.The point mutation,copy number variation and methylation level of EPS8L3 were low,and the level of gene overexpression was relatively high.2.The expression of EPS8L3 was upregulated in HCC tissues,and the expression levels are closely related to tumor pathological differentiation and prognosis.3.After the silence of EPS8L3,the abilities of proliferation,colony formation,migration and invasion of HCC cells were all weakened,and the cell cycle was blocked.After overexpression,the above-mentioned abilities were enhanced and the cell cycle progressed.4.After the silence of EPS8L3,the proliferative and pulmonary colonized abilities in vivo were weakened,while overexpressing EPS8L3,the proliferative ability in vivo was enhanced.5.EPS8L3 downregulated the expression of p21/p27 and upregulated the expression of MMP2,but has no significant effect on cell stemness and hepatocyte differentiation.6.EGFR dimerization and EEA1-mediated internalization were inhibited after the knockdown of EPS8L3,and the activation of EGFR-ERK pathway was weakened,but EPS8L3 was not significantly associated with SOS1 and ABI1.Conclusion:The expression of EPS8L3 was upregulated in HCC tissues,and the expression was correlated with tumor pathological differentiation and prognosis.EPS8L3 promoted the cell cycle progression by inhibiting the expression of p21/p27 and enhanced the proliferative ability,while promoting the expression of MMP2 and enhanced the abilities of migration and invasion.Moreover,EPS8L3 could enhance the activation of EGFR-ERK signaling pathway by promoting the EGFR dimerization and EEA1-mediated internalization,thereby affecting the abilities of proliferation and metastasis,but this process did not depend on the formation of a complex with SOS1 and ABI1.The above results indicated that EPS8L3 played an important role in the development of HCC,and its involvement in the regulation of EGFR-related signaling pathway was expected to become a potential target for EGFR targeted drug research,providing new insights for clinical tumor therapy.