GIT1-Mediated Epithelial-Mesenchymal Transformation Promotes The Invasion And Metastasis Of Hepatocellular Carcinoma

Background and Objective:Hepatocellular carcinoma（HCC）is the most common type of liver cancer and one of the common causes of cancer-related deaths.In recent years,the diagnosis and treatment technology of HCC has been continuously improved,but due to the insidious onset,high recurrence and metastasis of HCC,the overall prognosis of HCC patients is still poor.G protein-coupled receptor（GPCR）-kinase interacting protein 1（GIT1）is a multifunctional scaffold protein that plays an important role in cell adhesion and migration.Studies have correlated GIT1 with HCC,however,these correlations have not been fully elucidated.Therefore,we aimed at evaluating the clinical significance of GIT1 expression in HCC patients,and to investigate its role and potential mechanisms in HCC progression.Methods:The expression level of GIT1 in HCC and other gastrointestinal tumors were determined by using the public databases Oncomine and TCGA.According to the median value of GIT1 expression,TCGA HCC patients were divided into GIT1^high and GIT1^low expression groups.Then,the differentially expressed genes between the two groups were analyzed,and the threshold was set to|Log₂FC|>2 and P<0.05.Then,the Gene Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG） enrichment analysis were performed to analyze the potential biological functions of differentially expressed genes.Next,we used Western blot,quantitative reverse transcription-polymerase chain reaction（qRT-PCR）,and immunohistochemical staining to detect the expression of GIT1 in HCC tissues and adjacent tissues.Next,the expression of GIT1 was evaluated in different HCC cell lines.Through lentivirus,the synthetic GIT1 overexpression plasmid was used to transfect the HCC cell line with the lowest GIT1 expression,and the synthetic short hairpin RNA（shRNA）plasmid was used to transfect the HCC cell line with the highest GIT1 expression.The up-and down-regulated GIT1 cell lines were utilized for in vitro and in vivo functional experiments.The CCK-8 and clone formation experiment were performed to evaluate the effect of GIT1 on the proliferation of HCC cells;The wound healing and Transwell invasion assay were performed to evaluate the effect of GIT1 on the migration and invasion of HCC cells;The subcutaneous transplantation tumor experiment was used to measure the impression of GIT1 on tumor growth.In addition,Western blot was used to detect changes in the expression of epithelial-mesenchymal transition（EMT）-related proteins in HCC cell lines after up-regulation or down-regulation of GIT1,and explored the specific mechanisms.Finally,the immunohistochemical staining was used to detect the expression of GIT1 in tumor tissue microarrays of 158 patients with HCC,and the Kaplan-Meier and Cox regression methods were used to analyze the relationship between GIT1 and the clinicopathological characteristics and prognosis of HCC.Results:（1）Through the analysis of database data,it was found that the expression of GIT1 was elevated in HCC and related to the staging and prognosis of HCC.The differentially expressed genes between the GIT1^high and GIT1^low expression group were analyzed,and a total of 4163 differential genes were obtained.Further,GO and KEGG enrichment analysis showed that GIT1 might be involved in the process of translation initiation,RNA decomposition,protein targeting to cell membrane and endoplasmic reticulum,and was related to endocytosis,ribosome,actin cytoskeleton regulation,and protein processing in endoplasmic reticulum.（2）The results of Western blot,q RT-PCR,and immunohistochemistry showed that the expression of GIT1 in HCC tissues was higher than that in adjacent tissues.（3）The expressions of GIT1 in MHCC97-H,Huh7,Hep G2,and PLC/PRF/5 cells were detected.It was found that MHCC97-H has the high GIT1 expression levels while Huh7exhibited low GIT1 expression levels.GIT1 expression in MHCC97-H cells was knocked-down by transfecting GIT1-sh RNA lentiviral vector.Meanwhile,GIT1 was overexpressed in Huh7 cells with GIT1 cDNA lentiviral vector.Then the transfection efficiency was verified by Western blot.（4）The results of CCK-8 analysis showed that GIT1 overexpression significantly enhanced cell proliferation.The clonogenic assay showed that colony formation in Huh7 cells was significantly improved after GIT1 overexpression.Furthermore,the wound healing and Transwell invasion assays showed that GIT1 upregulation accelerated HCC cell migration and invasion.In addition,the results of in vivo transplantation tumor experiments showed that GIT1 could promote the growth of HCC.（5）Western blot was used to detect the expression of EMT-related proteins in MHCC97-H and Huh7 cells.The results revealed that upon GIT1 overexpression,E-cadherin was suppressed,while Vimentin was elevated relative to the negative control.Conversely,GIT1 knockdown led to elevated E-cadherin levels while Vimentin levels were inhibited.（6）Western blot was used to detect the changes in the activation level of the signaling pathway in the HCC cells,and the results revealed that p-ERK1/2 levels were suppressed after GIT1 knock-down in MHCC97-H.On the contrary,p-ERK1/2 protein levels were enhanced by GIT1 overexpression in Huh7 cells,indicating that GIT1 might promote HCC progression by activating ERK1/2 signaling.Then,the pathway inhibitor（SCH772984）was used to inhibit ERK1/2 signal transduction.There was a marked reduction in p-ERK1/2 levels in HCC cells with high expression of GIT1,leading to elevated E-cadherin levels and suppressed Vimentin levels.（7）Correlation analysis data suggested that the expression of GIT1 was related to tumor size（P=0.023）and embolus（P=0.002）.And the results of tissue microarray analysis showed that the expression of GIT1 was correlated with tumor size and embolus.Multivariate Cox model analysis revealed that GIT1 was an independent prognostic factor for cumulative recurrence（P=0.009）and overall survival（P<0.001）in HCC.The Kaplan-Meier survival curve showed that high GIT1 expression levels indicated poor prognosis for patients with HCC.Conclusion:（1）GIT1 expression is elevated in HCC tissues than that in adjacent non-tumor tissues,and enhance the proliferation,invasion,and migration potential of HCC cells.（2）GIT1 can activate the transduction of the ERK1/2 signaling pathway and may regulate the EMT changes of HCC cells through the ERK1/2 pathway.（3）The expression of GIT1 is related to tumor size and embolus,and GIT1 expression is an independent prognostic factor for the cumulative recurrence and overall survival of HCC.