| Objectives: Eukaryotic translation initiation factor 5A2(EIF5A2)is a newly discovered candidate oncogene in recent years.At present,the expression and specific biological role of EIF5A2 in intrahepatic cholangiocarcinoma are unclear.The purpose of this study is to clarify the expression of EIF5A2 in intrahepatic cholangiocarcinoma tumor tissue and explore the correlation between EIF5A2 expression and clinicopathological characteristics and prognosis of intrahepatic cholangiocarcinoma,To study the molecular mechanism of EIF5A2 promoting the progression of intrahepatic cholangiocarcinoma,so as to further reveal the pathogenesis of intrahepatic cholangiocarcinoma.Methods: Clinical paraffin tissue samples were collected,including 191 cases of intrahepatic cholangiocarcinoma tissue(141 cases of tissue chips,50 cases of clinical samples),32 cases of normal liver tissue,and 12 cases of intrahepatic cholangiocarcinoma tissue.The expression of EIF5A2 in intrahepatic cholangiocarcinoma tissue,paracancerous tissue,normal liver tissue,and intrahepatic cholangiocarcinoma tissue was detected by immunohistochemistry(IHC),The expression of EIF5A2 in tumor tissue and adjacent tissues of 8 matched intrahepatic cholangiocarcinoma was detected by Western blot;According to the immunohistochemical semi-quantitative scoring method,the immunohistochemical results were scored,combined with the clinicopathological data of tissue chips,126 cases with complete follow-up data were analyzed,and the relationship between EIF5A2 and the clinicopathological characteristics of intrahepatic cholangiocarcinoma and the impact on the overall survival rate and cumulative recurrence rate of patients with intrahepatic cholangiocarcinoma were discussed.The expression level of EIF5A2 m RNA and protein in HIBEpi C,RBE,HCCC9810 and HUCCT1 cells was detected by QPCR and Western Blot.The location of EIF5A2 in cells was observed by immunofluorescence(IF).The expression of knockdown EIF5A2 gene was stably transfected by HCCC9810 cell line,and the expression of EIF5A2 was stably transfected in HUCCT1 cell line.CCK-8 experiment and Ed U cell proliferation experiment were used to evaluate the cell proliferation ability,Flow cytometry was used to detect the changes in the cell cycle of ICC cell lines after knockdown and overexpression of EIF5A2,and scratch test and Transwell migration and invasion test were used to observe the changes in the migration and invasion ability of tumor cells.The stable transfection strains HCCC9810-sh RNA1 and HCCC9810-sh NC(negative control)with knockdown EIF5A2 were selected for transcriptome sequencing(RNA-seq),and the differential genes were analyzed by GO and KEGG enrichment to explore the potential biological function of EIF5A2 in the development of intrahepatic cholangiocarcinoma and the signal pathway that can participate in the regulation,and immunoprecipitation(Co-IP)was used to study whether there is interaction between EIF5A2 protein and MTA1 protein;Instantaneous transfection(si RNA)knocks down the downstream protein MTA1 of EIF5A2 in HUCCT1-EIF5A2 cells.The effects of knockdown of MTA1 on cell proliferation,invasion and migration,PI3K/AKT/mTOR signal pathway and Cyclin D1,Cyclin D3,MMP2 and MMP9 were detected;HUCCT1-EIF5A2 cells were treated with PI3K/AKT/mTOR signal pathway inhibitor LY294002,and the effects of PI3K/AKT/mTOR signal pathway inhibition on cell proliferation,invasion and migration and the expression of corresponding protein molecules were detected;Western blot was used to detect the effect of EIF5A2 on epithelial-mesenchymal transition(EMT)of intrahepatic cholangiocarcinoma cells.In the nude mouse animal model,the stable transfected cell lines HCCC9810-sh NC,HCCC9810-sh RNA1,HUCCT1-Vector,HUCCT1-EIF5A2 were selected to establish the subcutaneous tumorigenic model,and the volume and weight of the subcutaneous tumor in nude mice were recorded and analyzed.Western blot was used to detect the expression of EIF5A2,MTA1,Cyclin D1,Cyclin D3,MMP2,MMP9 protein in the tumor tissue after subcutaneous tumorigenesis in nude mice.Results: Immunohistochemical results showed that EIF5A2 was mainly expressed in the cytoplasm and nucleus of tumor cells.EIF5A2 was highly expressed in 98 cases(51.3%)of 191 cases of intrahepatic cholangiocarcinoma,6 cases(18.8%)of normal liver tissue,and 1 case(8.3%)of intrahepatic cholangiocarcinoma;Immunohistochemical staining was performed on 141 paraffin tissue samples(including cancerous tissue and paracancerous tissue)in tissue microarray.The expression of EIF5A2 in cancer tissue was higher than that in paracancerous tissue,normal liver tissue and intrahepatic bile duct tissue in 62 cases(44.0%)and 35 cases(24.8%);The total protein extracted from fresh cancer tissue and para-cancer tissue was detected by Western blot.The results showed that the expression of EIF5A2 in cancer tissue was higher than that in para-cancer tissue(P<0.05).The clinicopathological analysis of EIF5A2 and intrahepatic cholangiocarcinoma showed that the expression of EIF5A2 was significantly correlated with microvascular/bile duct invasion(P=0.044),tumor differentiation(P=0.014),lymphatic metastasis(P=0.033),and TNM stage(P=0.002),but not with age,sex,cirrhosis,ALT,Child-Pugh grade,CA19-9,tumor diameter,and tumor number;Univariate analysis showed that the increased expression of EIF5A2 was a high risk factor for low overall survival rate and high cumulative recurrence rate.Multivariate analysis showed that EIF5A2 was an independent risk factor for predicting the prognosis of patients with intrahepatic cholangiocarcinoma.CCK8 and Ed U cell proliferation experiments showed that after knocking down EIF5A2,the proliferation ability of HCCC9810-sh RNA1 group was significantly inhibited compared with that of HCCC9810-sh NC group,and the cell proliferation ability was enhanced after overexpression of EIF5A2 in HUCCT1 cells;Flow cytometry cell cycle detection showed that EIF5A2 could help intrahepatic cholangiocarcinoma cells span G0/G1 phase and promote tumor cell proliferation;Scratch test and Transwell migration and invasion test showed that the overexpression of EIF5A2 enhanced the migration and invasion ability of intrahepatic cholangiocarcinoma cells.RNA-seq analysis showed that after knocking down EIF5A2,the differential gene GO was enriched in the functions of cell migration,cell differentiation,growth factor activation,transcription factor and DNA binding,and KEGG was enriched in the signal pathways of Wnt,VEGF,PI3K/AKT,mTOR and IL-17;The cell phenotype experiment found that EIF5A2 can significantly promote the proliferation,migration and invasion of intrahepatic cholangiocarcinoma cells.Combined with the results of GO analysis,Western Blot verified seven proteins affected by EIF5A2 and closely related to proliferation,invasion and metastasis.It was found that EIF5A2 regulates the expression of MTA1 through transcription in intrahepatic cholangiocarcinoma.Co-IP results showed that there was no interaction between EIF5A2 and MTA1 proteins,In addition,EIF5A2 relies on MTA1 to activate the PI3K/AKT/mTOR signal pathway and then affect the proliferation,migration and invasion of cancer cells.In HUCCT1-EIF5A2 cells,the cell proliferation,invasion and migration are inhibited after the MTA1 is knocked down,and the expression of p-PI3 K,p-AKT,p-mTOR,Cyclin D1,Cyclin D3 and MMP2,MMP9 is decreased.After the inhibition of PI3K/AKT/mTOR signal pathway in HUCCT1-EIF5A2 cells,the ability of EIF5A2 to promote the proliferation,invasion and metastasis of HUCCT1 cells is significantly inhibited,The expression level of corresponding protein molecules decreased;In addition,Western blot analysis confirmed that EIF5A2 regulates the epithelial-mesenchymal transition(EMT)of intrahepatic cholangiocarcinoma cells by up-regulating the transcription factors Snail2 and Twist1,but has no significant effect on the expression of the transcription factor Snail.The results of subcutaneous tumorigenesis in nude mice showed that the subcutaneous tumor of the nude mice silencing EIF5A2 was smaller and lighter,and the overexpression of EIF5A2 promoted the growth of subcutaneous tumor in nude mice;Western blot test was used to detect the difference of protein expression in the subcutaneous tumor tissue of nude mice,which verified that EIF5A2 regulates the expression of MTA1 in vivo and affects the protein expression level of Cyclin D1,Cyclin D3,MMP2,MMP9.Conclusions: The high expression of EIF5A2 in intrahepatic cholangiocarcinoma is closely related to microvascular/bile duct invasion,tumor differentiation,lymphatic metastasis and TNM stage.The patients with high expression of EIF5A2 have low overall survival rate and high cumulative recurrence rate,which are independent risk factors for the prognosis of patients with intrahepatic cholangiocarcinoma.The expression of EIF5A2 in intrahepatic cholangiocarcinoma cell line was higher than that in human intrahepatic cholangiocarcinoma cell line.The expression of EIF5A2 was the highest in intrahepatic cholangiocarcinoma cell line HCCC9810,and the expression was low in RBE and HUCCT1 cells,mainly localized in the cytoplasm and nucleus;High expression of EIF5A2 can promote intrahepatic cholangiocarcinoma cells to cross G0/G1 phase and promote proliferation,and enhance the migration and invasion ability of intrahepatic cholangiocarcinoma cells.EIF5A2 transcription regulates the expression of MTA1 and activates the PI3K/AKT/mTOR signal pathway to induce the up-regulation of Cyclin D1,Cyclin D3,MMP2 and MMP9,thus promoting the proliferation,invasion and metastasis of intrahepatic cholangiocarcinoma cells.EIF5A2 also regulates the epithelial-mesenchymal transition(EMT)of intrahepatic cholangiocarcinoma cells by up-regulating the transcription factors Snail2 and Twist1,but has no significant effect on the expression of the transcription factor Snail.High expression of EIF5A2 can promote the growth of subcutaneous tumor in nude mice,increase the expression of MMP2 and MMP9 in subcutaneous tumor,and enhance the invasion and metastasis of intrahepatic cholangiocarcinoma. |
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