| Background and Objective:Hepatocellular carcinoma(HCC)is one of the ten most common malignant tumors in the world and the second leading cause of cancer death from malignant tumors[1–2].In recent years,although surgical resection and comprehensive treatment of liver cancer have made some progress,the overall five-year survival rate of liver cancer has not been significantly improved,which is mainly due to the invasion and distant metastasis of liver cancer[3–4].Therefore,elucidating the molecular biological mechanisms of invasion and metastasis of hepatocellular carcinoma(HCC)has become the focus of research on the prevention and treatment of HCC.Eukaryotic Translation Elongation Factor 1 Alpha 1,eEF1A1 is an important protein that triggers the initiation of protein translation and elongation.Studies have confirmed that eEF1A1 can participate in the regulation of cell growth,cell cycle regulation and apoptosis and other cellular physiological processes[5].In addition,it is reported in the literature that eEF1A1 expression is significantly increased in a variety of tumor cells including liver cancer[6],and the high expression of eEF1A1 is closely related to tumor cell proliferation,invasion and migration[7-11].However,the detailed mechanism of eEF1A1 in regulating the invasion and metastasis of liver cancer is still not fully understood,and further research is needed.HOXB9 is a member of the homeobox genes(HOX)family.Studies have reported that HOXB9 is highly expressed in breast cancer,lung cancer,colon cancer and gastric cancer[12-15].In addition,studies have also found that the high expression of HOXB9 is related to tumor angiogenesis,invasion,metastasis and poor prognosis[13,15].HOXB9 can promote tumor invasion by inducing epithelial to mesenchymal transition(Epithelial-Mesenchymal Transition EMT),and accelerate the DNA damage response to resist chemotherapy drugs and radiotherapy[12,15].Our previous studies have shown that HOXB9 is significantly highly expressed in HCC and participates in the process of HCC cell invasion and metastasis[16].However,the relationship between the expression of eEF1A1 and HOXB9 in liver cancer and whether there is an interaction between the two to affect the invasion and metastasis of liver cancer has not been reported yet.This study first detected the expression of eEF1A1 and HOXB9 in liver cancer tissues,and analyzed the correlation between their expressions and clinicopathological parameters of liver cancer patients;secondly,the expression of eEF1A1 and HOXB9 in liver cancer cells was clarified,and the expressions of eEF1A1 and HOXB9 in liver cancer cells were explored through experiments in vivo and The influence of eEF1A1 and HOXB9 on the invasion and migration ability of hepatocellular carcinoma cells;finally explore the specific mechanism of eEF1A1and HOXB9 affecting the invasion and metastasis ability of liver cancer cells.Methods:1.The m RNA and protein expression levels of eEF1A1 and HOXB9 in 156cases of liver cancer tissues and corresponding adjacent tissues were analyzed by q RT-PCR,immunohistochemistry and Western blot,and the expression of eEF1A1and HOXB9 in liver cancer and the clinicopathological parameters of liver cancer patients were analyzed.Its relevance to prognosis.2.The expression of eEF1A1 and HOXB9 in normal liver cells(HL-7702)and a variety of liver cancer cell lines(MHCC97h,HCCLM3,SMMC7721,Huh7 and Hep G2)were detected by q RT-PCR and Western Blot experiment methods;plasmids that interfere with eEF1A1 and HOXB9 were constructed,And screen out the eEF1A1 and HOXB9 plasmids with the best interference effect;after transfecting the sh-eEF1A1 plasmid in the above liver cancer cells,the Western Blot method was used to detect the expression of HOXB9,and the Transwell test was used to detect the changes in the invasion and metastasis ability of liver cancer cells after transfection.;Simultaneously transfect the HOXB9 overexpression plasmid into hepatocellular carcinoma cells that stably down-regulate the expression of eEF1A1,and detect the changes in the invasion and metastasis ability of hepatocarcinoma cells in each experimental group by Transwell and RTCA experiments.3.The protein immunoprecipitation experiment was used to detect whether the eEF1A1 protein and HOXB9 protein were combined,and the Western Blot experiment method was used to detect the changes in the total protein and nuclear protein expression of STAT1 after changing the expression of eEF1A1 in liver cancer cells;the expression of the liver cancer HCCLM3 with stable down-regulation of eEF1A1 expression The cells were transfected with STAT1 overexpression plasmid at the same time,and the protein expression changes of eEF1A1,HOXB9 and STAT1were detected by Western Blot experiment method,and the invasion and metastasis ability of liver cancer cells in each experimental group were detected by Transwell experiment;in liver cancer that overexpressed eEF1A1 stably Hep G2 cells were transfected with sh-STAT1 plasmid at the same time,and the protein expression changes of eEF1A1,HOXB9 and STAT1 were detected by Western Blot experiment method,and the invasion and metastasis ability of liver cancer cells in each experimental group were detected by Transwell experiment..4.After the expression or activity of STAT1 was changed,the protein expression of hoxb9 was detected by Western blot;the promoter binding region of STAT1 in hoxb9 was predicted by bioinformatics method,and the interaction between them was verified by luciferase reporter gene and chromatin immunoprecipitation.Results:(1)The overexpression of eef1a1 and hoxb9 is closely related to poor clinical prognosis.(2)eEF1A1 and HOXB9 are highly expressed in liver cancer cell lines,and after down-regulating the expression of eEF1A1 in liver cancer cells,the protein expression of HOXB9 also decreases,and at the same time reduces the invasion and migration ability of liver cancer cells;Down regulation of eEF1A1 expression in HCC cells resulted in the decrease of invasion and migration of HCC cells,which could be reversed by overexpression of HOXB9.(3)Co IP experiments confirmed that eEF1A1 and hoxb9 did not bind directly.In addition,changing the expression of eEF1A1 can regulate STAT1 total protein and nuclear protein.Further studies showed that STAT1 was the key protein of eEF1A1regulating the expression of HOXB9.(4)The protein expression of HOXB9 can be regulated by changing the expression or activity of STAT1 in hepatoma cells;Bio-medical analysis predicted the binding region of STAT1 and HOXB9 promoter,luciferase reporter gene and chip experiment further proved that STAT1 binds to the C region of HOXB9 promoter and upregulates the transcription level of HOXB9.Conclusion:In this study,we found that the expression of eEF1A1 and HOXB9 was up-regulated in HCC tissues,and there was a positive correlation between the expression of eEF1A1 and HOXB9.Overexpression of eEF1A1 and HOXB9 was significantly associated with poor prognosis of HCC patients.In addition,in vivo and in vitro experiments confirmed that reducing the expression of eEF1A1 can reduce the invasion and migration of HCC cells by down regulating the expression of HOXB9.Finally,we confirmed that eEF1A1 could increase the expression of HOXB9 by enhancing STAT1 mediated transcription activity of HOXB9.These results suggest that eEF1A1 can regulate the expression of HOXB9 through STAT1signaling pathway,thus promoting the invasion and migration of HCC cells,which provides a new therapeutic target for the prevention and treatment of HCC. |
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