The Role Of CXCR4 And EEF1A1 Interactions In The Metastasis Of Renal Cell Carcinoma

[Objective]Renal Cell Carcinoma(RCC) is a common malignant tumor of urinary system and when diagnosed about one-third of patients have come to the late stage. Metastatic renal cell carcinoma cannot be effectively treated and five-year survival rate is less than 20 percent, seriously affecting the health of people. CXCR4 is a chemokine, playing an important physiological and pathological features in the body. A large number of domestic and international research have evidenced that CXCR4 is related with tumor metastasis. The common sense now is that its activation can activate multiple downstream signaling pathways which influence the development and progression of tumors. This study is aimed to explored the interacting proteins and DNA in renal cancer cells and to rich CXCR4 pathway promoting metastasis of renal cell carcinoma.[Methods]1. CXCR4-EGFP expression lentiviral has been produced and it was transfected to get kidney cancer cell lines ACHN which was overexpressed of CXCR4, and verified transfection efficiency by fluorescence microscopy, quantitative real-time PCR and western blot.2. Protein immunoprecipitation, chromatin immunoprecipitation were used to explore the interacting proteins and DNA in renal cancer cells ACHN. Spectrometry and sequencing detected technology were used to find the protein and DNA which is interacted with CXCR4 in ACHN.3. Verify the results of mass spectrometry. Mass spectrometry obtained CXCR4 binding proteins eEF1A1(Eukrryotic Translation Elongation Factor 1 Alpha 1, eEF1A1). SiRNA corresponding was designed to lower the expression level. Produce the interference lentiviral and transfect the ACHN which has been transfected CXCR4 lentiviral. Finally we get the ACHN cell lines which have stable low expression of eEF1A1.4. Cell proliferation assays, apoptosis experiments, cell migration assays were use to explore the relationship between eEF1A1 with renal cell carcinoma metastasis. In the ACHN cells transfected EGFP lentivirus, eEF1A1 interference ACHN-EGFP cells, the ACHN cells transfected CXCR4-EGFP lentivirus, eEF1A1 interference ACHN-CXCR4-EGFP cells, CCK8 proliferation assay, apoptosis by flow cytometry, Transwell cell migration experiments wer used to study the effects of eEF1A1 on renal cancer cell and by comparing the high expression of CXCR4 with knockdown of eEF1A1 expression the interaction between CXCR4 and eEF1A1 on renal function in cancer cells was explored.5. 98 cases of renal cell carcinoma tissue were selected to build tissue chip. Through immunohistochemical staining, expression level of eEF1A1 was statisticed. Combined with the prognosis of patients with renal cell carcinoma, the relationship between prognosis of RCC and expression level of eEF1A1 was explored.[Results]1. ACHN renal carcinoma cell line overexpressing CXCR4-EGFP fusion protein was successfully constructed. Transfected ACHN cells could be seen the green fluorescence in over 90% cells under a fluorescence microscop and quantitative real-time PCR showed that transfecting CXCR4-EGFP virus increase the expression of CXCR4 in 7499 times. Western blot showed the fusion protein abundantly expressed.2. Immunoprecipitation was done and protein interacted CXCR4 was get and was analysed by mass spectrometry. The mass spectrometry analysis indicated the presence of about 50 KDa position bands might be eEF1A1. This conclusion was further validated by western blot by using eEF1A1 antibody in 50 KDa nearby position bands. In renal carcinoma cell line the interaction of ACHN CXCR4 and eEF1A1 was confirmed.3.Interfering RNA eEF1A1 was designed through gene database query. The interfering sequence was GCACCAUGAAGCUUUGAGUGAAGCTT GCUUCACUCAAAGCUUCAUGGUGCTT. This sequence was constructed lentiviral shRNA interference and transfected ACHN and ACHN-CXCR4 cells. Real-time quantitative PCR and western blot validated that the interference efficiency was about 70%. The expression level of interference group eEF1A1 downward 70% and then the stable eEF1A1 interference cell lines ACHN were successfully constructed.4.The experiments confirmed that cell function after interference of eEF1A1 slightly increased apoptosis, slowed down proliferation, and had no significant changes in migration. After the interference eEF1A1 in the high expression of CXCR4 ACHN cell lines increased apoptosis than before, proliferation slowed more but no significant change in migration. The eEF1A1 may enhance renal cancer cell proliferation, and some mechanism may be achieved through the interaction with CXCR4. EEF1A1 has no significant effect on migration.5. Tissue microarray showed poor prognosis of renal cell carcinoma with high expression of eEF1A1.[Conclusion]CXCR4 and eEF1A1 promote cell proliferation in renal cancer cell line ACHN. Their interaction adds to this trend. High expression of eEF1A1 in renal cell carcinoma indicates the poor prognosis.