| Background and Objective CXC chemokine receptor 4 (CXCR4), which is widely expressed in human malignant tumors including renal cell carcinoma (RCC), p lays a critical role in tumor angiogenesis, proliferation and organ-specific migration with its ligand stromal cell derived factor-1 (SDF-1). Recent researches have shown that the subcellular distribution of CXCR4, as well as its expression level, was a key influence factor on the biological characteristics and the prognosis of the tumor. In our previous work, we found that CXCR4 presented varied subcellular distributi on patterns in the RCC pathological tissue specimens from different sources, which was predominantly plasma membrane expressed in the primary tumors while being chiefly intracellular localized in the metastases. Furthermore, CXCR4 could be tran sferred into the cytoplasm so far as the nucleus of the renal cancer cells under con tinuous incubation with SDF-1 in vitro. However, the mechanism wKat drives the tr anslocation, especially the nuclear localization, of CXCR4 together with its biologic al function has not been well described. Herein, we explored the factors involved i n CXCR4 subcellular distribution, particularly its nuclear localization, and demonstra ted its related biological behavior changes of the renal cancer cells.Methods 1) The plasmids, which contained the CXCR4 Mut-EGFP fusion gen e with the mutant CXCR4 nuclear localized signal sequence created by overlap exte nsion polymerase chain reaction, were constructed to transfect the renal cell lines A 498 and ACHN. Then the nuclear as well as the subcellular distribution state of pr otein CXCR4 Mut-EGFP in the cells were thoroughly observed under microscopes. 2) The renal cancer cells lines (A498 and ACHN) which stably expressed protein CXCR4-EGFP and protein CXCR4 Mut-EGFP were established respectively before t he biological functional differences in proliferation, migration, invasion, cell-cycle an dapoptosis between the cells expressing distinguished CXCR4 proteins were compar ed. And then, the proteins in specific conjunction with CXCR4 based on the results from mass spectrometry and GFP-Trap method, were screened by literature reviewi ng to identify the ones potentially impacted on the subcellular distribution state of CXCR4.3) As non-muscle myosin ?A (NM? A) was selected for the subsequent research, the expression levels of NM?A and CXCR4 in different subcellular area were tested by western-bolt for the CXCR4 nuclear positive renal cancer cells after they were incubated with SDF-1, and the nuclear envelope distribution of CXCR4 was ensured by immunohistofluorescence. Finally, we assessed the changes of CX CR4 subcellular distribution and the renal cancer cell biological function under NM ?A suppressor, and analyzed the effect of CXCR4 localization at different subcellul ar area on migration and invasion characteristics of the cells.Results 1) The CXCR4 Mut-EGFP plasmids with mutant CXCR4 NLS sequenc e were successfully constructed and CXCR4 would be absence at the nuclear with any mutant basic amino acid in NLS. The wild protein CXCR4-EGFP was expresse d at the cell membrane, cytoplasm, nuclear envelope and nuclei, while the CXCR4 Mut-EGFP was expressed at the cell membrane, cytoplasm, nuclear envelope except the nuclei.2) The CXCR4 nuclear positive as well as the CXCR4 nuclear negativ e renal cancer cell lines were also established. And the former cell lines showed gr eater capability in migration and invasion (P<0.05) but similar performance in cell-cycle and apoptosis when comparing with the latter ones. Furthermore, it was indic ated that NM?A was potential protein involved in the regulation process of CXCR 4 subcellular distribution among the candidate proteins.3) The expression levels of NM?A at different subcellular area would not be disturbed by SDF-1 stimulation. However, CXCR4 expression level would decrease in total, increase in the nuclei a nd remain unchanged in the cytoplasm after SDF-1 stimulation. And it was confirm ed CXCR4 protein would also be expressed at the nuclear envelope by immunohist ofluorescence. Furthermore, the expression level of CXCR4 could be reduced in nuc lei and raised at the cytoplasm or nuclear membranes. In addition, the capability in migration but invasion of the cells could be insignificantly iimproved by increasing the cytoplasm membrane expression level of CXCR4 in the CXCR4 nuclear negati ve renal cancer cells.Conclusion 1) CXCR4 nuclear localization were determined by the integrity of its' NLS sequence. And any mutant basic amino acid in NLS would lead to the n uclear absence of CXCR4.2) The CXCR4 nuclear positive renal cancer cells shows different biological performance as the CXCR4 nuclear negative ones, and the for mer showed greater capability in migration and invasion.3) CXCR4 could be expre ssed at the nuclear envelope, but the relevent function was not clear yet.4) NM? A was involved in regulation of CXCR4 subcellular distribution, and inhabiting NM ?A was helpful for membrane and nuclear envelope localization of CXCR4.5) Th e immigration and invasion abilities of the renal cancer cells were affected by CXC R4 subcellular distribution status, they could be improved by increasing CXCR4 ex pressing level on the membranes.6) The renal cancer cells biological function was influenced by CXCR4 distributed at different subcellular area, and the mechanism i nvolved is worth further investigation. |
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