Study On The Nuclear Localization Sequence Of CXCR4 In Renal Cell Carcinoma

[Objective]Study has found that the CXCR4 transfering to nuclear was in relation to the metastasis of renal cell carcinoma. The experiment is aimed to explore the nuclear localization sequence of CXCR4 which transfered to nuclear under the stimulation of SDF-1, in order to lay the theoretical basis of finding materials which can inhibit metastasis of renal cell carcinoma, and further enrich the content of signaling mechanism of SDF-1/CXCR4 axis.[Methods]The online software PSORTâ…¡Prediction was used to predict the nuclear localization sequence of CXCR4. With EGFP-CXCR4 as the template, we obtained the fragment with the nuclear localization sequence of CXCR4 deleted through the methods of over-lapping PCR and enzyme digestion etc. Then the fragment was connected to pGEM-Teasy vector. After proved to be correct, the product was enzyme digested, as well as EGFP-N1 respectively. The products were connected together, then the mutant of EGFP-CXCR4 with NLS deleted was constructed successfully. The mutant, as well as EGFP-CXCR4 and EGFP-N1 which were set up to be the control groups, was transfected to Human embryonic kidney 293 cells (HEK-293). And other groups were established as the control with no SDF-1 added. After the stimulation of SDF-1 for 24 hours, these plasmids' localization in the cells was observed by confocal microscopy, through which we could determine the accuracy of the software predicting the nuclear localization sequence of CXCR4.[Results]1. The software indicated that the NLS of CXCR4 might lied in the amino acids 146 to 149,named as RPRK, and the sequence of cDNA was "AGGCCAAGGAAG".2. The mutant of EGFP-CXCR4 with NLS deleted was constrcted successfully through the test of enzyme digestion and sequencing, with no base-pair mutation and reading frame shift.3. The results observed by confocal microscopy showed that after transfected to Human embryonic kidney 293 cells, the EGFP-CXCR4 plasmid transfered to nuclear after the stimulation of SDF-1 for 24 hours. However, the mutant of EGFP-CXCR4 with NLS deleted didn't transfer to nuclear but to cytoplasm with the same condition.[Conclusion]1. The result that the NLS of CXCR4 might lied in the amino acids 146 to 149,named as RPRK, is consist with the outcome predicted by the sofeware.2. Blocking the nuclear localization sequence of CXCR4 may help to inhibit the metastasis of renal cell carcinoma.3. The experiment provided a theoretical basis to further study of blocking CXCR4 nuclear localization sequence and thus inhibiting metastasis of renal cell carcinoma.