| Objective To investigate the role of IL-8 on EMT in breast cancer and the underlying mechanisms.Methods 1.Western blotting and semi-quantitative RT-PCR was adopted to detect the expression of IL-8 receptors CXCRA, CXCRB in human breast cancer cell lines MCF-7, SK-BR-3 and MDA-MB-231; After the treatment of human recombinant cytokine IL-8 in breast cancer cell lines MCF-7, SK-BR-3 and MDA-MB-231, the changes in cell morphology were observed under an optical microscope; and EMT markers E-cadherin, Vimentin and Fibronectin were detected by Western blotting.2.Semi-quantitative RT-PCR, real-time quantitative PCR (real-time fluorescence quantitative PCR, QRT-PCR) and Western blotting was adopted to detect the mRNA and protein level of EMT transcription factor Snail after IL-8 treatment in human breast cancer cell line MCF-7 and SK-BR-3.3.Breast cancer cell line MCF-7 was differently treated with the inhibitors for signaling pathways at the downstream of IL-8 (MAPK/ ERK1/2, PI3K/AKT, JAK/STAT3 ) and IL-8, and EMT markers Vimentin was detected by Western blotting. Next,Western blotting was adopted to detect the expression of p-AKT after breast cancer cell lines MCF-7 and SK-BR-3 treated with IL-8. Furthermore, breast cancer cell line MCF-7 and SK-BR-3 were differently treated with PI3K/AKT signaling pathway inhibitor and IL-8. Scratch test was utilized to detect the migration of breast cancer cell and Boyden chamber invasion assay was utilized to detect invasiveness. While semi-quantitative RT-PCR and QRT-PCR for mRNA level of Snail,Western blotting for protein level of EMT marker E-cadherin, Vimentin, Fibronectin and Snail.4.Co-immunoprecipitation was applied to enrich Snail and the proteins that interact with Snail in breast cancer cell line SK-BR-3. After the proteins in immune complexes were separated by SDS-PAGE and silver staining for the gel,the gel was cut into three parts of average size and sent to Beijing Huada company for mass spectrometry (LC-MSMS) testing. Analysed the LC-MSMS results for the proteins that interact with Snail. To inquiry the funtions and features of the proteins screened out in NCBI database, and detect the mutual binding capacity between Snail and the proteins screened out in web PEPSITE.5.1mmunohistochemical assay(IHC)was applied to detect the expression of IL-8, CXCRB, E-cadherin, Vimentin, Snail, C14orfl66 and TUBA1B in the specimens of benign breast hyperplasia, in situ breast cancer and breast cancer with lymph node metastasis.6.Established breast cancer xenograft model in nude mouse,with intratumoral injection of IL-8, to investigate the role of IL-8 on the growth and liver,lung metastases of breast cancer in nude mice. Furthermore, the expression of EMT marker, Snail, C14orfl66 and TUBA1B in the xenografts were verified by IHC.Results 1.Western blotting and semi-quantitative PCR showed that both IL-8 receptor CXCRA and CXCRB were detected in breast cancer cell line MCF-7, whereas SK-BR-3 and MDA-MB-231 only CXCRB; With optical microscope,we observed that IL-8 promoted EMT in MCF-7, SK-BR-3 as cell morphology became elongated and intercellular adhesion decreased after IL-8 treatment, while not in MDA-MB-231; Western blotting showed that IL-8 upregulated the expression of mesenchymal markers Vimentin and Fibronectin,downregulated the expression of epithelial markers E-cadherin only in MCF-7 and SK-BR-3.The results demonstrated IL-8 promoted EMT in breast cancer cell line MCF-7, SK-BR-3,but not in MDA-MB-231. Therefore,breast cancer cell lines MCF-7 and SK-BR-3 were chosen as experimental object in follow-up tests.2.Semi-quantitative PCR, QRT-PCR and Western blotting showed that IL-8 significantly upregulated the mRNA and protein expression of EMT transcription factor Snail.3.The results showed that IL-8 might promote EMT in breast cancer cells by activating PDK/AKT signaling pathway. As IL-8 upregulated the expression of mesenchymal marker Vimentin in MCF-7 and this effect was blocked by the PI3K/AKT signaling pathway inhibitors. So as to scratch test and Boyden chamber invasion assay:IL-8 enhanced the migration and invasion of breast cancer cells, and this effect was blocked by PI3K/AKT signaling pathway inhibitor. Similarly, IL-8 upregulated the expression of Snail, Vimentin, Fibronectin and reduced the expression of E-cadherin,which could also be blocked by PI3K/AKT signaling pathway inhibitor.These results further demonstrated that IL-8 promoted EMT and upregulated the expression of EMT transcription factor Snail in breast cancer cells by activating PI3K/AKT signaling pathway.4. Analysis of the LC-MSMS data preliminarily showed that C14orfl66 and TUBA1B interacted specifically with Snail. From NCBI database we found out that these two proteins could promote cancer invasion and metastasis. Web software PEPSITE detection showed that C14orfl66,TUBA1B interacted closely with Snail. Furthermore, the results of semi-quantitative RT-PCR and Western blotting indicated that IL-8 upregulated the mRNA and protein expression of C14orfl66 and TUBA1B, and PI3K/AKT signaling pathway inhibitor blocked this effect.5. The results of IHC showed that the expression of IL-8, CXCRB, Vimentin, Snail, C14orfl66,TUBA1B in specimens of benign hyperplasia, in situ breast cancer and breast cancer with lymph node metastasis increased successively, and on the contrary for the epithelial marker E-cadherin.6. Breast cancer xenograft experiment in nude mice showed that intratumoral injection of IL-8 significantly increased the tumor volume,weight and increased metastatic nodules in liver and lung. HE staining showed that liver and lung metastasis were significantly more obvious in the mouse injected with IL-8. Finally, the results of IHC for the xenograft tumor indicated that IL-8 upregulated the expression of Vimentin, Snail, C14orfl66 and TUBA1B significantly, while E-cadherin downregulated.Conclusion 1.IL-8 promoted EMT and upreguled the expression of EMT transcription factor Snail in breast cancer cell line MCF-7 and SK-BR-3.2.Co-immunoprecipitation preliminarily demonstrated that C14orfl66, TUBAIB interacted with Snail and IL-8 promoted EMT,upregulated the expression of Snail,C14orfl66 and TUBAIB by activating PI3K/AKT signaling pathway in breast cancer cells.3.The expression of IL-8, CXCRB, Vimentin, Snail, C14orfl66 and TUBA1B in specimens of breast cancer with lymph node metastasis were significantly higher than specimens of in situ breast cancer and benign hyperplasia. IL-8 promoted the growth and liver,lung metastasis of xenograft tumor in nude mice, and upregulated the expression of Vimentin, Snail,C14orfl66 and TUBAIB, downregulated the expression of E-cadherin. |
PDF Full Text Request