Differential Molecular Verification Of Interactions Between Nasopharyngeal Carcinoma Cells And Lymphatic Endothelial Cells

Background: Nasopharyngeal carcinoma(NPC)is one of the most common malignant tumors in the south of China,which is prone to lymph node metastasis.The interaction among the cells in the tumor microenvironment may play an important role in the lymphatic metastasis process.Our previous research showed that growth and migration capacity of human lymphatic endothelial cells(HLEC)was promoted when cultured in the conditioned medium(CM)of NPC cell line 5-8F(5-8F-CM),which is with high lymph node metastatisis potential.In this previous study,5-8F cells were co-cultured with HLEC through Live Cell Station,and it was shown that the migration capacity of 5-8F was significantly enhanced after co-culture.And next,after sorting these two co-cultured cells by flow cytometry,screening of potential pathways through the RNA-Seq,protein antibody chip detection and bioinformatics analysis.The results showed that the different molecules were mainly enriched in TGF-beta signaling pathway,cell adhesion,cell chemokines,angiogenesis factors and so on.And our results also showed that there were differences in the expression of some miRNAs,including miR-17-92 cluster members in the cells after co-culture.Objective: Our present study aimed to establish a cell-interaction model co-culture of LECs with 5-8F cells,verifying the difference expression molecules and investigating their possible roles in the lympatic metastasis of NPC.Method: Conditioned medium(CM)of LEC(LEC-CM)and 5-8F-CM were prepared after respective culture of LEC cells and 5-8F cells for 24 h.And next,5-8F cells were cultrured in LEC-CM,and LECs were cultured in 5-8F-CM.Subsequently,realtime-PCR and western blot were used to detect the expression of TGF-beta and some metastasis-related molecules in these post-CM-cocultured 5-8F and LECs cells.In the following experiments,two co-culture methods,conditioned medium co-culture and transwell co-culture,were used to detect the expression of miR-17-92 cluster members in cells and supernatants.Finally,target gene prediction and bioinformatics analysis of miR-17-92 were carried out.Result: 1.Realtime-PCR and Western Blot were used to detect the expression of the genes and proteins in LEC and 5-8F cells before and after respective cultured in 5-8F-CM or LEC-CM.It was found that the expression of TGF-beta and some metastasis-related molecules(CTGF,Fractalkine,DCN,IGF-2R,MIF)in LEC cells were significantly reduced,nevertheless,the expression of these molecules were significantly upregulated in 5-8F cells after CM co-culture.2.The expression of E-cadherin was detected at the transcriptional level and protein level.It was found that after CM co-culture,the expression of E-cadherin mRNA and protein in LEC and 5-8F cells was significantly down-regulated,while the expression of beta-catenin,MMP-9 and Vimentin were upregulated at transcriptional level.3.Most of the miR-17-92 cluster members in 5-8F and LEC cells and their supernatant were significantly upregulated after CM co-culture.4.In co-culture using transwell,when 5-8F cells were inoculated in the lower chamber,with LECs being cultured in the upper chamber,the expression of the miR-17-92 cluster members significantly increased in both these cells and their supernanants.And when LECs were inoculated in the lower chamber,with 5-8F cells beingin the upper chamber,the expression of the miR-17-92 cluster members also significantly increased in 5-8F cells and LECs,but in the supernatants of these cells,significances were shown in the downregulation of most of the miR-17-92 cluster members.5.GO analysis found that the predicted target genes of miR-17-92 were mainly involved in biological processes such as cellular process,biological regulation,metabolic process,stress reaction,developmental process,cellular component organization or biogenesis.And KEGG Pathway analysis found that the target genes were significantly enriched in endocytosis pathway,the MAPK signaling pathway,the Wnt signaling pathway,the pathway of Proteoglycans in cancer,the pathway of non-small cell lung cancer,PI3K-Akt signaling pathway,adherens junction pathway,apoptosis pathway and TGF-beta signaling pathway,etc.Conclusion: 1.After cultured in LEC-CM,the key genes of the TGF-beta pathway in 5-8F cells were up-regulated,which may be associated with the enhancement of their migration ability.On the other hand,the downregulation of key genes in the TGF-beta pathway in LECs after cultured in 5-8F-CM may promote the proliferation of LECs.2.Nasopharyngeal carcinoma cells 5-8F could stimulate LEC cells to secrete miR-17-92,and the upregulation of miR-17-92 cluster may be related to the proliferation of lymphatic endothelial cells.