| [Objective] We explored the role of CD38and its possible mechanism in nasopharyngeal carcinoma (NPC) side population (SP) cells by identificating and resreaching the biological function of SP cells.[Methods] MofloTM XDP High-Performance Cell sorter was used to sort the SP cells in NPC cell lines. Flow cytometry, microRNA chip, quantitative real-time PCR and Western Blotting were used to illuminate NPC SP cells biological characteristics, CD38expression level and its possible mechanism on the levels of mRNA, microRNA and protein.[Results]1. Identification and biological characteristics of the SP cells in NPC cell lines.We used the characteristic of efflux fluorescent dye Hoechst33342to identify the SP phenotype in four kinds of NPC cell lines including HK-1, HNE1, HNE2and C666-1. Results suggested that HK-1and C666-1cell lines contained a higher proportion of SP cells,5.2±0.72%and0.46±0.07%respectively. HNE1and HNE2cell lines contained a lower proportion of SP cells,0.01%and0.02%respectively. We chose HK-1cell line which showed the highest percentages of SP cells to accomplish following experiments. SP cells had the ability of efflux Hoechst33342via ABC transporter. Verapamil, an ABC transporter inhibitor, could well block the fluorescent efflux and reduce the percentage of SP cells.To further test the biological characteristics of SP cells, we sorted SP and NSP (non-side population cells, NSP) cells by using flow cytometry. We found cancer stem cells (CSCs) markers, CD133, OCT4, ALDH1Al and CD38whose expressions were significantly higher in SP cells. In addition, SP cells had similar biological characteristics with CSCs, such as capacities of greater cell proliferation and self-renewal, invasion and migration ability and cell cycle mainly in GO/G1phase.2. The microRNAs differentially expressed profiling of NPC HK-1SP cells and NSP cells.We performed microRNA microarray analysis (Agilent Human miRNA microarray18.0) to investigate the miRNAs expression in NPC HK-1SP cells and NSP cells. The results showed that84miRNAs were differentially expressed in SP cells and NSP cells among the tested1887miRNAs. Five miRNAs (hsv2-miR-H10, miR-572, miR-3656, miR-638, miR-2861) were up-regulated in SP cells and79microRNAs (miR-374b-5p, miR-140-5p, miR-1470, et al) were down-regulated in SP cells. In these different expressed miRNAs,94.05%miRNAs were down-regulated in SP cells. Then, qRT-PCR verified that microRNAs such as miR-634, miR-664, miR-140-5p, miR-4317, miR-3646and miR-582-5p were differentially expressed between NPC SP and NSP cells. Using the webset TargetScan (http://www.targetscan.org), we found that many molecules in these differentially expressed miRNAs were related with some CSC markers. For example, CD38was regulated by miR-634, miR-664, miR-140-5p. CD24was regulated by miR-4317, miR-3646, miR-582-5p. CD133was regulated by miR-30a. CD44was regulated by miR-532. CD90was regulated by miR-125b. Meanwhile, we tested the cell surface and total protein expression levels of several cancer stem cells markers in NPC SP and NSP cells by flow cytometry. We found that CD44, CD24, CD38, CK19highly expressed on the surface of SP cells and that CD133, CD24, CD90, CD38, CD21highly expressed in SP cells intracellular. The results suggested that they were negatively correlated between these different expressed microRNAs and protein levels of CSCs markers. Based on the above results, we found that CD38expression was significantly different between SP and NSP cells. So we conjectured that CD38-mediated pathway might play an important biological function in SP cells.3. CD38exerts biological effects by synergizing with ZAP70pathwayCD38interacts with CXCR4, CXCL12, CD31, CD49D, MMP-9, ZAP70and other molecules through a variety of ways and regulates the tumor progression. We detected the mRNA expression level of molecules involved in CD38network and found CXCR4, ZAP70, CD31, CD49D, MMP-9showed up-regulated expression in SP cells. Among these molecules, CXCR4was11times up-regulate in SP cells (t=5.355, p=0.0003), CD31was3.3times (t=3.559, p=0.0061), CD49D was1.5times (t=2.754, p=0.0362), MMP-9was3.2times (t=2.602, p=0.0482), ZAP70was1.3times (t=5.584, p<0.0001), respectively. Then, we found that CD38and its downstream molecule ZAP70were highly expressed on protein level in SP cells by Western Blotting method. Our results suggest that CD38might exert biological effects by synergizing with ZAP70.[Conclusion](1) We successfully identified the rates of SP cells in NPC cell line HK-1and C666-1were5.2±0.72%and0.46±0.07%respectively, and verified the SP cells had similar biological characteristics with CSCs.(2) We established microRNA expression profiling in NPC SP and NSP cells successfully, and found that miR-634, miR-664, miR-140-5p and so on were down-regulated and hsv2-miR-H10, miR-572, miR-3656, miR-638, miR-2861and so on were up-regulated in SP cells.(3) We found the expression of CD38was different between SP and NSP cells. CD38might synergize with ZAP70pathway to exert its biological effects. |
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