| It was documented that nasopharyngeal carcinoma (NPC) is endemic in Southern China. All over the World,80%of NPC cases occured in China. In the past two decades, NPC remains the main cause of cancer deaths in Guangxi Zhuang Autonomous Region, and the third cause of deaths in malignancy, accounts for11.63%of all cancer deaths. Exploring the pathogenesis of NPC is of great significance.The aetiology of NPC involves three major factors:genetic susceptibility, Epstein-Barr virus (EBV) infection and exposure to chemical carcinogens, which follow typical multiple gene-environment interaction model. Unlike other human malignancy, activation or over-expression of oncogenes is not a main factor during the development and progression of NPC. However, the inactivation of tumor suppressor genes (TSGs) is more common. Promoter methylation has been proposed as a mechanism for inactivation of TSGs in human malignancies. The epigenetic changes influenced cellular pathways including EBV latent infection, cell cycle control, DNA repair system, apoptosis and tumor invasion and metastasis in NPC. Epigenetic changes play a crucial role in the tumourigenesis of NPC.In previous study, we have estabilished a global hypermethylation profile in NPC by a high-throughput method-cDNA microarray. In which the transcription of FSTL1gene were up-regulated almost20folds after pharmacologic inhibition of DNA methyltransferase in both CNE2and HONE1cells. We also identied that the transcription of FSTLl was significantly downregulated in NPC compared with the normal controls. These results indicated that FSTL1could be a target gene for epigenetic siliencing in NPC.In this study, we aimed to explore the mechanism of FSTL1epigenetic inactivated in NPC; to identify the biological functions of FSTL1in the tumorigenesis of NPC; to investigate the function of FSTLl in the regulation of macrophages and in the immune evasion of NPC cells. The results will address the role of FSTL1as a functional tumor suppressor gene in NPC, and its role in immune regulation, which in turn provide basic experimental data and theoretic evidences for disvovering diagnostic molecular markers and theraputic targets for NPC.First, we analyzed the methylation status of FSTL1gene in NPC cell lines and primary tumors by methyl-CpG domain-based capture (MBDCap) sequencing, and bisulfite genomic sequencing (BGS). Genomic DNA from7NPC primary tumors and4NPC cell lines were sheared to fragment double strand DNA, and the methylated DNA were captured by MBD protein-biotin-magnetic beads, then the captured DNA were used to construct SOLiD fragment libraries and sequenced on a SOLiD system and performed a genome-wide scale methylation profiling. The methlation profiling showed that FSTL1is hypermethylation in NPC. The promoter and first exon of FSTL1including29CpG sites were covered in the BGS.60%-80%of the29CpG sites were hypermethylated in2NPC cell lines (CNE2and HONE1),2NPC primary tumors, while normal nasopharyngeal epithelia showed was almost free of methylation. These results indicated that FSTL1is inactivated by promoter hypermethylation, might act as a functional tumor suppression gene in NPC.To explore the biological function of FSTL1in NPC, the full-length coding sequence of FSTL1was amplified by RT-PCR, and constructed to a mammalian expression vector pCMV-Tag3A. The pCMV-Tag3A-FSTL1plasmid was identified by double endonuclease digestion and Sanger sequencing. The results supported that the construction of expression vector was successful. pCMV-Tag3A-FSTL1plasmid was transfected into CNE2cells. After that, FSTL1stable transfectance of:FSTL1-CNE2was selected. The transcription and protein of exogenous FSTL1was confirmed by RT-PCR and Western-blot, respectively.Next, we performed a series of in vitro and in vivo experiments to analyze the biological function of FSTL1in NPC. The results showed that:1. Either the ectopic expression of FSTL1or the treatment of recombinant human FSTL1protein inhibits NPC cell proliferation;2. Ectopic expression of FSTL1could impede NPC cell migration and invasion;3. Ectopic expression of FSTL1could inhibit the colony formation of NPC cells;4. Ectopic expression of FSTL1could induce NPC cell cycle arrest;(72.03±1.27)%of FSTL1-CNE2cells were detected in G0/G1phase, while the proportion of empty vector-CNE2cells was (63.68±0.91)%(P=0.001);(1.34±0.83)%of FSTL1-CNE2cells were in S phase, while the corresponding proportion of empty vector-CNE2cells was (18.31±5.20)%(P=0.001);(26.62±1.59)%of FSTL1-CNE2were in G2/M phase, while the proportion of empty vector-CNE2was (18.01±4.28)%(P<0.05);5. ectopic expression of FSTL1could induce apoptosis in NPC cells;(37.13±3.13)%of FSTL1-CNE2cells was shown apoptosis, while (19.05±2.18)%in empty vector-CNE2; Cleaved Caspade-3fragments were detected by Western-blot in FSTL1-CNE2;6. Ectopic expression of the FSTL1could suppress the tumorignesis of NPC cells in nude mice;FSTL1-CNE2cells inoculated in nude mice grew slower than empty vector-CNE2; Tumor volumes from FSTL1-CNE2and empty vector-CNE2cells reached (91.35±16.06) mm3and (329.09±89.90) mm3respectively two weeks after injection,(P<0.05); the average weight of the tumor mentioned above were (0.155mg±0.037)g and (0.378mg±0.212) g for FSTL1-CNE2cells and empty vector-CNE2, respectively (P<0.05); the tumorigenesis suppression rate of FSTL1was worked out as59%. These data suggested that FSTL1might be a potential TSG in NPC.To evaluate whether secreted FSTLl could be a serological biomarker for NPC, we detected FSTL1in serum from NPC patients and normal controls by ELISA. The results suggested that there are no significant differences in FSTL1levels between two groups.FSTL1is reported to regulate the function of macrophage as a pro-inflammation cytokine. Then, we analyzed the expression of FSTL1and products of macrophage in NPC biopsies by immunofluorescence staining, chronic inflammation nasophayngeal tissue was used as normal control. FSTL1, one of the macrophage marker:CD68, macrophage products TNF-a and IL1were evaluated in15NPC tissues and15NP inflammation tissues. Consistent with our previous study, that the expression level of FSTLl was significantly lower in NPC primary tumors. Although the number of macrophage did not decreased in NPC stroma, the expression levels of two secretory products of macrophage, TNF-a and IL1, were significantly decreased, which suggests that downregulation of FSTL1might inturn suppresses of macrophage function.To further demonstrate that FSTL1may play an important role in the regulation of macrophage, we induced the human macrophage from peripheral blood mononuclear cell by recombinant human macrophage colony stimulating factor (rhM-CSF) as the first step. On the7th days after treatment, macrophage was successfully induced, which confirmed by morphological observation and immunofluorescence staining of CD14, a macrophage specific marker.Furthermore, we treated the macrophages by the recombination human FSTL1protein, and evaluated the levels of the macrophage products, IL1β and TNF-a in, in the supernatant by ELISA. We found that IL1β and TNF-a secreted by macrophage were upregulated gradually under the treatment of FSTL1. The levels of IL1β and TNF-a were higher that those in empty control group at the7th day after treatment.In conclusion, FSTL1is a novel candidate TSG in NPC; epigeneticly inactivating FSTL1in NPC cells could promote cell proliferation and inhibit the functions of macrophages, which in turn lead to the immuno evasion of NPC cells. |
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