| Objective:Nasopharyngeal carcinoma is one of common head and neck cancers,which is insidious onset.Most patients are found late,and the prognosis is not good.Treatment of nasopharyngeal cancer is mainly by radiotherapy.Although there is some progress in treatment,for 5-year survival rate of the patients,it is not significantly improved.So far,the pathogenesis of nasopharyngeal carcinoma is not clear enough.Long non-coding RNA has an important adjustment function to life activity.We focus on the study of the relationship between long non-coding PCAT7 and the nasopharyngeal carcinoma.To study the expression changes of PCAT7 in nasopharyngeal carcinoma,and to clarify the effects of PCAT7 on the biological activity of nasopharyngeal carcinoma cells and explore the potential molecular mechanisms.Methods:The first part:First of all,the content of the PCAT7 was detected through the quantitative reverse transcription polymerase chain reaction(qRT-PCR)in tissues of the experimental group and the control group,which collected from 50 patients with NPC and its adjacent normal tissues respectively.After the research in human tissue,further study at a cellular level was then carried out.Cultivating NPC cell lines CNE-2,HNE-1,C666-1,NONE-1,SUNE-1,CNE-1 as the experimental group,human nasopharyngeal epithelial cell NP-69 as the non cancerous cell control group,through the qRT-PCR experiments,to prove expression changes of the PCAT7 at a cellular level in the two groups.Two cell lines,CNE-1 and SUNE-1,which changed more obvious were selected for the subsequent experiments.Three sequences targeted PCAT7 was designed and transfected SUNE-1 and CNE-1 cells.qRT-PCR detected the interference efficiency of siRNA.The interference efficiency of siRNA-1 was most obvious and selected for the subsequent experiments.Cell proliferation ability was estimated in vitro by CCK-8 assay and colony-formation assay.Subsequently,we construct animal model of NPC.The tumor size of nude mouse with or without PCAT7 interference was monitored once four days to explore the effect of PCAT7 on tumor growth.The second part:Bioinformatics software miRDB was used to predict the potential target miRNAs of PCAT7 and then though biotin-avidin pull down assay to further verification.The miRNA interaction with PCAT7 was selected for further study.qRT-PCR detected the expression changes of miRNA after the silencing of IncRNA PCAT7.The regulation relationship between PCAT7 and miRNA was confermed by the dual-luciferase report assay.CCK-8 assay examined the effect of PCAT7 and miRNA on NPC cells proliferation.In order to further explore the role of miRNA in nasopharyngeal carcinoma cells proliferation,we predicted the potential target genes of miRNA using bioinformatics software TargetScan,and then validated by dual-luciferase report assay.qRT-PCR and IHC detected the expression of miRNA and its target gene in 50 cases of nasopharyngeal carcinoma.Western blot detected the effect of PCAT7 and miRNA on target gene.CCK-8 assay examined the effect of PCAT7 and miRNA on NPC cells proliferation.Results:The first part:PCAT7 levels were determined by qRT-PCR and found to be higher expressed in NPC tissues compared with corresponding non cancerous tissues(N = 50).PCAT7 was also found to be up-regulated in six NPC cell lines,including CNE-2,HNE-1,C666-1,HONE-1,CNE-1 and SUNE-1 cells,compared with a normal human oral keratinocytes cell line NP69),and PCAT7 expression was much higher in CNE-1 and SUNE-1 cells than other NPC cells CNE-2,HNE-1,C666-1 and HONE-1),thus,we performed the following experiments in CNE-1 and SUNE-1 cells.Additionally,we also discovered that higher PCAT7 expression implied a worse survival rate in NPC patients.Next,we explored the role of PCAT7 on NPC cell growth.CNE-1 and SUNE-1 cells were transfected withsiRNA PCAT7.The qRT-PCR results indicated that sh-PCAT7-1 was the most effective one to knock down PCAT7,we then chose it for the representation of sh-PCAT7.The CCK8 results showed that sh-PCAT7 suppressed CNE-1 and SUNE-1 cells proliferation and colony forming.Subsequently,we explored the effect of PCAT7 silencing on tumor growth in vivo.The results of animal models of nasopharyngeal carcinoma revealed that the tumor size in the sh-PCAT7 group was smaller compared with sh-NC group in 36 days after injection.The tumor weight of sh-PCAT7 group was also significantly less than that of in sh-NC group.Additionally,immunohistochemistry(IHC)analysis identified that the tumors isolated from CNE-1/sh-PCAT7 and SUNE-1/sh-PCAT7 cells revealed less Ki-67 staining areas than those of from control cells.In summary,we confirmed that silencing PCAT7 could repress tumor growth in vitro and vivo.The second part:we predicted the miRNAs that might interact with PCAT7 using miRDB.Biotin-avidin pull-down assay indicated PCAT7 could pull down miR-134-5p,but can't markedly pull down other potential targeted miRNAs.Hence,miR-134-5p was selected for further study.qRT-PCR detected the expression of miR-134-5p,our result demonstrated that miR-134-5p was lowly expressed in NPC tissues(n=50).The expression of PCAT7 and miR-134-5p showed a significant negative correlation as analyzed by Pearson correlation analysis.The dual-luciferase assay indicated that miR-134-5p could directly bind with PCAT7 at the special recognition sites.The results of CCK-8 revealed miR-134-5p inhibited NPC cells growth,while when co-transfection of miR-134-5p and pcDNA3.1-PCAT7,the growth-inhibitory role of miR-134-5p in NPC was reversed,and the growth expedited role of PCAT7 was also hampered by miR-134-5p.These results indicated that PCAT7 promoted NPC cell growth via acting as a ceRNA for miR-134-5p.To explore the effects of miR-134-5p in the mechanisms and functions of PCAT7,we selected direct target genes for miR-134-5p using TargetScan and found that ELF2 may be the effective one.Our result suggested miR-134-5p could inhibit the protein expression of ELF2.In addition,dual-luciferase assay confirmed that ELF2 was a direct target gene of miR-134-5p.The results of IHC showed ELF2 was down-regulated in NPC tissues.The over-expression of ELF2 could promote the NPC cells proliferation.While,the growth expedited role of ELF2 was hampered by miR-134-5p.Our results demonstrated miR-134-5p functioned as a tumor suppressor on NPC by directly targeting ELF2.' Further,our data also imply that PCAT7's oncogenic roles are largely in part through spongeing miR-134-5p,and then activating ELF2.Conclusion:In summary,our results revealed that PCAT7 expression was up-regulated in NPC tissues and cells,and its over-expression was closed associated with poor prognosis and might be a negative prognostic factor for NPC cancer patients.Knockdown of PCAT7 exerted tumor-suppressive roles via repressing NPC cell proliferation in vitro and tumor growth in vivo.Furthermore,PCAT7-mediated oncogenic functions was partly through acting as a ceRNA for miR-134-5p,releasing the miR-134-5p's inhibitory role for its target gene ELF2.These results suggested that PCAT7 might be acted as oncogene,a potential diagnostic marker,and a novel therapeutic target for NPC. |
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