Identification Of Long Non-coding RNA Expression Profile And The Effect And Molecular Mechanism Of FOXCUT In Nasopahryngeal Carcinoma

Background:Nasopharyngeal carcinoma（NPC）is a type of head and neck carcinoma that is very common in Southern China and Southeast Asia.Although most primary NPC patients are sensitive to radiotherapy and chemotherapy,there remain a significant percentage of NPC recurrence and distant metastasis.The poor prognosis of patientis with NPC is largely the result of the tumor recurrence and distant metastasis after therapy.Moreover,the sensitivity and specificity of the detection methods used are not enough.Therefore,it is necessary to explore the molecular basis of NPC and to look for promising diagnostic markers and therapeutic strategies.Long noncoding RNAs（lncRNAs）are encoded by a vast less explored region of the human genome,has the specificity of tissue and cells,and may hold missing drivers of cancer and have gained attention recently as a potentially crucial layer of cancer cell regulation.Although many lncRNAs are expressed at low levels in cells and tissues,lncRNAs play important roles in various biological processes.According to existing reports,very few studies have been conducted on the expression profile of IncRNAs,and on the function and potential mechanism of lncRNAs in NPC.Only several lncRNAs associated with NPC,such as MALAT-1,HOTAIR and H19,have been reported.FOXCUT is a novel IncRNA（NR₁25804.1）located on chromosome 6p25 and upstream of the FOXC1 promoter.It is closely associated with breast cancer and oral squamous cell carcinoma,affecting their occurrence and development,but its effect in NPC has never been studied.Forkhead box C1（FOXC1）is a member of the Fox transcription-factor family and is overexpressed in multiple malignant tumors and involved in both tumor development and healthy biological processes.Recent studies have shown that FOXC1 can regulate cell proliferation and metastasis by inducing epithelial-mesenchymal transition（EMT）in NPC.However,the function of the FOXCUT-FOXC1 pair in NPC has not been described.Objective:To uncover complete long non-coding RNA expression profiles in NPC and chronic nasopharyngitis（CNP）tissues by high throughput sequencing and and predict target gene of differentially expressed lncRNAs.Moreover,to preliminarily study the expression of lncRNA FOXCUT in NPC cells and tissue,and analyze its functions in NPC cells.Explore the regulation relationship between lncRNAs and mRNAs,and to discuss the correlaion of lncRNA FOXCUT with EMT related molecular markers.Method:（1）High throughput sequencing technology was used to detect the levels of lncRNAs in four patients with NPC and four with CNP.（2）Real-time PCR was performed to validate the result of sequencing.（3）Antisanse and up/downstream strategies were used to predict of lncRNAs and mRNAs associations.（4）ROC curve was constructed to evaluated the diagnostic value of the lncRNAs indentified.（5）Employing real time PCR method to detect the expression levels of lncRNA FOXCUT in NPC cells and tissues.（6）A chi-square test or Fisher’s exact test was used to analyze statistical differences in clinical characteristics.（7）Human NPC cells were transfected into siRNA by lipofectamineTM3000.CCK-8,wound-healing assay,xCELLigence system were used to detect the reversal effect of overexpression of lncRNA FOXCUT in NPC cells.（8）Employing real-time PCR method to detect the expression levels of mRNA FOXC1 in NPC cells and tissues.（9）Real-time PCR and Western blot methods were employed to detect the regulation relationship between lncRNA FOXCUT and mRNA FOXC1.（10）NPC cell 5-8F was transfected into siRNA FOXCUT by lipofectamineTM 3000,real time PCR and western blot methods were used to observe the EMT related molecular markers expression.Results:（1）According to the gene expression profiles we obtained,a total of 296 lncRNAs and 2589 mRNAs were differentially expressed between NPC and CNP tissues.（2）The relative fold-change in their expression was consistent with the sequencing results.（3）Based on the antisense prediction method,49 lncRNAs were found to be complementarily paired with mRNAs.Based on up/downstream analysis,33 lncRNAs were found to be complementarily paired with mRNAs.Long non-coding RNA FOXCUT（ENST00000628509）and mRNA FOXC1（ENST00000380874）have upstream pairing.（4）The area under the ROC curve for lncRNAs ENST00000418244,ENST00000505700,ENST00000480284,ENST00000424518,ENST00000628509 was 0.831,0.764,0.620,0.827 and 0.873,respectively,indicating that they have potential diagnostic value for NPC.（5）Real time PCR verified that lncRNA FOXCUT are overexpressed in both NPC cells and biopsied NPC tissues.（P<0.05）（6）Upregulated FOXCUT expression was associated with advanced lymph node classification（P=0.016）and distant metastasis（P=0.017）,whereas no significant correlation between FOXCUT expression and age or sex.（7）The proliferation and migration of NPC 5-8F and CNE2 cells were inhibited after down-regulation of IncRNA FOXCUT expression.（p<0.05）（8）Real time PCR verified that IncRNA FOXC1are overexpressed in both NPC cells and biopsied NPC tissues.（p<0.05）（9）Our results showed that FOXC1 expression was inhibited by FOXC1 siRNAs.Western blot analysis shows that FOXC1 protein expression to be reduced following si-FOXC1.Notably,transfection with FOXC1 siRNAs had no effect on FOXCUT expression levels based on RNA expression analysis.FOXCUT expression was attenuated by～60%following transfection with FOXCUT siRNA,with co-suppression of FOXC1 mRNA levels by-50%.（10）Downregulated the expression of FOXCUT in 5-8F cells of NPC could decrease the expression of EMT related molecular markers β-catenin、N-cadherin and VEGF-A,but had no effect on the expression of E-cadherin.Conclusions:There are IncRNAs molecules expressed differently in NPC and CNP tissues.High throughput sequencing technology is an effective method to screen the IncRNAs in NPC tissues.Our study indicated that IncRNA FOXCUT may play an important role in promoting the proliferation and migration of NPC cells.Our results also suggested that FOXC1 mRNA levels are potentially regulated by the IncRNA FOXCUT.Meanwhile,there may be a correlation between the expression of IncRNA FOXCUT and mesenchymal markers.Inhibition of FOXCUT expression can partially reverse the EMT pathway in NPC.Moreover,IncRNA FOXCUT is likely to be used as a target of NPC diagnostic and therapy.