Study On TNC Expression And Its Function And Molecular Mechanism In Nasopharyngeal Carcinoma

Part I.The expression of TNC in nasopharyngeal carcinoma and its clinical significanceObjective To explore the expression level of tenascin-C(TNC)in nasopharyngeal carcinoma(NPC)and its correlation with the basic clinical and pathological characteristics of patients by bioinformatics analysis and immunohistochemical(IHC)staining.Methods The whole-genome expression chips related to NPC tissue from the Gene Expression Omnibus(GEO)database were screened.Then,the GEO2 R web tool was used to analyze the difference in TNC messenger RNA(m RNA)levels between NPC tissues and normal control tissues.What’s more,one NPC tissue chip was purchased to detect the expression distribution of TNC in NPC tissues and normal control tissues by IHC staining.At last,the relationship between TNC expression and patient’s age,gender,and TNM stage was analyzed with TNC staining score.Results The datasets GSE53819(including the whole genome expression profile of 18 cases of NPC tissue and 18 cases of nasal mucosal epithelial tissue)and GSE103611(including the whole genome expression profile of 24 cases of locally advanced nasopharyngeal carcinoma(LA-NPC)with metastasis tissues and 24 cases of LA-NPC without metastasis tissues).T-test analysis of GSE53819 m RNA data showed that the TNC m RNA level in NPC tissues was significantly higher than that in normal control tissues(12.14±0.011 vs.11.66±0.056,p<0.05).The NPC tissue chip included 115 cases of NPC tissues and 35 cases of nasopharyngitis(NPG)tissues.None of them received any anti-tumor treatment before biopsy.Among them,NPC patients have complete clinical data,including age,gender,and Tumor-Node-Metastasis(TNM)stage.χ2 test analysis showed that the positive expression rate of TNC in NPC tissues was significantly higher than that in NPG tissues(86.0% vs.12.9%,p<0.001).T-test analysis of GSE103611 demonstrated that TNC m RNA level in LA-NPC with metastasis tissues was significantly higher than that in LA-NPC without metastasis tissues(1.97±0.381 vs.1.83±0.209,p<0.05).What’s more,the high TNC expression rate in stage T3-4 NPC tissues was significantly higher than that of stage T1-2 tissues(73.7% vs.41.0%,p<0.01);the high expression rate of TNC in stage N2-3 NPC tissues was significantly higher than that of stage N0 tissues(82.5% vs.38.5%,p<0.01);the high expression rate of TNC in tissues of M1 NPC tissues was significantly higher than that of stage M0 tissues(92.6% vs.53.4%,p<0.001).In addition,TNC expression level was not significantly related to patient’s gender(62.2% vs.60.3%,p=0.32)or age(patients with NPC were between 24 and 78 years old,with an average age of 49.47±7.32;<50 years old:64.7% vs.>50 years old: 60.9%,p=0.07).Conclusion TNC m RNA and protein expression levels in NPC tissues were significantly higher than those in normal control tissues.And TNC m RNA expression levels were positively correlated with NPC metastasis.The high expression of TNC was correlated with the TNM stage of NPC patients,which possibly be one of the poor prognostic factors of NPC patients.Part II.The influence of TNC on the malignant biological behaviors of nasopharyngeal carcinoma cell epithelial-mesenchymal transition(EMT)Objective To explore the relationship between tenascin-C(TNC)expression and the malignant biological behaviors of nasopharyngeal carcinoma(NPC)cells such as epithelial-mesenchymal transition(EMT),proliferation and invasion.Methods NPC cell line 5-8F with high metastasis and 6-10 B cell line with low metastasis were selected as the research objects;short hairpin RNA(sh RNA)targeting TNC gene was transduced into the cell by lentiviral vector to inhibit the expression of TNC gene and construct TNC expression knock-down stable cell lines.Cell Counting Kit-8(CCK-8)and 5-Ethynyl-2’-deoxyuridine(EDU)experiments were used to compare the in vitro proliferation ability of NPC cells;wound healing experiments and transwell chamber migration experiments were used to detect the in vitro migration ability of NPC cells;transwell chamber invasion experiments were used to detect the in vitro invasion ability of NPC cells;Quantitative Real-time PCR(q RT-PCR)and western blot experiments were used to detect the expression level of TNC in NPC cells and its relationship with the expression of EMT-related molecular markers;the nude mouse tumor model was used to detect the in vivo proliferation ability of NPC cells.Results TNC expression knock-down and negative control 5-8F and 6-10 B cell lines were constructed,which were named 5-8F-sh TNC,5-8F-sh NC,6-10B-sh TNC and6-10B-sh NC,respectively.CCK-8 and EDU experiments results showed that5-8F-sh TNC and 6-10B-sh TNC cell had weaker proliferation ability than sh NC cells;wound healing and transwell chamber migration experiments results demonstrated that 5-8F-sh TNC and 6-10B-sh TNC cell had lower migration ability;the transwell cell invasion experiment results showed that the invasion ability of 5-8F-sh TNC and6-10B-sh TNC cells was weakened.q RT-PCR experiment results showed that E-Cadherin m RNA levels which was marker of epithelial cell was increased in5-8F-sh TNC and 6-10B-sh TNC cells,and N-Cadherin,Vimentin and Slug m RNA levels which were markers of mesenchymal cell were decreased;Western blot experiments confirmed that TNC expression in 5-8F-sh TNC and 6-10B-sh TNC cells was significantly reduced,E-Cadherin expression level was increased,and the expression levels of N-Cadherin,Vimentin and Slug were decreased.Tumorformation experiments in nude mice confirmed that the tumor model constructed by5-8F-sh TNC and 6-10B-sh TNC cells had faster tumor growth rate and the final volume of the tumors were increased.Conclusion Down-regulation of TNC expression inhibited the in vitro proliferation,migration,invasion,EMT and in vivo proliferation of NPC cells.TNC may induce EMT in NPC cells.Part III.TNC regulates the epithelial-mesenchymal transition of nasopharyngeal carcinoma cells through the m TOR pathwayObjective To explore the potential molecular mechanism of tenascin-C(TNC)inducing epithelial-mesenchymal transition(EMT)of nasopharyngeal carcinoma(NPC)cells.Methods Weighted gene co-expression network analysis(WGCNA)was used to construct a TNC co-expressed gene network on the NPC messenger RNA(m RNA)data set GSE53819 and the core TNC co-expression gene was screened for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)by gene set enrichment analysis(GSEA).Western blot(WB)experiment was used to verify the signal pathways enriched in the GSEA results.After adding corresponding signal pathway agents to 5-8F-sh TNC and 6-10B-sh TNC cells,western blot experiment was used to test the effect of signal pathway agents on the pathway;Cell Counting Kit-8(CCK-8)experiment was used to detect the effect of signal pathway agents on 5-8FThe effect of sh TNC and 6-10B-sh TNC on the proliferation ability in vitro;the effect of signal pathway agents on the migration ability of 5-8F-sh TNC and 6-10B-sh TNC in vitro was detected by wound healing experiments;the signal pathway agents were detected by transwell cell invasion test Influence on the invasion ability of5-8F-sh TNC and 6-10B-sh TNC in vitro.Results The TNC co-expressed gene network was successfully constructed,among which the core gene set was enriched in functions and signal pathways such as EMT and m TOR.Western blot experiments confirmed that the expression levels of p-P70S6 K and p-AKT in 5-8F-sh TNC and 6-10B-sh TNC cells were significantly reduced.After adding m TOR signaling pathway agonists,CCK-8 experiments confirmed that the in vitro proliferation ability of 5-8F-sh TNC and 6-10B-sh TNC cells were enhanced,which was still lower than that of 5-8F-sh NC and 6-10B-sh NC cells;wound healing experiments confirmed that the in vitro migration ability of 5-8F-sh TNC and6-10B-sh TNC cells was enhanced,which was still lower than that of 5-8F-sh NC and6-10B-sh NC cells;transwell cell invasion experiments confirmed that the in vitro invasion ability of 5-8F-sh TNC and 6-10B-sh TNC cells was enhanced,which was still lower than that of 5-8F-sh NC and 6-10B-sh NC cells.Conclusion Down-regulation of TNC expression inhibited the m TOR pathway of NPC cells.TNC regulates the proliferation,invasion and metastasis and EMT process of NPC cells partially by activating the m TOR pathway.