Primary Study Of The Perineural Invasion Related Genes Of Salivary Adenoid Cystic Carcinoma By RNA-Seq

Perineural invasion(PNI)is a notorious biological characteristic of SACC that distinguishes it with other head and neck tumors.In recent years,many scholars have emphasized that we should study all the tumor gene interactions at the whole genome level so as to have a comprehensive and systematic understanding of tumors.Traditional gene chips rely on the genome information that we already know,but it is also the greatest limitation of the technology.RNA-seq can reveal unknown transcripts,gene fusion,and genetic polymorphisms,and the chips can only detect clear known targets.In the case of deep sequencing,RNA-seq is more effective than gene chip in the detection of high and low richness transcripts.RNA-seq has been widely applied to differential gene expression analysis,new transcripts discovery,alternative splicing studies,etc.,because of its obvious advantages over traditional sequencing methods.And with the development of RNA-seq and the reduction of the cost of sequencing,the application of this technology is becoming more and more extensive.In order to investigate the perineural invasion(PNI)related genes,we use RNA-Seq technique to analyse the level of gene expression in single culture group and co-culture group of salivary adenoid cystic carcinoma(SACC)and Schwann cells(SCs).According to the PNI related gene results,our preliminary experiments and literature review,we think that the NT-3/TrkC axis may regulate the tumor-nerve interactions,and mediate the migration of SCs towards SACC cells to promote the occurrence of PNI.Therefore,we used ELISA,RT-PCR,Western immunoblot,wound healing test,transwell perineural invasion assay and three-dimensional(3D)co-culture assay to demonstrate that NT-3/TrkC signaling pathway plays an important role in PNI of SACC.We also conducted a clinical Immunohistochemical staining study of SACC and followed up the patients to explore the biological mechanism of PNI in SACC.The first part Experiment PURPOSE: To investigate the perineural invasion(PNI)related genes,the level of gene expression in single culture group and co-culture group of salivary adenoid cystic carcinoma and Schwann cells were analyzed by RNA-Seq technique.METHODS: Two groups of salivary adenoid cystic carcinoma cells were analyzed for gene expression by co-culture system with rat Schwann cells.And two groups of Schwann cells were analyzed for gene expression by co-culture system with SACC cells.Then,cluster analysis,GO(gene ontology)function enrichment analysis,KEGG(Kyoto encyclopedia of genes and genomes)pathway enrichment analysis were used to analyze gene function,and qRT-PCR was used to verify the 6 key-genes.Differential expression analysis of two conditions was performed using the edgeR package(3.12.1).P-value of 0.05 and log2(Fold change)of 1 were set as the threshold for significantly differential expression.RESULTS: The results showed that a total of 395 genes were differentially expressed in two SACC groups(p?0.05,|log FC|?1.0),135 genes were up-regulated(34.2%)and 260 genes were down regulated(65.8%);1239genes were differentially expressed in two Schwann cells groups(p?0.05,|log FC|?1.0),757 genes were up-regulated(61%)and 482 genes were down regulated(39%).GO enrichment analysis results showed that these PNI related DEGs involved in angiogenesis,extracellular matrix,cell proliferation,apoptosis,epithelial morphogenesis,cell migrationand cell motility;KEGG pathway enrichment analysis displayed that it participated in histidine metabolism,interactions of tumor necrosis factor,chemotactic factor,cytokine receptor,PI3K-Akt signaling pathway,focal adhesion and ECM-receptor interaction.qRT-PCR was used to validate 6 key-genes,and the results were consistent with RNA-Seq.CONCLUSIONS: Differentially expressed genes related to perineural invasion(PNI)of SACC were obtained,which provides important experimental basis for elucidating the mechanism of PNI of SACC,and also provides a new strategy and target for treatment of perineural invasion.The second part Experiment PURPOSE: The purpose of the present study was to investigate whether the NT-3/TrkC axis mediated the interaction between the salivary adenoid cystic carcinoma(SACC)cells and the Schwann cells(SCs),and whether SCs was involved in the early perineural invasion(PNI)process actively via the NT-3/TrkC axis.METHODS: The role of the NT-3/TrkC axis in the process of tumor-nerve interactions was measured under the conditions of NT-3 stimulating and TrkC blocking in SACC cell line.ELISA,RT-PCR and Western blotting were used to detect the expression of NT-3 and TrkC in tumor-nerve interaction.Wound healing test,invasion and three-dimensional(3D)co-culture assays were performed to evaluate the motor ability characteristics between the interaction of SACC-83 cells and SCs.SPSS17.0 software package was used to analyze the results of the experiment.RESULTS: In the co-cultured SACC-83 cells,the expression of NT-3 was rised markedly when compared with the solely cultured SACC cells.In the co-cultured SCs,the expression of TrkC was also increased.Through the tumor-nerve interaction,the migration and invasion ability of co-cultured SACC-83 cells and SCs increased significantly.Moreover,before the tumor attacked the nerve,the NT-3/TrkC axis firstly mediated the migration of SCs towards SACC-83 cells.Under the stimulation of NT-3,the motor ability of SACC-83 cells and SCs improved and the apoptosis of SACC-83 cells was slightly inhibited.In addition,blocking TrkC by K252 a could markedly inhibit these effects.CONCLUSIONS: Our results indicated that the NT-3/TrkC axis regulated the tumor-nerve interactions,and mediated the migration of SCs towards SACC cells to promote the occurrence of PNI.Blocking the NT-3/TrkC axis might be an effective molecule-targeted treatment for anti-PNI therapy for SACC.The third part Experiment PURPOSE: To analyze the expression of NT-3 and TrkC in SACC specimen,and to study the correlation between the expression of NT-3/TrkC and PNI,and explore the biological mechanism related to the prognosis of SACC patients.METHODS: Immunohistochemistry was applied to detect the expression of NT-3 and TrkC in 78 primary SACC specimens and 25 normal parotid tissue specimens.Through consulting a large number of cases material and telephone follow-up,and then analyzed,contrast,and summarized.SPSS17.0 software package was used to analyze the results of the experiment.RESULTS: The elevated expression of NT-3(88.5%)and TrkC(92.3%)was detected in SACC specimens,and was significantly correlated with PNI,distant metastasis and clinical stages(P<0.05).Additionally,high expression of NT-3 was significantly associated with poor prognosis in patients with SACC(P<0.05).CONCLUSIONS: The expression of NT-3 and TrkC in SACC tissues is positively correlated with PNI,distant metastasis and clinical staging.The expression level of PNI related NT-3 molecules is negatively correlated with the prognosis of SACC patients.