| Objectives:Cancer is common in oral malignant tumor in China,and the ratio of tongue squamous cell carcinoma is the highest.In recent years,with the improvement of people’s living standard,the incidence of human tongue cancer is increasing year by year,and the age of the disease is gradually decreasing.The most effective way to treat tongue squamous cell carcinoma is still based on surgical treatment,radiation therapy and comprehensive chemotherapy.The pathogenesis and prognostic factors of tongue squamous cell carcinoma are not yet very clear.Tongue tissue is rich in lymphatic circulation and blood circulation,and complex anatomical structure in oral and maxillofacial region.Tongue squamous cell carcinoma often occurs at the early stage of cervical lymph node metastasis,which increases the difficulty of treatment.The 5 year survival rate of tongue cancer patients wandered about 50%,while the 1 year survival rate of recurrent patients decreased to 60%(stage II)and 32%(stage Ⅳ).The factors that influence the prognosis of tongue squamous cell carcinoma are age,sex,tissue differentiation,invasion depth,TNM classification,treatment methods,regional recurrence and lymph node micrometastasis.However,studies have shown that the clinical factors can not fully explain the biological behavior of tongue squamous cell carcinoma.It is still necessary to further explore the mechanism of tongue cancer development and prognosis from molecular level.Therefore,fully studying the mechanism of growth and metastasis of tongue squamous cell carcinoma and finding molecular markers related to its diagnosis,treatment and prognosis are of great clinical significance for the treatment of tongue squamous cell carcinoma.The purpose of this study is to provide theoretical basis for the study of Hu R as a prognostic marker of tongue cancer.Through further analysis of the transcriptome sequencing of Hu R expression,we sought to identify the key molecules what Hu R regulate and play a role in the development and progression of malignant tumors.We provided theoretical support for Hu R as a potential target for molecular approaches to tumor therapy.To detect transcriptome analysis of squamous cell carcinoma tissues and adjacent normal tissues of squamous cell carcinoma of tongue with different levels of Hu R by transcriptome sequencing technique.To compare the differentially expressed genes and transcription structure variants between tongue squamous cell carcinoma tissues and normal tissues of patients with high Hu R expression and low expression.By molecular level validation,genes specifically altered in patients with high expression of Hu R were identified.And functional annotation and biological pathway analysis of genes specific for Hu R involvement were further analyzed.Methods:(1)Mmunohistochemical staining(S-P)was used to detect the expression of Hu R,COX-2 and VEGF-C in 83 cases of squamous cell carcinoma of tongue and the adjacent normal tissues,and the correlation between the three and their clinicopathological features were analyzed.(2)By using Kaplan-Meier method and Cox proportional hazards regression model,to analysis of the relationship between the expression and operation in tongue cancer Hu R,COX-2,VEGF-C after survival,progression free survival,and to explore the prognostic value of three for squamous cell carcinoma of the tongue.(3)17 cases of squamous cell carcinoma of tongue proved by pathology in Affiliated Hospital of Jining Medical College and Jining First People’s Hospital were collected from October 2016 to April 2017.After the repair,the slice and the HE staining,all the tumor specimens were evaluated blindly by two pathologists.The type of tissue was tongue squamous cell carcinoma and the tumor cells in the tissue were not less than 70%,and 3 cases with strong and weak Hu R expression were screened out.(4)Trizol method was used to extract the total RNA of tumor tissues and paracancerous tissues of patients with squamous cell carcinoma of the tongue,and the Agilent 2100 analyzer was screened.The quality control analysis showed that RIN reached more than 7.0,and there was basically no degradation and integrity of RNA.(5)Illumina(Hi Seq 2500/4000)sequencing: Completed by Huada gene science and Technology Co.,Ltd.(6)Data analysis: 1)Using the filter software SOAPnuke,the raw data(raw reads)obtained from the sequencing was filtered.Clean reads was filtered by filtering reads with low quality,joint contamination,and unknown base N content.2)Applying HISAT software,clean reads was compared to the reference genome.3)Detection of new transcripts prediction,SPINDEL detection and differential splicing gene.4)According to the requirements of the samples,the differentially expressed genes were detected,and the differentially expressed genes were analyzed by cluster analysis and functional enrichment analysis.Results:(1)83 cases of tongue squamous cell carcinoma,positive rate of Hu R cytoplasmic expression,positive rate of Hu R nuclear expression,the positive rate of COX-2 cytoplasmic expression and positive rate of VEGF-C nuclear expression was 67.47%(56/83),74.70%(62/83),67.47%(56/83)and 62.65%(52/83),and adjacent normal tissue was 0% 13.3%(11/83)and 45.78%(38/83),10.84%(9/83).The differences were statistically significant.Further statistical analysis suggested that the positive expression of Hu R and COX-2 in cytoplasm and the positive expression of VEGF-C in nucleus were significantly correlated with lymph node metastasis and pathological staging in patients with squamous cell carcinoma(P<0.05),and the strong positive expression of nuclear VEGF-C was closely related to the differentiation degree of tongue squamous cell tumor(P<0.05).However,there was no correlation between the expression of nuclear Hu R and the clinicopathological features(age,sex,tumor size,tumor differentiation,lymph node metastasis,postoperative pathological staging)in patients with squamous cell carcinoma of the tongue.(2)There was a positive correlation between the levels of cytosolic Hu R,COX-2 and VEGF-C in squamous cell carcinoma of tongue(P<0.05).(3)The positive rate of Hu R and COX-2 and the 5-year survival rate and disease-free survival of VEGF-C positive group were significantly lower than that of the control group(P<0.05).The 5-year survival rate and disease-free survival of the positive expression group were significantly lower than those of the three negative control group(P<0.05).(4)Multivariate analysis of the Cox regression model showed that the independent prognostic factors of tongue squamous cell carcinoma included the expression of cytoplasmic Hu R,the pathological staging and lymph node metastasis(P<0.05).The risk of death in the cytoplasm Hu R positive group was 6.7 times that in the negative expression group.And the risk of death in patients with lymph node metastasis was 3.2 times greater than that in patients without lymph node metastasis.(5)Sequenced general results: The results showed that Q30 was higher than 85%,and the average rate of each sample was 88.70%.Meanwhile,the average ratio between samples showed that the data were comparable.(6)New transcript prediction: The transcriptome was reconstructed using String Tie for each sample,and then cuffcompare was used to compare the reconstructed transcript with the reference annotation information to obtain a new transcript.To prediction coding potential for new transcripts using CPC software.Finally,a total of 44876 new transcripts were detected,of which 35421 were predicted to encode transcripts,and 9455 were non-coding transcripts.(7)SNP and INDEL detection: A large number of SNP were found in 10 tongue squamous cell specimens,and these SNP were distributed in different regions.The incidence of prior conversion was always significantly higher than that of several other variants.The distribution of exons and introns in SNP and INDEL loci accounted for about 90% of the total,but not more than 10% in the intergenic spacer region.(8)Differential splicing gene detection: Among the specimens,the number of variable splicing numbers from many to less was SE,RI,A3 SS,A5SS,MXE,respectively,in which SE accounts for the largest proportion.The tumor tissues and paracancerous tissues of Hu R were compared.The corresponding DSG Gene Ontology function accounts for the largest proportion in biological processes,secondly,it was cell component and molecular function.Similar trends were also seen in other samples and samples.(9)Differentially expressed gene detection: A large number of differentially expressed genes were found between the tumor tissue and the adjacent tissue of a single sample.Among the three groups with high expression of Hu R,6426(52.72%),6805(71.71%),and 2739(57.55%)genes were up-regulated,which exceeded the percentage of down regulated genes.But,among the two groups with low expression of Hu R,2360(48.30%)and 6004(50.51%)genes were up-regulated,and the proportion of down regulated genes was basically the same.(10)Differentially expressed genes,GO,functional analysis: Among the two differentially expressed genes involved in 162 biological processes,16,16,13 and 13 participate in cell transformation,single organism process,biological regulation and metabolic processed respectively.Among 113 differentially expressed genes involved in cell components,16,16,12,and 12 were involved in cell,cell parts,cell membranes,organelles,respectively.Among 38 differentially expressed genes involved in molecular function,17,10,3 and 3 participate in helix,catalytic activity,signal transduction activity and transport activity respectively.(11)Differentially expressed genes,Pathway functional analysis:KEGG biological pathway classification and enrichment analysis were carried out according to the results of differential gene detection.Of the 34463 total differentially expressed genes,26 genes were involved in the differential expression of Lo_Tu and Hi_Tu groups.These differentially expressed genes were involved in signal transduction pathways,organophosphate and phosphate metabolism pathways.The differentially expressed genes in the Lo_Tu and Hi_Tu groups were involved in cell metabolism and death,cell transport and metabolism.Most of enrichment pathway differential genes were still down-regulation gene.Conclusions:(1)The expression of Hu R and COX-2 and the strong expression of VEGF-C in tongue squamous cell carcinoma tissues are closely related with tumor progression and lymph node metastasis.(2)The expression of Hu R in cytoplasm is positively correlated with the expression of COX-2 and VEGF-C,and there is a correlation between the expression of the three proteins.(3)The expression of cytosolic Hu R is an independent prognostic factors in patients with squamous cell carcinoma of the tongue.(4)It is preliminarily speculated that Hu R can be used as a potential molecular target for anti-tumor therapy.(5)Overexpression of Hu R results in altered gene expression in patients.Compared with tumor tissues adjacent to the tumor and low expression of Hu R,downregulation of genes is more than up-regulated in tumor associated genes.GO enrichment analysis and KEGG analysis of differentially expressed genes show that these genes are involved in specific functions and signaling pathways,suggesting that Hu R may play a unique pathogenic role in the development of tongue cancer.(6)Many genes expressed in patients with high expression of Hu R include genes involved in apoptosis,signal transduction pathways,cellular immunity,and so on.(7)Transcriptome based high throughput sequencing analysis can identify differentially expressed genes in patients at different levels of Hu R expression.All these provide experimental basis and theoretical basis for the further study of the pathogenesis and development of Hu R in tongue cancer. |
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