Expression Profile Of CircRNAs And Biological Function Of CircARNTL2 In Tongue Squamous Cell Carcinoma

Background and purposes: With the rapid advancement of high-throughput sequencing,a number of circ RNAs have been discovered.It has been reported that circ RNAs play important roles in variety of human tumors.However,few circ RNAs have been found in the tumorigeneisis of Tongue Squamous Cell Carcinoma(TSCC).The goal of this study is to characterize an expression profiling of circ RNAs and investigate the biological behavior effects of circ ARNTL2 in TSCC.Materials and methods: circ RNAs differential expression in 6 pairs of TSCC tissues and paracancerous tissues was analyzed by high-throughput sequencing and bioinformatics technology.Four genes that were up-regulated significantly in cancer tissues and included in circ Base database were selected and verified by RT-q PCR in20 pairs of TSCC samples.Subsequently,RT-q PCR was used to explore the expression of circ ARNTL2 in TSCC cell lines.The circ ARNTL2 characteristics were detected by Random 6 mers primers and Oligo d T primers reverse transcription assay,RNase R assay,Sanger sequencing.RNA nucleus and cytoplasm isolation assay detected the expression of circ ARNTL2 in the nucleus and cytoplasm.The correlation between circ ARNTL2 expression and clinicopathological factors in TSCC patients was analyzed with chi-square test.The receiver operating curve(ROC)was used to evaluate the value of circ ARNTL2 as a tumor biomarker in the diagnosis of TSCC.si RNAs were designed for transient transfection in CAL27 and SCC9,while the most remarkable knock-down si RNA was chosen to functional experiments in vitro.After circ ARNTL2 interference,CCK8 assay was used to detect the effect the proliferation ability.The ability to clone was examined by plate clone formation experiment.The cell viability was investigated by living and dead cells fluorescence staining.Wound healing assay and Transwell migration assay were explored to the TSCC cell migration ability.Transwell invasion assay was detected cell invasion ability.Cell apoptosis and cell cycle were tested by flow cytometry.Western Blot technique was used to examine the expression of related proteins.Results: A total of 276 circ RNAs(fold change≥2,p<0.05)were differentially expressed by high-throughput sequencing results,including 68 up-regulated and 208down-regulated expression.RT-q PCR indicated that circ ARNTL2 was most significantly up-regulated in TSCC tissues(p=0.0009)and higher in CAL27 and SCC9 cells(p<0.05)compared with paracancerous tissues.Furthermore,Random 6mers primers and Oligo d T primers reverse transcription assay found that expression of circ ARNTL2 was higher with Random 6 mers primers than Oligo d T primers(p<0.001).RNase R assay denoted that circ ARNTL2 was resistant to RNase R digestion.Sanger sequencing confirmed the backsplice sequence of circ ARNTL2.RNA nucleus and cytoplasm isolation assay verified that the higher expression of circ ARNTL2 existed in cytoplasm rather than nucleus.Chi-square test showed that circ ARNTL2 expression was correlated with TNM stage(p=0.015)and lymph node metastasis(p=0.014).ROC curve analysis suggested that AUC was 0.8115,sensitivity was 72.73%,specificity was 88.64%.After Silencing circ ATNTL2 the abilities of proliferation,migration,and invasion were declined(p<0.05),while the rate of cell apoptosis was enhanced(p<0.05),and S phase was arrested(p<0.05)in CAL27 and SCC9.What’s more,Interference with circ ARNTL2 increased the expression of cleaved caspase-3 protein(p<0.05).In the meantime,the expression of E-cadherin protein increased and the expression of N-cadherin decreased(p<0.05).Conclusion: In this study,the expression profile of circ RNAs in TSCC was analyzed.The up-regulation of circ ARNTL2 in TSCC tissues and cell lines was discovered.The circular characteristics and location of circ ARNTL2 were confirmed.as a potential biological marker,circ ARNTL2 may be involved in TNM stage and lymph node metastasis and had a certain diagnosis value.circ ARNTL2 promoted the proliferation,migration and invasion,inhibited apoptosis and affected cell cycle.Therefore,we supposed that circ ARNTL2 plays an oncogenic role in the development of TSCC.