Screening And Bioinformatics Analysis Of CircRNA Associated With Cervical Lymph Node Metastasis In Laryngeal Squamous Cell Carcinoma

Objective To analyze the altered expression profiles of circular RNA(circRNA)and messenger RNA(mRNA)in laryngeal squamous cell carcinoma(LSCC)samples with positive cervical lymph node metastasis and metastatic lymph node samples,then construct a circRNA-miRNA-mRNA network to comprehensively explore the regulatory mechanism of circRNA in cervical lymph node metastasis of LSCC.Methods 1.Tumor samples and cervical lymph node samples were extracted from LSCC patients with cervical lymph node metastasis(n=2)and LSCC patients without cervical lymph node metastasis(n=1).Total RNA was extracted from different specimens and checked for quality.2.With log2 |fold change|>1,adjusted P<0.05 as the standard,ceRNA chip high-throughput sequencing technology was applied to analyze the altered expression profiles of circRNA and mRNA in the LSCC samples with positive cervical lymph node metastasis and metastatic lymph node samples.3.Through seed sequence matching,circRNA-miRNA regulatory pairs and mRNA-miRNA regulatory pairs were established to obtain intersection miRNAs,and correlation coefficient analysis was performed on circRNAs and mRNAs in the intersection set to construct the circRNA-miRNA-mRNA network based on the ceRNA principle.4.The DAVAID software was used to analyze the GO and KEGG pathway enrichment of target genes in the ceRNA regulatory network.Results 1.CeRNA chip analysis showed that there were 8032 circRNAs and 3205 mRNAs significantly differentially expressed in the LSCC samples with cervical lymph node metastasis,of which 3603 circRNAs and 1404 mRNAs were up-regulated,and 4429 circRNAs and 1801 mRNAs were down-regulated.And a total of 4280 circRNAs and 1536 mRNAs were significantly differentially expressed in the metastatic lymph node samples,of which 922 circRNAs and 636 mRNAs that were up-regulated,and 3358 circRNAs and 900 mRNAs that were down-regulated.2.The ceRNA regulatory network of the LSCC tissues with metastatic lymph node contains 250 circRNA-miRNA regulatory pairs and 225 mRNA-miRNA regulatory pairs,including 159 circRNAs(29 up-regulated circRNAs and 130 down-regulated circRNAs),179 mRNAs(21 up-regulated mRNAs and 158 down-regulated mRNAs);the ceRNA regulatory network of metastatic lymph node tissues included 115 circRNA-miRNA regulatory pairs and 106 mRNA-miRNA regulatory pairs,containing 60 circRNAs(all of them were down-regulated),78 mRNAs(1 up-regulated mRNA,77 down-regulated mRNAs).3.A total of 8 circRNAs co-existed in the two sets of ceRNA network(including hsa_circ₀024329,hsa_circ₀061347,hsa_circ₀074574,hsa_circ₀024328,hsa_c irc₀074576,hsa_circ₀068500,hsa_circ₀024325,hsa_circ₀051131),and their expressions were all significantly down-regulated.4.The GO enrichment analysis showed that the target genes were mainly enriched in exosomes and cell membranes,while KEGG pathways enrichment analysis indicated that they may be involved in the regulation of cervical lymph node metastasis of LSCC through calcium ion signaling pathway.Conclusion The expression profiles of circRNA were significantly different in the LSCC samples with metastatic lymph node and metastatic lymph node samples,which may be involved in cervical lymph node metastasis of LSCC patients through complex signaling pathways and biological processes,and it is expected to be a useful biomarker for evaluating potential lymph node metastasis and formulating the molecular targeted therapy.