CircRNA₁02411 Promotes Cell Invasion And Migration Through Let-7b-5p-mediated Regulation Of CCR7 In Head And Neck Squamous Cell Carcinoma

Objective: The mechanism on pathogenesis and metastasis of head and neck squamous cell carcinoma?HNSCC?remains unclear so far,and there is no effective treatment method for HNSCC.There have been more and more studies on gene alterations in the process of HNSCC,and new molecular diagnostic and therapeutic targets have been sought through further related gene research.Circular RNA?circ RNA?is a class of non-coding RNA?nc RNA?with covalently closed circular structures that shows great potential in gene regulation.Many studies have demonstrated that circ RNA plays a crucial role in the occurrence and development of cancer by regulating post-transcriptional gene expression levels.However,the function and regulation mechanism of circ RNA in HNSCC remains unclear.Our previous studies have demonstrated that chemokine receptor 7?CCR7?is involved in the metastasis of HNSCC.Moreover,let-7b-5p can inhibit the invasion and migration of tumor cells through binding to CCR7.The purpose of this study was to identify the differences in expression patterns of circ RNAs in HNSCC and to analyze the biogenesis patterns and intracellular distribution of circ RNAs.And that,we analyzed the relationship between the sequences of circ RNAs,let-7b-5p and CCR7.Finally,circ RNA₁02411 was selected for further study,and the role of circ RNA₁02411 in metastasis of HNSCC and its regulatory mechanisms were explored.Methods: Real-time polymerase chain reaction?q RT-PCR?was used to detect the expression of let-7b-5p in cancer tissues and para-cancerous tissues in 15 cases of HNSCC.The expression level of CCR7 m RNA was detected in the high expression group of let-7b-5p in cancer tissue,and then circ RNA microarray analysis was performed to screen the differential expression profile of circ RNA.Circ RNA-micro RNA-CCR7 interaction was predicted by bioinformatics analysis to determine the let-7b-5p/CCR7-related circ RNA,and then the chip results were verified by q RT-PCR.Dual luciferase reporter genes were constructed and assayed to confirm the interaction of CCR7-associated circ RNA-let-7b-5p.QRT-PCR was used to detect the expression of CCR7-related circ RNA in HNSCC cell lines.Western blot was used to detect the expression of CCR7 protein in the cell lines,and the cell line was confirmed with highly expression of CCR7-related circ RNA and CCR7 protein.Small interfering RNA?si RNA?was constructed for the position of the circ RNA splicing sequence,the circ RNA sequence that avoided splicing sites and the position of linear sequence of circ RNA,and then q RT-PCR was used to detect the expression levels of circ RNA and linear sequence after transfection.We selected the sequence with the best interfering efficiency to construct si RNA plasmid.The selected cell lines were divided into four transfection groups: si-NC control group,si-circ RNA₁02411 group,si-NC+let-7b-5p inhibitor group and si-circ RNA₁02411+let-7b-5p inhibitor group.The expression level of CCR7 m RNA and protein in the transfected cells was detected by q RT-PCR and Western blot analysis,respectively.And that,MTT assay,scratch test and Transwell assay were used to detect the changes of cell proliferation,migration and invasion after transfection.Results: Compared with para-cancerous tissues,the expression level of let-7b-5p and CCR7 m RNA were highly expressed in cancer tissues of 5 patients.Further microarray analysis revealed that 287 circ RNAs were differentially expressed between cancerous and paracancerous tissues,of which 146 circ RNAs were up-regulated and 141 circ RNAs were downregulated in cancer tissues.Based on the results of circ RNA microarray analysis,a competitive endogenous RNA network consisting of 68 circ RNAs,38 micro RNAs and CCR7 was constructed using Cytoscape software.Circ RNA₁02411-let-7b-5p-CCR7 axis was further discovered in the ce RNA network.The results of q RT-PCR showed that circ RNA₁02411 and CCR7 m RNA were up-regulated in cancer tissues,and luciferase reporter assay results showed that circ RNA₁02411 can be directly targeted by let-7b-5p.The expression levels of circ RNA₁02411 and CCR7 protein in PCI-37 B cells was higher than that in PCI-37 A cells.Furthermore,the expression level of CCR7 m RNA and protein in si-circ RNA₁02411 transfected group was lower than that in si-NC control group,and the abilities of cell proliferation,migration,and invasion were correspondingly decreased.Compared with si-NC+let-7b-5p inhibitor transfected group,the expression level of CCR7 m RNA and protein in si-circ RNA₁02411+let-7b-5p inhibitor transfected group was significantly decreased,and the ability of cell proliferation,migration,and invasion were also weakened accordingly.Conclusion: Circ RNA₁02411 serves as micro RNA sponge to inhibit let-7b-5p activity,thereby promoting CCR7 expression to enhance cell proliferation,migration and invasion in the process of occurrence and metastasis of head and neck squamous cell carcinoma.