Hsa?circ?0000839 Regulates The Effect Of CCR7 On Proliferation,invasion And Migration Of Head And Neck Squamous Cell Carcinoma Through MiR-320a-3p And Let-7b-5p

Objective: Head and neck squamous cell carcinoma(HNSCC)is the most common malignant tumor in head and neck region.The characteristic of regional lymph node metastasis is one of the main reasons for poor prognosis of patients with HNSCC.It is of great significance to elucidate the molecular mechanism of invasion and metastasis and to search for clinical intervention factors for clinical targeted therapy of HNSCC.CircRNA is a kind of non-codingRNA without 5 'end cap and 3' Poly A tail.A large number of researches have shown that circRNA can act as miRNA "sponge" and participate in the regulation of tumor biological function through the adsorption of miRNA.In addition,circRNA can also bindRNA binding proteins,act as protein scaffolds and encode polypeptides,and participate in the occurrence and development of tumors.MicroRNAs are a group of small endogenous non codingRNAs with a length of19-25 nucleotides.They play a key role in biological regulation by recognizing and binding to a specific 3 '-UTR sequence of mRNA,promoting its degradation or inhibiting its expression at the post transcriptional level.Let-7b-5p is a member of let-7 family.In cancer research,let-7b-5p can participate in the regulation of tumor cell cycle and inhibit the development of lung cancer,breast cancer,ovarian cancer and so on.MiR-320a-3p is a member of miR-320 family.Studies have shown that miR-320a-3p may inhibit the radioresistance of non-small cell lung cancer by inhibiting ? methylation of HIF1 PTEN.In addition,miR-320a-3p can inhibit the proliferation and metastasis of cervical cancer,colon cancer,liver cancer and other tumors,and inhibit the development of tumor.Chemokine receptor CCR7 is a member of G-protein coupled receptor family.It can promote cell invasion and migration by interacting with its two ligands CCL19 and CCL21.In the research of lymph node metastasis mechanism of head and neck squamous cell carcinoma,we confirmed that CCR7 can regulate the invasion and migration of HNSCC cells by regulating the expression of downstream PI3 K / Akt / m TOR,Cdc42,RAC,P70S6 K,MAPK family,Pyk2 and Stat3.In this study,we mainly test and verify the regulation of hsa?circ?0000839,let-7b-5p and miR-320a-3p on the biological function of HNSCC cells,the relationship between let-7b-5p,miR-320a-3p and CCR7,and the regulation of hsa?circ?0000839 on let-7b-5p and miR-320a-3p,thus provideing a new target and direction for the diagnosis and treatment of HNSCC.Methods: 1.Expression of let-7b-5p and miR-320a-3p in HNSCCReal-time PCR was used to detect the expression of let-7b-5p in HNSCC cell lines PCI-37 A and PCI-37 B,and the expression of miR-320a-3p in HNSCC cell lines Scc9,Scc15 and human normal keratinocyte Hok.The expression levels of let-7b-5p and miR-320a-3p were compared in the adjacent,cancerous and metastatic lesions from HNSCC patients and TCGA database.2.Expression of hsa?circ?0000839 in HNSCCFive pairs of HNSCC tissue from patients were detected by microarray analysis,and the circRNAs with higher expression levels in cancer tissue was screened.Hsa?circ?0000839 expression in HNSCC lesions and adjacent tissues,as well as in Scc9 and Scc15 were detected by Real-time PCR.3.Effects of let-7b-5p and miR-320a-3p on the biological function of HNSCCThe expression of let-7b-5p and miR-320a-3p in PCI-37 A,PCI-37 B,Scc9 and Scc15 were regulated by trasfection withRNA oligo: mimics,inhibitor and negative control.CCK8 cell proliferation assay,wound healing assay and transwell invasion and migration assay were used to detect the changes in cell proliferation,invasion and migration ability of each transfection group.4.Regulatory effect of let-7b-5p and miR-320a-3p on CCR7 expressionThe expression levels of let-7b-5p and miR-320a-3p in PCI-37 A,PCI-37 B,Scc9 and Scc15 cells were regulated by cell transfection technology.The CCR7 mRNA and protein expression levels were detected by Real-time PCR and Western blot assay respectively.5.Validation of targeted binding of let-7b-5p and miR-320a-3p with CCR7Let-7b-5p and miR-320a-3p were predicted by bioinformatics to target CCR7,and the binding site between let-7b-5p and CCR7 mRNA 3 '-UTR sequence was verified by dual luciferase reporter gene.6.In vivo verification of regulatory effect of let-7b-5p on CCR7Constructing a stable let-7b-5p overexpression PCI-37 B cell line by lentiviral vector transfection.The cells were divided into let-7b-5p overexpression group,negative control group and blank control group.Balb/c-nu nude mice were subcutaneously inoculated with cells for tumor formation experiment.The growth rate of tumor tissue in each group was observed,and the tumor size was recorded every 3 days.After 21 days of inoculation,the nude mice in each group were sacrificed to obtain tumor tissue and compare the volume and mass.The let-7b-5p and CCR7 mRNA expression levels were detected by Real-time PCR assay,and CCR7 protein was detected by Western blot assay.7.Effect of hsa?circ?0000839 on proliferation,invasion and migration of HNSCC cellsSi-circ?0000839,si-NC and blank control group were transfected into Scc9 and Scc15 cells to regulate the expression level of hsa?circ?0000839.CCK8 cell proliferation assay,wound healing assay and transwell invasion and migration assay were used to detect the changes in cell proliferation,invasion and migration ability of each transfection group.8.Hsa?circ?0000839 regulates the expression of miR-320a-3p and CCR7The expression of hsa?circ?0000839 in Scc9 and Scc15 cells was downregulated by cell transfection.The CCR7 mRNA and protein expression levels were detected by Real-time PCR and Western blot assay respectively.9.Validation the binding of hsa?circ?0000839 with miR-320a-3p and let-7b-5pBioinformatics prediction analysis showed that let-7b-5p and miR-320a-3p were targeted of hsa?circ?0000839,double luciferase reporter gene was used to verify the binding site between hsa?circ?0000839 and miR-320a-3p,and FISH in situ hybridization was used to verify the co-localization of hsa?circ?0000839 and miR-320a-3p.Results: 1.Expression of let-7b-5p and miR-320a-3p in HNSCC tissues and cell linesReal-time PCR results showed that the expression levels of let-7b-5p and miR-320a-3p in HNSCC tissues were lower than those in adjacent tissues.Analysis of TCGA database shows that the expression level of CCR7 in the primary head and neck squamous cell carcinoma is higher than that of normal tissues,and the expression level of CCR7 in different tumor stages and different stages is higher than that of normal tissues.The expression level of let-7b-5p in PCI-37 A cells was higher than that in PCI-37 B cells,and the expression level of miR-320a-3p in Scc9 and Scc15 cells was lower than that in Hok cells.2.Expression of hsa?circ?0000839 in HNSCC tissue and cellsMicroarray analysis showed that the expression level of hsa?circ?0000839 in cancer tissues was higher than that in adjacent tissues.Real-time PCR detection in tissue samples also verified this result.The expression level of hsa?circ?0000839 in Scc9 and Scc15 cells was significantly higher than that in Hok cells.3.Effects of let-7b-5p and miR-320a-3p on the biological function of head and neck squamous cell carcinomaAfter let-7b-5p or miR-320a-3p mimics and inhibitor were transfected into HNSCC cells,the expressions of let-7b-5p and miR-320a-3p were correspondingly up-and down-regulated.Wound healing assay,transwell migration assay indicated that let-7b-5p and miR-320a-3p over expression significantly decreased the migration ability of HNSCC cells,while let-7b-5p and miR-320a-3p low expression significantly increased the migration ability of HNSCC cells.Transwell invasion assay showed that over expression of let-7b-5p and miR-320a-3p could decrease the ability of HNSCC cell invasion,while knockdown of let-7b-5p and miR-320a-3p led to a significant increase in cell invasion ability.CCK-8 results showed that the proliferation ability of HNSCC cells decreased after up-regulating the expression of let-7b-5p and miR-320a-3p,but increased after down regulating let-7b-5p and miR-320a-3p expression.4.Regulatory effect of let-7b-5p and miR-320a-3p on CCR7 expressionOver expression of let-7b-5p and miR-320a-3p could decrease the CCR7 expression levels both mRNA and proten,but the CCR7 expression levels were increased after down regulation of let-7b-5p and miR-320a-3p.5.Validation of targeted binding of let-7b-5p and miR-320a-3p with CCR7Bioinformatics prediction analysis showed that let-7b-5p and miR-320a-3p had binding sites with CCR7,and dual luciferase reporter gene verified that let-7b-5p had binding sites with CCR7 mRNA 3'-UTR sequence.6.Verification of regulatory effect of let-7b-5p on CCR7 in vivoLet-7b-5p overexpression lentiviral vector was constructed to infect PCI-37 B.The cells in blank control group,NC control group and let-7b-5p overexpression group were used for tumor formation in nude mice.The results showed that let-7b-5p overexpression group significantly decreased tumor growth rate,tumor volume and mass compared with the control group.Compared with the control group,the expression level of let-7b-5p was significantly up-regulated in let-7b-5p overexpression group,while the expression level of CCR7 mRNA and protein was decreased.7.Effects of hsa?circ?0000839 on proliferation,invasion and migration of HNSCC cellsAfter transfection of si-circ?0000839,the expression level of hsa?circ?0000839 was decreased.Transwell migration assay and wound healing assay indicated that low expression of hsa?circ?0000839 could decrease the migration ability of HNSCC cells.The results of transwell invasion assay showed that the invasion ability of HNSCC cells was significantly decreased after the expression of hsa?circ?0000839 was decreased.CCK-8 assay showed that low expression of hsa?circ?0000839 could decrease the proliferation ability of HNSCC cells.8.Hsa?circ?0000839 regulates the expression of miR-320a-3p and CCR7After low expression of hsa?circ?0000839 in HNSCC cells,the CCR7 mRNA and protein expression levels were significantly reduced,and miR-320a-3p expression levels increased.9.Validation the binding of hsa?circ?0000839 with miR-320a-3p and let-7b-5pBioinformatics prediction analysis showed that there were interaction sites between hsa?circ?0000839 and miR-320a-3p and let-7b-5p.Double luciferase reporter gene verified the binding site between miR-320a-3p and hsa?circ?0000839.FISH in situ hybridization showed that miR-320a-3p and hsa?circ?0000839 were co-located in HNSCC cells.Conclusion: MiR-320a-3p and let-7b-5p can inhibit the abilities of HNSCC cells proliferation,invasion and migration,and have a negative regulatory relationship with CCR7.Hsa?circ?0000839 can promote the proliferation,invasion and migration of HNSCC cells,and may act as "sponge" of miR-320a-3p to regulate its effect on CCR7.