Role And Mechanism Of Circ Flna In Migration Of Laryngeal Squamous Cell Carcinoma

Laryngeal squamous cell carcinoma(LSCC)is the most common head and neck malignancy accounting for more than 95% of laryngeal cancer.Most of LSCC patients who are diagnosed at early stage may benefit from surgery,followed by radiotherapy and/or chemotherapy.However,the 5-year overall survival rate of patients with LSCC who are asymptomatic in the advanced stage remains lower than approximately 50%.Therefore,an understanding of the driven-element and molecular mechanisms of tumorigenesis in LSCC is crucial.Circular RNAs(circ RNAs),named by covalently the closed-loop structures,play critical roles in regulation of development of human disease,especially in tumor genesis and progression.Previous studies have confirmed that circular RNA can regulate the genes expression by sponging mi RNAs.Moreover,the circular structure of circular RNA has greatly improved its stability,and it has tissue and disease specificity.Therefore,it is expected to become a potential biological marker for disease diagnosis and treatment.In this study we first tested the expression of circ FLNA in LSCC tissues,tumor adjacent tissues and LSCC cell lines and analyzed the relationship between circ FLNA expression and lymph node metastasis in patients with LSCC;And on this basis,we further investigated the biological mechanism of circ FLNA involved in the migration of LSCC cells through in vitro experiments.Part 1 The expression level of circ FLNA in laryngeal squamous cell carcinomaObjective: To investigate the expression of circ FLNA in laryngeal squamous cell carcinoma tissues and cell lines.Methods: 1.Collecting LSCC tissues,normal tissues adjacent to cancer,LSCC cell lines and normal squamous epithelium cell lines.q RT-PCR was used to detect the expression of circ FLNA in LSCC tissues and cell lines.2.Analyze the correlation between circ FLNA expression and clinicopathological characteristics of LSCC patients.Results: 1.circ FLNA was present in LSCC tissues Designed and synthesized reverse primers for the junction of circ FLNA,and the PCR templates were complementary DNA(c DNA)and genomic DNA(g DNA).The result of agarose gel electrophoresis showed sing c DNA as a template,but not g DNA,could amplify the fragment at the circ FLNA junction.LSCC tissues were treated with RNase R.q RT-PCR showed that the expression of linear FLNA in the RNase R treatment group was reduced by 87% compared with the control group,while circ FLNA was just reduced by 36%.2.circ FLNA was upregulated in LSCC tissues and cell lines q RT-PCR showed that the expression of circ FLNA in LSCC tissue was significantly higher than that in adjacent nomal tissues.Additionally,the expression of circ FLNA was significantly increased in SCC-2 and Hep2 cells.3.circ FLNA expression was related with lymph node metastasis in LSCC tissues The clinical characteristics of 39 LSCC patients were analyzed,statistical analysis showed that the expression of circ FLNA in LSCC tissues was not significantly correlated with the patient’s age,gender,tumor size,clinical stage,differentiation,smoking and drinking,but was positively related to lymph node metastasis.q RT-PCR result showed that compared with LSCC tissues without lymph node metastasis(n = 25),circ FLNA was higher in LSCC tissues with lymph node metastasis(n = 14).Summary: circ FLNA expression was upregulated in LSCC tissues and cell lines and related with lymph node metastasis.Part2 The relationship between circ FLNA upregulation and migration of laryngeal squamous cell carcinomaObjective: To investigate the role of circ FLNA in LSCC cell migration and the underlying mechanism.Method: 1.circ FLNA over expression vector pc DNA3.1-circ FLNA or interfering RNA sh-circ FLNA was transfected into LSCC cells.Transwell migration experiments were performed to detect cell migration ability.2.Western blot was used to detect the expression of FLNA,MMP-2 and MLK1 protein levels in LSCC cells with circ FLNA knocked down or overexpressed.3.q RT-PCR was used to detect the expression of FLNA in LSCC cells with circ FLNA knocked down or overexpressed.4.LSCC cells was transfected the liner FLNA knockdown vector or circ FLNA overexpression vector,respectively,or co-transfected them together.Transwell migration experiments were carried out to detect cell migration ability.Results: 1.Overexpression of circ FLNA promoted LSCC cell migration Transfection with pc DNA3.1-circ FLNA markedly increased circ FLNA level in LSCC cells.Transwell migration assays showed that the overexpression of circ FLNA significantly promoted Hep2 cell and SCC-2 cell migration ability.2.Knockdown of circ FLNA inhibited LSCC cell migration LSCC cells were transfected with sh RNA against circ FLNA or its negative control.q RT-PCR analysis showed that shcirc FLNA transfection caused considerable downregulation of circ FLNA level in Hep2 cell and SCC-2 cells.Transwell migration assays revealed that knockdown of circ FLNA inhibited Hep2 cell migration.3.circ FLNA promoted cell migration by upregulating FLNA protein expression Overexpression of circ FLNA substantially increased the MMP-2 and FLNA protein level in Hep2 cell;however,it did not affect MKL1 protein expression.In contrast,knockdown of circ FLNA in Hep2 cell markedly decreased MMP-2 and FLNA protein level.Depletion of FLNA in Hep2 cells decreased Hep2 cell migration compared with negative control.The migration ability was partly rescued following the overexpression of FLNA compared with that following the overexpression of circ FLNA alone.q RT-PCR analysis showed that overexpression or knockdown of circ FLNA expression in Hep2 cells did not affect FLNA m RNA expression.Summary: 1.Overexpression of circ FLNA promoted LSCC cell migration.2.circ FLNA promoted cell migration by upregulating FLNA protein expression.Part 3 circ FLNA participates in the migration of laryngeal squamous cell carcinoma by sponging mi R-486-3pObjective: To explore whether circ FLNA regulated LSCC cell migration by sponging mi R-486-3p.Method: 1.RNA22,mi Randa and RNAhybrid was used to predict mi RNAs which could interact with circ FLNA.2.Culturing Hep2 cells and detecting the relationship between circ FLNA and mi R-486-3p by luciferin reporter and oligo-pull down following q RT-PCR in Hep2 cells.3.Targetscan was used to predict the target gene of mi R-486-3p;Luciferase reporter assay and western blot were used to detect whether FLNA is the target gene of mi R-486-3p;4.RT-q PCR was used to detect the expression of FLNA in LSCC tissues and cell lines.5.The TCGA database was used to analyze the prognostic relationship between FLNA and LSCC patients.mi R-486-3p was overexpressed or knocked down in LSCC cells and western blot was used to detect the protein level of FLNA.6.LSCC cell lines were transfected with sh FLNA or mi R-486-3p mimics respectively,or co-transfected both together.Or pc DNA3.1-circ FLNA and mi R-486-3p mimics were transfected into LSCC cells separately or together.Western blot was used to detect the protein levels,and transwell migration assay was used to detect cell migration ability.Results: 1.circ FLNA functioned as a sponge of mi R-486-3p in LSCC cell and upregulated FLNA protein level.we identified the mi RNA-binding sites in the circ FLNA sequence by using three target prediction programs,mi Randa,RNA22 and RNAhybrid.circ FLNA contained sequences complementary to mi R-34a-5p,mi R-92b-5p,mi R-296-3p,mi R-486-3p,mi R-661,mi R-574-5p,mi R-760,mi R-486-3p,mi R-1226-5p,mi R-1271-3p,mi R-1275 and mi R-1287-5p.q RT-PCR result showed that mi R-486-3p,mi R-547 and mi R-1275-5p were significantly enriched in the circ FLNA-overexpressed precipitates.However,luciferase assays showed that co-transfection with circ FLNA-luciferase-reporter vector and mi R-486-3p,but not mi R-574-5p or mi R-1275-5p,significantly decreased the luciferase activity mediated by wild-type circ FLNA sequence.2.FLNA was a target of mi R-486-3p in LSCC cells Targetscan indicated that mi R-486-3p binding site exists in the 3’UTR of the FLNA gene;Luciferase activity showed co-transfected of mi R-486-3p mimic and FLNA wild-type 3’UTR decreased luciferase activity.Western blot showed that overexpression of mi R-486-3p could reduce the expression of FLNA protein while knockdown of mi R-486-3p could increase the expression of FLNA protein.3.Circ FLNA/mi R-486-3p/FLNA pathway regulated the LSCC cell migration Sh FLNA,or mi R-486-3p mimics were transfected into LSCC cells respectively,or co-tranfected them together.Western blot results showed that when sh FLNA and mi R-486-3p mimics were co-transfected into Hep2 cells,MMP-2 and FLNA protein levels were significantly reduced compared with the single transfection group;Transwell migration experiments also showed that knockdown of FLNA or overexpression of mi R-486-3p enhanced the inhibition of mi R-486-3p overexpression on cell migration.pc DNA3.1-circ FLNA and mi R-486-3p mimics were transfected separately or together into LSCC cells.Western blot showed that co-transfection of mi R-486-3p partially reversed the increase in MMP-2 and FLNA protein expression caused by overexpression of circ FLNA.Transwell migration experiments also confirmed that overexpression of circ FLNA in Hep2 cells can significantly promote cell migration and reverse the inhibitory effect of mi R-486-3p overexpression on cell migration.4.FLNA m RNA was up-regulated in LSCC tissues and correlated with poor prognosis q RT-PCR showed that FLNA expression was higher in LSCC tissue and cell lines,and much higher in LSCC patients with lymph node metastasis.This result was consistent with the TCGA database analysis.In addition,analysis of the TCGA database revealed that high expression level of FLNA was correlated with poor prognosis in LSCC patients.Summary: 1.The increased level of circ FLNA regulated the LSCC cell migration by targeting mi R-486-3p/FLNA axis.2.FLNA m RNA was up-regulated in LSCC tissues and correlated with poor prognosis.Conclusion: 1.circ FLNA is up-regulated in laryngeal squamous cell carcinoma,and highly expressed circ FLNA is associated with lymph node metastasis.2.Up-regulated expression of circ FLNA promotes migration of laryngeal squamous cell carcinoma.3.circ FLNA functioned as a sponge of mi R-486-3p to affect FLNA protein expression and participate in the migration of laryngeal squamous cell carcinoma.