Study On The Role Of HMGB1 In Laryngeal Cancer Cell Migration Via FLNA

Introduction: Laryngeal cancer is a common malignant tumor in head and neck tumors,which seriously endangers human life and health.Clinical epidemiological investigation shows that invasion and metastasis are the main causes of death in laryngeal cancer patients.Therefore,it is of great scientific significance and potential clinical value to study the molecular mechanism of laryngeal carcinoma cell migration.High mobility group box-1(HMGB1)is a highly conserved nuclear protein.By binding to DNA,HMGB1 changes the structure of chromosomes,thus participating in nuclear replication,transcription,chromatin remodeling and other activities.HMGB1 has been shown to participate in lots of tumor migration such as breast cancer,rectal cancer,liver cancer,lung cancer,prostate cancer,cervical cancer and ovarian cancer.In previous study,we found that HMGB1 might play an oncogenic role in laryngeal carcinoma(data not shown),but the specific mechanism was still unclear.Filamin A(FLNA)is an important cytoskeleton protein in mammalian cells.As a scaffold protein,FLNA can interact with more than 90 kinds of proteins.Most importantly,FLNA affects cell adhesion and migration by interacting with vimentin.It is reported that FLNA is closely related to the occurrence and development of many kinds of tumors.Interestingly,FLNA has dual regulation function on cancer cell migration and metastasis,which might be related to its subcellular localization: FLNA inhibits and promotes cancer cell migration and metastasis when located in nuclear and cytoplasm,respectively.Two binding sites of HMGB1 in FLNA promoter region were reported.Thus,we hypothesize that HMGB1 participates in laryngeal carcinoma cell migration by regulating FLNA and the effect is related to the subcellular localization of FLNA.In the study,we planned to detect and analyze FLNA expression and its association with clinical data.Meanwhile,we employed human laryngeal carcinoma cell line Hep-2 as study object and detected the effect of HMGB1 on FLNA,illuminated the influence of HMGB1 on laryngeal carcinoma cell migration via FLNA.These will provide the scientific basis for further investigating the clinical significance of HMGB1 and FLNA in the occurrence and development of laryngeal cancer.Materials and methods: Materials: Laryngeal carcinoma and its corresponding para-cancerous tissues,human embryonic kidney cell line HEK293,human laryngeal carcinoma cell line Hep-2.Methods: Cell culture,cell transfection,cell and tissue RNA and protein extraction,cDNA synthesis and real-time PCR,Western Blot,immunofluorescence,transwell and scratch test.Result: 1.Real-time PCR showed that the expression of FLNA mRNA in 19(63.3%)of 30 laryngeal carcinoma tissues was reduced,the average mRNA level in laryngeal cancer tissues was significantly lower than that in adjacent tissues(P<0.01).FLNA mRNA level was also significantly lower in laryngeal cancer Hep-2 cells than that in HEK293 cell(P<0.05).2.Statistical analysis results displayed that FLNA expression level was significantly correlated with lymph node metastasis,TNM stage and differentiation(P<0.05),but not with age and sex(P>0.05).3.Western Blot results showed that FLNA was significantly downregulated both at mRNA and protein levels in Hep-2 cells transfected by HMGB1-siRNA compared to control group(P<0.05),indicating that HMGB1 could negatively regulate FLNA in laryngeal cancer cells.4.Immunofluorescence detection results demonstrated that FLNA was mainly distributed in the nuclear of Hep-2 cells transfected by HMGB1-siRNA compared to control group and the ratio of FLNA in nucleus to that in cytoplasm was increased.5.Scratch and transwell detection results showed that migration distance,number and ability of Hep-2 cells transfected by HMGB1-siRNA were significantly increased compared to NC groups(P<0.05).Meanwhile,migration distance,number and ability of Hep-2 cells co-transfected by HMGB1-siRNA and FLNA-siRNA were significantly decreased compared to controls(P<0.05).These suggested that FLNA knockdown could reverse the effect of HMGB1 on laryngeal cancer migration.6.Western Blot results revealed that E-cadherin level was significantly lower in Hep-2 cells transfected by FLNA-siRNA compared to control group(P<0.01),but N-cadherin and Vimentin protein levels were significantly higher(P<0.05).Conclusion: 1.FLNA is down regulated in laryngeal carcinoma,suggesting that it plays a tumor suppressive role in laryngeal carcinoma.2.FLNA has the ability to inhibit the migration of laryngeal cancer cells and inhibit epithelial-mesenchymal transition process in laryngeal carcinoma cells.3.HMGB1 promotes the migration of laryngeal cancer cells through FLNA.4.HMGB1 inhibits the expression and nuclear translocation of FLNA in laryngeal carcinoma.