The Mechanism Of HMGB1/NF-κB Signaling Pathway On Invasion And Metastasis Of Laryngeal Squamous Cell Carcinoma

5.7%-7.6%of systemic malignant tumors and the incidence rates of head and neckmalignant tumors spectrum of second.40%patients were diagnosed until stage Ⅲ orⅣ of tumor progress. For the treatment of laryngeal cancer, terminal cancer patientshave to prolong their life at the sacrifice of laryngeal function. Therefore, earlydetection and treatment can effectively improve the patient’s life quality and survivalperiod. At present, the study of laryngeal cancer has entered into gene level. Previousstudies have found multiple genes have a close relationship with laryngeal cancerdevelopment, such as EGFR, COX-2, VEGF, STAT3, Ccbe1, Survivin, c-myc, p53,p16, Cyclin D1, Jab and others. However, high expression of HMGB1gene whichinvolved in the process of multiple tumor development can be detected in a widevariety of tumor. Excessive expression of HMGB1can lead to the excessiveproliferation and distant metastasis of tumor cells.ObjectiveTo detect the biology behavior change of laryngeal cancer after HMGB1genesilencing, we analyzed HMGB1gene and protein expression patterns in laryngealcancer tissue and laryngeal cancer cell lines, and we also used siRNA targeting todisrupt the HMGB1gene in laryngeal cancer cell lines. Furthermore, to find the newtarget of diagnostic and treatment in laryngeal cancer, we studied the regulatorymechanism of HMGB1in the proliferation, invasion or distant metastasis of laryngealcancer cells, and also investigated the specific mechanism of HMGB1in drugresistance of laryngeal cancer cells.Method1. The HMGB1protein expression of was studied by immunohistochemistry inmetastatic laryngeal carcinoma and no metastatic laryngeal carcinoma, and thecorrelation between HMGB1expression and laryngeal carcinoma distant metastasis was also analyzed.2. The HMGB1protein expression in Hep-2and Hep-2/v cell lines by using cellimmunofluorescence, RT-PCR and Western Blot.3. The siRNA expression vector which contains HMGB1gene sequence wassynthesised and transferred into Hep-2and Hep-2/v cell lines by lipofection.4. The HMGB1gene and protein expression in Hep-2cell line was analyzed byRT-PCR and Western Blot, and the HMGB1protein expression in Hep-2/v cellline was analyzed by Western Blot.5. The RAGE and NF-κB protein expression was detected in transfected Hep-2cells by Western Blot, and the MDR1protein expression in transfected Hep-2/vcells was detected in the same way. The MMP-2and MMP-9protein activity intransfected Hep-2cells were detected by gelatin zymography.6. The migration of Hep-2cell in vitro was detected by wound healing test, and theability of clone formation was detected by colony formation assay. The migrationand invasion of Hep-2cell was detected by Transwell.7. The proliferation of the transfected Hep-2cells was detected by CCK-8and cellcycle test, and the HMGB1and MDR1protein expression were detected by cellimmunofluorescence.Results1. There were high protein expression(92.86%) of HMGB1in all carcinoma tissues,94.44%in laryngeal cancer specimens with lymphatic metastasis and79.17%inlaryngeal cancer specimens without lymphatic metastasis, respectively.(χ2=14.45，P＜0.05).2. Cell immunofluorescence showed that HMGB1expressed in both nucleus andcytoplasm of Hep-2and Hep-2/v cell lines, and it gave priority to nuclearexpression in Hep-2cell line while it gave priority to cytoplasmic expression inHep-2/v cell line.3.61.39%of HMGB1mRNA expression was inhibited by siRNA-hmgb1in Hep-2cell line, and also47.69%and41.38%of HMGB1protein expression wereinhibited in Hep-2and Hep-2/v cell lines by siRNA-hmgb1. Respectively.4. RAGE and NF-κB protein expression decreased by52.44%(P＜0.01) and30.59%(P＜0.05) after the transfection with siRNA-hmgb1. 5. MMP-9protein activity decreased obviously by94.50%,84.71%and66.79%(P＜0.01) at24h,48h and72h after transfection with siRNA-hmgb1. MMP-2protein activity also decreasedsignificantly by57.96%(P＜0.01),39.25%(P＜0.01) and26.34%(P＜0.05) at24h,48h and72h after transfection withsiRNA-hmgb1.6. The cell migration ability of Hep-2cell at6h were44.144.48,23.056.47and21.243.45,respectively. The cell migration ability of Hep-2cell at12h were93.60.39,85.142.4and51.482.63,respectively. Positive control group hada significantreduction within12h-24h, compared with another two groups (P＜0.01).7. The numbers of cell clones in negative control group were681.7±41.14,384.3±15.76and250.0±23.46,respectively. However, cell clones in positivecontrol group were503.0±8.19,317.7±17.82and174.3±4.63. The differencesbetween the two groups were statistically significant(P＜0.05).8. The numbers of penetrating membrane were36.675.24/HP in positive controlgroup, while128.7023.75/HP in negative control group, t=3.783，P＜0.05.9. Cell proliferation ability in positive control group decreased by20.18%and21.92%, respectively at24h and48h after the transient transfection for Hep-2/vcells.10. G1phase cell proportion in positive control group increased significantly afterthe transient transfection for Hep-2/v cells, which had significant differencecompared with another two groups (P＜0.05). Hep-2/v cells could induce aneffective cell cycle arrest at G0/G1phase by siRNA-hmgb1.11. MDR1protein expression was effectively reduced after Hep-2/v cells transfectedinto siRNA-hmgb1.Conclusion1. High HMGB1protein was expressed in laryngeal cancer specimens, and thelevel of HMGB1protein is higher in laryngeal cancer specimens with lymphaticmetastasis than laryngeal cancer specimens without lymphatic metastasis. Ourresults proves that HMGB1protein involve in development and metastasis oflaryngeal carcinoma. 2. SiRNA-hmgb1inhibits migration ability and invasiveness of laryngealcarcinoma in vitro.3. SiRNA-hmgb1inhibits cell proliferation ability of laryngeal carcinoma.4. SiRNA-hmgb1can reduce drug resistant of laryngeal drug-resistance cell line.5. HMGB1gene may be molecular target for the treatment of laryngeal carcinoma.