The Effect Of Hepatitis B Virus X Protein Binding Protein On The Prognostic And Biological Behavior Of Head And Neck Squamous Cell Carcinoma And Mechanism

Objectives:Head and Neck squamous cell carcinomas(HNSCC)is the 7th most common cancer reported worldwide in 2018,with high malignancy and susceptibility to early metastasis.Head and neck cancer involves a wide range of sites,mainly divided into squamous cell carcinomas(SCC)of oral,nasal,pharyngeal,laryngeal and other mucosal sites origin.Currently,surgery is the main treatment,supplemented by radiotherapy and chemotherapy.Since there is no extremely effective treatment,most patients still have recurrence and metastasis after surgery,and the survival rate in the middle and late stages is even lower.Biomarkers can be used for prognostic assessment in the clinical setting and also for estimating disease risk and screening for occult primary cancers,and effective cancer markers can be used for early diagnosis and developing clinical treatment strategies.Researchers have done a lot of research to find effective markers for head and neck squamous cancers.Hepatitis B X-interacting protein(HBXIP)was discovered in 1998 by Melegari M et al.using yeast two-hybrid technique from the hepatoma cell line Hep G2.HBXIP is a newly discovered multifunctional regulatory oncoprotein,and its important role in tumor development is of great interest.HBXIP is a newly discovered multifunctional regulatory oncoprotein in recent years,and its important role in tumor development is of great interest.Since it plays different regulatory roles,studying its molecular mechanism will lay the foundation for better development of tumor therapeutic drugs in the future.Therefore,this study firstly analyzed HBXIP expression using public databases.Next,the prognostic role of HBXIP in clinical cases was analyzed by detecting its expression in clinical tumor pathology tissues.Then,the role of HBXIP in the biological function of the cell line was observed in a tongue squamous cell carcinoma cell line by overexpression and gene silencing of HBXIP,and the phosphorylation of target proteins in the PI3K/Akt cellular pathway was examined to investigate whether it is possible to play a role through this cellular pathway.Finally,a tongue squamous carcinoma cell line was transplanted on nude mice organisms to observe tumorigenesis and to explore the role of HBXIP in cancer development and progression and whether it could be a therapeutic target for head and neck squamous carcinoma.Methods:1.The oncology public databases Oncomine^TM and UALCAN were first applied to analyze the m RNA expression differences of specific gene HBXIP in head and neck squamous cancer tissues and normal tissues,and 15 head and neck squamous cancer datasets with a total of 84 studies were included in the study.2.In this study,221 pathological specimens undergoing surgical resection of head and neck squamous carcinoma from 2006-2016 at the National Cancer Institute,Japan,and 38 normal tissue specimens were collected,and these formalin-fixed,paraffin-embedded tissues were made into tissue microarrays and stained for antibodies to HBXIP protein using immunohistochemistry.These patients were followed up and observed for 2-74 months(median observation time 34 months),and clinical information of these patients was analyzed for survival to determine the prognostic role of this protein in patients with head and neck cancer tumors.3.The tongue squamous carcinoma cell line TSCCa was selected and the cell lines were transfected by plasmid overexpression and gene silencing.Firstly,the HBXIP plasmid was transfected into the cell line,and the cells were divided into experimental group(transfected with p EGFP-N1-HBXIP plasmid),control group(untransfected group)and control group(vector group,p EGFP-N1).Secondly,the cells were divided into experimental group(HBXIP?7 and HBXIP?8)and control group si RNA-control using gene silencing technique si RNA to silence the gene HBXIP.In the study of biological functions of HBXIP,firstly,RT-PCR method and Western blotting method were used to detect the expression of HBXIP in tongue squamous Secondly,MTT assay,Transwell assay and scratch assay were used to detect the effects of HBXIP overexpression and gene silencing on the proliferation,migration and invasion of tongue squamous carcinoma cells;then secondly,Western blotting was used to detect the effects of HBXIP overexpression and gene silencing on the target proteins in PI3K/Akt signaling pathway AKT,p-AKT,PI3K,p-PI3K and S100A4 protein expression;finally,ELISA was applied to detect the expression of VEGF in the supernatant of each group of cells.4.24 BALB/c mice aged 6-8 weeks were selected and randomly divided into 4groups(each group was 6):control group(untransfected group,si RNA-control group si C),experimental group(HBXIP overexpression group,gene silencing group si#7).The cells of each group after transfection were injected subcutaneously into the ventral lateral thigh of nude mice at the same dose and observed after 5 weeks.The tumor-forming tissues were measured volumetrically to compare the tumor formation;q RT-PCR and Western blotting were applied to detect the m RNA of HBXIP and the expression of HBXIP protein;the expression of VEGF protein was detected in the homogenate supernatant of each group of tumor cells using the final application of ELISA.Results:1.The expression of HBXIP m RNA in head and neck cancer tissues was analyzed,and it was found that HBXIP m RNA was upregulated in head and neck cancer tissues in both public databases compared with normal tissues.The results were analyzed using Student's t-test and the differences were significant(P<0.05)for comparison.2.(1)Follow-up of 221 clinical cases for 2-74 months(median time 34 months),175 males and 46 females,median age 68 years(29-95 years);(2)Analysis of immunohistochemical results showed that HBXIP protein expression in head and neck cancer tissues differed significantly(P<0.05)in terms of clinical stage,tumor size and tumor recurrence;in the oral cancer subgroup analysis,HBXIP protein expression differed significantly(P<0.05)in terms of age(median age 66 years),clinical stage and tumor size;in the pharyngeal subgroup analysis showed significant differences in HBXIP protein expression only in terms of tumor recurrence(P<0.05);in the tongue cancer subgroup analysis showed significant differences with respect to clinical stage and tumor size(P<0.05);(3)Multivariate Cox analysis showed that in the total head and neck cancer cases,positive HBXIP protein expression,lymph node metastasis,tumor diameter greater than 4 cm and nerve invasion were significantly associated with overall survival(P<0.05);in the oral cancer subgroup,lymph node metastasis and nerve invasion were significantly associated with overall survival(P<0.05);in the pharyngeal subgroup,positive HBXIP protein expression and In the laryngeal subgroup,positive HBXIP protein expression and tumor diameter greater than 4 cm were significantly associated with overall survival(P<0.05);in the tongue cancer subgroup,positive HBXIP protein expression and nerve invasion were significantly associated with overall survival(P<0.05).(4)Kaplan-Meier analysis showed that there was a significant difference in overall survival(OS)among 221 HNSCC patients classified by positive or negative HBXIP protein expression(P=0.044);there was no significant difference in OS among104 patients in the oral cancer subgroup;however,in 109 patients in the pharyngeal cancer subgroup(P=0.028);there was also a significant difference in OS among 59patients in the tongue cancer subgroup(P=0.038);(5)In the analysis of 38 clinical cases,HBXIP protein and m RNA expression were elevated compared with their normal tissues,and the results were significantly different(P<0.001).3.(1)In the plasmid-transfected HBXIP overexpression cell line,the results of MTT assay showed that the number of surviving cells in the experimental group was higher than that in the control group,and the difference was statistically significant(P<0.05);the results of scratch assay showed that the migration rate in the experimental group was significantly higher than that in the control group(P<0.01);the results of Transwell assay showed that the number of invading cells in the experimental group was significantly;(2)In si RNA-transfected HBXIP gene silenced cell lines,cell proliferation differences were statistically significant(P<0.01)between the experimental and control groups at 96 h by Cell Titer-Glo Luminescent cell viability assay;the scratch assay results showed that the cell migration rate was significantly lower in the experimental group than in the control group after 24 h(P<0.01);Boyden chamber assay showed that the number of cell invasion was significantly lower in the experimental group than the control group after24 h(P<0.01);(3)Western blotting results showed that in plasmid-transfected HBXIP overexpression cell lines,relative to the control group,with HBXIP overexpression p-AKT,p-PI3K and S100A4 expression was increased in the experimental group(P<0.05);in si RNA-transfected HBXIP gene silencing cell lines,relative to the control group,with HBXIP gene silencing p-AKT,p-PI3K and S100A4 expression was increased in the experimental group with HBXIP gene silencing p-AKT,p-PI3K and S100A4 expression decreased in the experimental group,but statistical differences were only found in the si#7 experimental group(P<0.05);(4)The experimental results of ELISA to detect the amount of VEGF protein in the cell culture supernatant suggested that in plasmid-transfected HBXIP overexpressed cell lines and si RNA-transfected HBXIP gene silenced cell lines,the amount of VEGF protein was increased and decreased(si#7)compared to the control group(P<0.05 and P<0.01),respectively.4.(1)In the experiment of 24 BALB/c mice,the tumor volumes of grouped si#7 and si C groups,untransfected group and HBXIP overexpression group were(540.5±14.6)mm3 and(710.3±15.3)mm³,(1143.2±29.6)mm³ and(1649.3±29.3)mm³,respectively,with statistically significant differences between the compared groups(P<0.05);(2)RNA and protein were extracted from tumors of each group,and the expression of HBXIP m RNA and protein were increased in the overexpression group compared with the untransfected group,and decreased in the gene silencing group compared with the si RNA control group,and the differences were statistically significant in each group(P<0.05 and P<0.01);(3)ELISA results of VEGF protein content in tumor tissue homogenate supernatant suggested that the expression was increased in the overexpression group compared with the untransfected group and decreased in the gene silencing group compared with the si RNA control group,and the differences were statistically significant in each group(P<0.01 and P<0.05).Conclusions:HBXIP is not only associated with poor prognosis in patients with head and neck,pharyngeal and tongue cancers,but also can affect the proliferation,migration and invasive biological functions of tongue squamous carcinoma cells through overexpression and gene silencing of HBXIP,and has the potential to promote malignant biological functions by promoting AKT,PI3K phosphorylation and abnormal increase in S100A4 protein expression.In addition,multivariate analysis showed that positive HBXIP expression was a significant independent prognostic factor in patients with head and neck,pharyngeal and tongue cancers.In conclusion,the prognostic value of HBXIP expression found in this study provides an important experimental basis and an important biomarker for targeted therapy in patients with head and neck,pharyngeal and tongue cancers.