Role And Significance Of Matrix Metalloproteinases And Angiogenic Factors In Noninvasive Screening Of Oral Squamous Cell Carcinoma

BackgroundOral squamous cell carcinoma,also call OSCC,is the most common oral malignant tumor,accounting for 90%of oral malignant tumors^[1].OSCC is often diagnosed at an advanced stage.Although much progress has been made in prevention and treatment,the5-year survival rate of patients with OSCC is approximately 50%in the middle stage and less than 20%in the advanced stage^[2,3].Of all the measures to reduce OSCC mortality,disability and improve prognosis,early diagnosis and prevention are still the best methods.However,the etiology and pathogenesis of OSCC have not been clarified,which has greatly hindered the primary prevention and treatment of OSCC.The current"gold standard"for the diagnosis of OSCC is the clinical examination,and biopsy of suspicious lesions.However,biopsy will cause local trauma and bleeding,and it is easy to cause infection or disease spread.Whereas,there are few noninvasive and safe methods for early screening and early detection of OSCC.In noninvasive methods,tumor biomarkers in body fluids can be used to diagnose tumors,determine the causes and stages of tumors,and play a key role in their development and treatment.Therefore,it is important and urgent to search for noninvasive screening biomarkers of OSCC.Saliva as a diagnostic fluid is a new method of tumor early screening and health monitoring.With its advantages of noninvasive,simple,safe,reliable,easy to store and low cost,it has become a feasible method for predicting diagnosis,which is conducive to the research on early screening of OSCC,tumor prognosis and clinical targeted therapy.In recent years,the m RNA and protein of IL-1,IL-6,IL-8,CD44,MMP-1 and MMP-3 have been the research focus of OSCC saliva biomarkers,and have been reported as saliva biomarkers for oral cancer^[4-6].However,there is still a lack of clinical application research and commercial sensitive kits for clinical diagnosis are not mature.Matrix metalloproteinases（MMPs）are a family of zinc ion and calcium ion dependent proteins,secreted by stromal cells and tumor cells.Their main role is to degrade extracellular matrix and basement membrane to promote tumor invasion and metastasis.At the same time,it plays an important role in promoting the formation of angiogenic mimicry channels.Angiogenic factors are a kind of natural substances that promote angiogenesis.They are closely related to MMPs in the occurrence and development of tumors,and directly or indirectly participate in the processes of tumor growth and apoptosis,angiogenesis,invasion and metastasis,and play a key role in the initial steps of regulating carcinogenesis and invasion.The commercial MMPs and angiogenic factor detection kit used in this project is a microarray chip,which can rapidly and quantitatively detect a variety of proteins with a very small number of samples at the same time.It has the advantages of high efficiency,large flux,short time consumption and high accuracy.As a powerful tool with high sensitivity,specificity and stability,protein microarray can be used as a diagnostic aid in the screening of tumor biomarkers.ObjectiveThis topic aims to combine the public database resources,use protein chip technology to reveal the expression levels and differences of MMPs and angiogenic factors in oral squamous cell carcinoma tissues and saliva,so as to provide scientific experimental basis for noninvasive screening,diagnosis and prevention of OSCC.Material and MethodsThe research was divided into the following two parts:1.Expression and significance of MMPs in oral squamous cell carcinoma and saliva（1）Through bioinformatics analysis,we sorted out the gene chip data set GSE30784from the GEO public database,screened differentially expressed genes,conducted GO enrichment analysis and KEGG pathway analysis.Results from the GCBI database showed that most MMPs are up-regulated in head and neck squamous cell carcinoma.（2）Clinical OSCC tissue samples and Adjacent normal tissue（ANT）samples were collected for pathological diagnosis.（3）The expression levels of MMPs in OSCC tissues and ANT were detected by protein microarray technology.（4）Saliva samples of OSCC patients and healthy individuals（Control,CN）were collected,and the expression levels of MMPs in the saliva of OSCC patients and healthy individuals were detected by protein microarray technology.2.Expression and significance of angiogenic factors in oral squamous cell carcinoma and saliva（1）CD31 and CK18 immunohistochemical tests were performed on clinical OSCC and ANT.（2）Angiogenin（ANG）,Angiopoietin-2（ANG-2）,Epidermal Growth Factor（EGF）,Hepatocye growth factor（HGF）,Placenta growth factor（PLGF）,Vascular endothelial growth factor（VEGF）,Platelet-derived growth factor B（PDGF-BB）,basic fibroblast growth factor（b FGF）,Heparin-binding EGF-like growth factor,（HB-EGF）and Leptin in OSCC tissues and ANT were detected by RT-q PCR.（3）Protein expression of angiogenic factors in the OSCC tissues and ANT were detected by protein microarray technology.（4）Protein expression of angiogenic factors in the saliva of OSCC patients and healthy individuals were detected by protein microarray technology.（5）The mice OSCC model were established,and pathological analysis and CD31immunohistochemical tests were performed on mouse OSCC tissues and mouse normal tongue tissues,and comparative medical analysis was performed.Results1.Expression and significance of MMPs in oral squamous cell carcinoma and saliva（1）According to the results of bioinformatics analysis,there were 1267 genes with more than 2 times the difference in expression,among which 654 genes were up-regulated and 613 genes were down-regulated.Among them,the MMP1,MMP3,MMP7,MMP9,MMP10,MMP11,MMP12,MMP13,TIMP1 were significantly different in up-regulated genes.The GCBI database results showed that the majority of MMPs were up-regulated in head and neck squamous cell carcinoma.Based on the previous research results of our research group,MMPs were taken as the focus of attention.（2）The pathological diagnosis of clinical OSCC tissue samples showed that the ANT tissues were clearly stratified,well differentiated in all layers,with orderly arrangement of basal cells.However,the epithelial cells in OSCC tissue were arranged in disorder,with keratinocytes and intercellular Bridges,and cell nucleus were significantly enlarged of different sizes.A few cells showed nuclear division,which was highly differentiated squamous cell carcinoma.（3）The protein microarray results showed that the expressions of MMP1,MMP2,MMP3,MMP8,MMP9,MMP10,MMP13,TIMP1,TIMP2 in OSCC tissues were all higher than those in ANT,and TIMP4 was the opposite,which was statistically significant.（4）Results of saliva protein microarray showed that the expressions of MMP1,MMP3,MMP8,MMP9,MMP10,MMP13 and TIMP4 in the saliva of OSCC patients were up-regulated compared with healthy individuals（P<0.05）.2.Expression and significance of angiogenic factors in oral squamous cell carcinoma and saliva（1）Immunohistochemical results showed that compared with ANT,OSCC tissues had higher microvessel density（MVD）,and the epithelial cells proliferated rapidly.（2）RT-q PCR results showed that the expression of ANG,ANG-2,HGF,PLGF,VEGF,PDGF-BB and HB-EGF in OSCC tissues were up-regulated,while EGF and b FGF were down-regulated which was statistically significant compared with that in ANT.（3）Results of tissues protein microarray showed that the expressions of ANG,ANG-2,EGF,HGF,PLGF,VEGF in OSCC tissues were all higher than those in ANT,showing statistical significance.（4）Results of saliva protein microarray showed that the expressions HGF,PLGF were up-regulated in the saliva of OSCC patients compared with the healthy individuals（P<0.05）.（5）The pathological analysis results showed that the normal tongue tissue had mild hyperplasia of the tongue epithelium,the basal cells were arranged neatly and the epithelial nailing process was regular.The OSCC tissue induced by 4NQO showed abnormal morphology or multinucleated cells and a large number of keratinocytes,and the number and cross-sectional area of blood were increased.The immunohistochemical results showed that The number of blood vessels in the OSCC group was significantly higher than that in normal tongue tissue,with higher MVD.Conclusion1.In OSCC,the expression of MMP1,MMP3,MMP9,MMP10 and MMP13 at the gene level were all significantly up-regulated,as well as the protein levels in the pathological tissues and patients’saliva.Among them,MMP1,MMP3 and MMP13showed extremely low protein expression in the control group and high protein expression in the OSCC group,suggesting that MMP1,MMP3 and MMP13 may be potential biomarkers of OSCC.MMPs has the potential to become a noninvasive screening marker for OSCC.2.The expression of HGF and PLGF in OSCC tissues was significantly up-regulated at gene and protein levels,as well as their protein levels in the saliva of OSCC patients.It suggested that these angiogenic factors have the potential to be markers for noninvasive screening of OSCC.3.The establishment of mice OSCC model confirmed that angiogenesis in OSCC model was related to the occurrence of OSCC.This result is the same as our research on human OSCC,which indicates that the model can well simulate the occurrence and development of human OSCC and has a good similarity,and it provides a basis for further research in the later stage.