Preliminary Study On Expression And Function Of F13A1 In Oral Squamous Cell Carcinoma

Background: In recent years,oral cancer has made continuous progress in diagnosis and treatment,but survival rate of oral cancer patients has not been significantly improved.Some patients are easy to relapse after conventional treatment several years.More than 50% of patients with oral squamous cell carcinoma(OSCC)develop lymph node metastasis at diagnosis,which is one of the most common adverse prognostic factors in OSCC patients.Exosomes are nanoscale biological vesicles that released into surrounding body fluids after the fusion of the multivesicular body with the plasma membrane.It contains many different components,such as DNA,mRNA,non-coding RNA,protein and lipid,etc.As the carrier of intercellular communication materials,exosomes has important significance for the regulation of the biological function of receptor cells.In the previous experiment,our group collected 10 cases of normal human,OSCC without lymph node metastasis and OSCC patients with lymph node metastasis,and extracted exosomes from 3 groups of serum,and performed proteomic analysis.It was found that the A chain of Coagulation Factor XIII(F13),Coagulation Factor XIII A Chain(F13A1),was only detected in the OSCC patient metastasis group,and the other two groups could not be detected.It is suggested that there is a close relationship between F13A1 and OSCC metastasis.The function of this protein in OSCC deserves further study.OBJECTIVE: Based on previous proteomics results,we study the expression of F13A1 in blood samples of OSCC patients and normal subjects.We detect the expression of F13A1 in OSCC tissues and adjacent tissues by qPCR and immunohistochemistry,and analyze relationship between F13A1 and clinicopathological parameters,to analyze the relationship between F13A1 and clinical pathological parameters,and to explore its potential diagnostic and prognostic significance,and to speculate on the potential biological function of F13A1.CD163 was used as the marker of M2 macrophages to detect the expression of M2 macrophages in OSCC tissues and adjacent tissues,analyze the relationship between it and the clinicopathological parameters,and explore the correlation between the expression of F13A1 and CD163,so as to speculate the correlation between F13A1 and tumor immunity.We construct F13A1 overexpression,silencing and corresponding negative control cell lines to detect the phenotype of cells in each group,and to explore the biological function of F13A1 in OSCC.Methods: We collected serum and whole blood of healthy normal persons,OSCC patients with lymph node metastasis and non-metastatic OSCC patients were collected from 10 cases each.The above samples were from the first affiliated hospital of Guangxi Medical University and the Affiliated Stomatological Hospital of Guangxi Medical University.The expression of F13A1 mRNA in serum and whole blood was detected by qPCR.WB method was used to detect the expression of F13A1 protein in serum and serum exosomes.Cancer tissues and adjacent tissues of OSCC patients were cut out to save from Affiliated Stomatological Hospital of Guangxi Medical University.The expression of F13A1 was detected by qPCR and immunohistochemistry,45 cases were applied to qPCR detection,including 35 males and 10 females,with an average age of 50.98±13.05,and 43 cases were applied to IHC detection,including 32 males and 11 females,with an average age of 52.05±13.12,and we analyzed its relationship with clinicopathological parameters.The expression of CD163 in OSCC and adjacent tissues was detected,and we analyzed its relationship with clinicopathological parameters,and explored the correlation between the expression of F13A1 and CD163.Lentivirus transfection of SCC9 and Cal27 was used to construct F13A1 overexpression,silencing expression and corresponding negative control stable cell lines,and the phenotype of the transfected cells were observed.Results :(1)Serum qPCR results showed no significant difference in F13A1 mRNA expression between OSCC group,OSCC lymph node metastasis group,OSCC non-lymph node metastasis group and healthy control group.(2)QPCR results of whole blood showed that F13A1 mRNA expression was significantly increased in OSCC group(P < 0.001),OSCC lymph node metastasis group(P=0.001),and OSCC non-lymph node metastasis group(P < 0.001)compared with healthy control group,and the difference was statistically significant.(3)WB method failed to detect the expression of F13A1 in serum.In the detection of serum exosome,the serum exosome F13A1 was highly expressed in OSCC patients with lymph node metastasis group,while the expression of serum exosome F13A1 in OSCC patients without lymph node metastasis and normal persons was low or even absent.(4)No difference was found in qPCR of OSCC and adjacent tissues,and no correlation was found between each clinicopathological parameter.(5)OSCC tissues and tissue adjacent to carcinoma immunohistochemical experiment found that F13A1 protein expression in OSCC tissues increased,decreased expression in the tissue adjacent to carcinoma(P < 0.001),T3-4 F13A1 express a T1-2 group increased(P < 0.001),lymph node metastasis group organization F13A1 protein expression level in a group without lymph node metastasis group increased(P = 0.002),group III-IV F13A1 protein expression increased in the I-II period group(P < 0.001),the others no correlation between pathological parameters.(6)CD163 higher expression in OSCC tissues,and lower expressed in tissue adjacent to carcinoma(P < 0.001),CD163 expression in T3-4 group was higher than T1-2 group(P < 0.05),CD163 expression in lymph node metastasis group a group without lymph node metastasis group(P < 0.001),CD163 expression in stage III-IV group was higher than stage I-II group(P < 0.001).There was no impact on the rest of the pathological parameters.F13A1 and moderately correlated to the expression of CD163(R = 0.544,P < 0.001)(7)Proliferation,migration and invasion abilities of overexpression of F13A1 in Cal27 and SCC9 cells were increased,while the silenced cells was decreased,all P values were all less than 0.05.Conclusion :(1)Detection of F13A1 in blood samples has partial reference value in determination of OSCC occurrence and metastasis;(2)F13A1 protein may promote the unlimited proliferation and lymph node metastasis of OSCC,and is related to the M2-type polarization of macrophages;(3)F13A1 promotes the proliferation,invasion and migration of SCC9 and Cal27.