The Study Of Risk Factors And Pathogenic Mechanism Via Targeting Survivin In Carcinogenesis Of Oral Submucous Fibrosis

Background and ObjectivesOral submucous fibrosis(OSF),a kind of oral mucosa diseases,is regarded as the precancerous condition of oral squamous cell carcinoma (OSCC).It is predominantly seen in South and Southeast Asia,including India,Bangladesh,Pakistan and Taiwan.On the Chinese mainland,OSF is common in Hunan Province and Hainan Province.Its distribution is associated with chewing betel quid(BQ).The malignant transformation rate of OSF is 7～13%depending on the different study population mainly in India and Taiwan,and it is expected to rise in recent years.As far as we know,the carcinogenesis of OSF is a complex and multi-step process that results from multiple environmental factors and genetic susceptibility. However,its pathogenesis remains unclear.In view of this,the exact mechanisms of the malignant transformation of OSF needs to be investigated further.First of all,from the epidemiological point of view we used logistic regression analysis to investigate the risk factors for carcinogenesis of OSF so as to provide the evidence for treatment and self-prevention. Secondly,we chose Survivin and its related molecules as the delegates in our study.From the perspective of molecular biology,we explored the pathogenesis of the carcinogenesis of OSF reined to two aspects of blocked apoptosis and cell cycle disorders,which could provide a theoretical basis for early diagnosis and treatment.MethodsFirstly,a case-control study was performed between 42 patients with OSCC originating from OSF and 40 patients with OSF.The numeral relationship between the 13 interrelated factors and malignant transformation of oral submucous fibrosis was studied by the multivariate logistic regression analysis.Secondly,the expression of Survivin was analyzed by immunohistochemical SP method,reverse transcription polymerase chain reaction(RT-PCR) and Western blotting assay in 10 cases of normal oral mucosal epithelium,40 cases of OSF epithelium(10 cases of early stage,15 cases of moderately advanced stage,and 15 cases of advanced stage) and 42 cases of OSCC originating from OSF, respectively.Thirdly,we used immunohistochemical SP method and Western blotting assay to detect the expression of p-Survivin in those tissues,respectively.Fourthly,Western blotting assay was used to examine the expressions of Cyclin B1,p34^cdc2and p-p34^cdc2 in the tissues, respectively.Finally,Co-immunoprecipitation was used to confirm the relationship between the p34^cdc2 and Survivin. Results1.Logistic regression analysis indicated that the four factors including age,duration of BQ chewing,duration of cigarette smoking, and OSF accompanied by oral leukoplakia(OLK) or oral lichen planus(OLP) entered the logistic regression equation.2.The results of immunohistochemical SP method and RT-PCR showed that Survivin was not detected in normal oral mucosal epithelium; the rate of Survivin detection in OSCC originating from OSF(95.2%, 40/42) was significantly higher than that in OSF(47.5%,19/40)(P＜0.01); no statistically significant difference was found in the rates of Survivin expression among the early stage(30%,3/10),the moderately advanced stage(46.7%,7/15)and the advanced stage(60.0%,9/15) of OSF(P＞0.05).Western blotting analysis further confirmed the phase expression of Survivn protein in carcinogenesis of OSF.For patients with OSCC originating from OSF,Survivin mRNA expression was not correlated to age,tumor size,tumor location or TNM stage.3.The results of immunohistochemical SP method showed that the expression of p-Survivin was negative in normal oral mucosal epithelium; the positive staining rate of p-Survivin in OSCC originating from OSF (97.6%,41/42) was significantly higher than that in OSF(50.0%,20/40) (P＜0.01);however,the rate of p-Survivin detection showed no significant difference among the early stage(30.0%,3/10),the moderately advanced stage(46.7%,7/15) and the advanced stage(66.7%,10/15)of OSF(P＞0.05).Western blotting analysis showed gradually increasing expression of p-Survivin protein as the stage of OSF increased,which was consistent with the results of immunohistochemical SP method.4.Western blotting analysis indicated that p34^cdc2 and p-p34^cdc2 were not detected in normal oral mucosal epithelium;the expression of Cyclin B1 was very weak in normal oral mucosal epithelium;there were gradually increasing expressions of Cyclin B1,p34^cdc2 and p-p34^cdc2 as the stage of OSF increased;the expressions of Cyclin B1,p34^cdc2 and p-p34^cdc2 in OSF group were significantly higher than those in normal group(P＜0.05);the expressions of these molecules showed significant diferent(P＜0.05) between the OSF and the OSCC originating from OSF, but there was no significant difference among the early stage,the moderately advanced stage and the advanced stage of OSF(P＞0.05).5.Co-immunoprecipitation assay confirmed the combination of p34^cdc2 and Survivin.Conclusions1.This study identified that age,duration of BQ chewing,duration of cigarette smoking,and presence of OLK or OLP may be the risk factors in carcinogenesis of OSF.2.The over-expression of Survivin in OSF and OSCC originating from OSF is closely associated with malignant transformation of OSF. Survivin,as a promoting factor in carcinogenesis of OSF,takes part in the inhibition of apoptosis and mitotic interference in the course,which may be an important molecular mechanism in the carcinogenesis process of OSF.Survivin may be helpful in early diagnosis and therapy of carcinogenesis of OSF and may provide an index for diagnosis as well as a target for treatment.3.P34^cdc2-Cyclin B1 complexes,the important kinase of G₂/M phase transition in cell cycle,could promote Survivin Thr34 phosphorylation.It further validates that Survivin has functional activity in malignant transformation of OSF.4.From the perspective of the cell cycle,it was confirmed that cell cycle G₂/M checkpoint disorder resulted from the over-expression of Cyclin B1 and p34^cdc2 in OSF and OSCC originating from OSF,which may be another important molecular mechanism in the carcinogenesis process of OSF.