Ezh2 Expression And Biological Function In Tongue Squamous Cell Carcinoma Of The Research

ObjectiveThe purposes of the present study are:1. to evaluate the expression of enhancerof zeste homolog2(EZH2) in the human tongue squamous cell carcinoma (TSCC),three cell lines (Tca8113, SCC9, SCC25) and tissue specimens (TSCC and normaltongue). Based on the patients’ information and their follow-up data, the correlationof the EZH2level with patients’ clinicopathological characteristics and overallsurvival was further assessed to determine EZH2as a novel tumor marker for tonguecancer early diagnosis and prognosis prediction.2. to unravel the biological roles ofEZH2during the TSCC initiation and progression through monitoring the resultingphenotypic changes (migration, invasion, proliferation and apoptosis) of tonguecancer cells by EZH2inhibtion induced by DZNep and siRNA.Methods(1) Immunohistochemical staining was used to survey the expression of EZH2inTSCC samples and normal tongue specimens. The correlation of the EZH2level with patients’ clinicopathological characteristics and overall survivalwas analyzed with SSPS17.0software package.(2) Immunofluorenscence cell staining was used to detect the expression andlocalization of EZH2in three TSCC cell lines Tca8113, SCC9, SCC25.(3) Western Blot and Real-time quantitative polymerase chain reaction(Real-time PCR) were used to detect the expression of EZH2in TSCCspecimens, TSCC cell lines (Tca8113, SCC9, SCC25) and normal tonguespecimens.(4) Western Blot was used to monitor the expression changes of EZH2in Tca8113after DZNep treatment and specific siRNA transfection.(5) Wound healing assay, transwell, MTT assay and flow cytometry were usedto determine the effects of EZH2inhibitionon on the cell migration, invasion,proliferation and apoptosis of Tca8113.Results(1) Immunohistochemical staining results showed that EZH2was stronglypositive in42/52tongue carcinoma specimens, weakly positive in10/52tongue carcinoma specimens. It was weakly positive in9/12normal tonguetiusses and was negative in3/12normal tongue tiusses. The expression ofEZH2was signifincant higher in human TSCC specimens than that innormal tongue specimens (P <0.001). EZH2over-expression was found tobe associated with tumor grades (P=0.033), cervical lymph node metastasis(P=0.036) and the presence of local invasion (P=0.012), but not withgender, age, tumor size and clinical stage (P>0.05). The overall survivalrates in patients with EZH2high expression were significantly lower thanthose in patients with EZH2low expression (P=0.028).(2) Immunofluorenscence cell staining indicated that the expression of EZH2was strongly positive in cell nucleus.(3) Western Blot and Real-time PCR indicated the expression of EZH2mRNAin TSCC tiusses and TSCC cell lines (Tca8113, SCC9, SCC25) wassignificantly higher than that in normal counterparts.(4) The expression of EZH2in Tca8113was significantly inhibited after DZNeptreatment and siRNA transfection in vitro as determined by western blot.(5) Wound healing assay, transwell, MTT and flow cytometry indicated thattumor cell migration, invasion and proliferation, was remarlably suppressed while cell apoptosis was significantly enhanced after EZH2inhibition byDZNep and siRNA in vitro.Conclusions(1) The EZH2is aberrantly overexpressed in human TSCC specimens andTSCC cell lines. Its overexpression is found to be siginificantly associatedwith tumor grades, cervical lymph node metastasis and the presence of localinvasion, suggesting that it is a valuable potential biomarker for humantongue cancer.(2) EZH2is involved in cell migration, invasion, proliferation, and apoptosis inTSCC and it may be a potential therapeutic target against tongue cancer.