The Molecular Mechanisms Of SFRP2Gene Methylation On The Promotion Of Oral Squamous Cell Carcinoma(OSCC)

Oral squamous cell carcinoma (OSCC) is the sixth most common human malignancy worldwide. Despite advances in therapy, the5-year survival rate after treatment is still poor. In previous studies, smoking was recognized as an important risk factor of OSCC development, cancer relapse after treatment and the occurrence of second primary cancer. Moreover, the results from epigenetic surveys revealed that the carcinigens of tobacco could affect the levels of DNA methylation in OSCC patients. It is well known that the changed levels or patterns of DNA methylation, such as reduced levels of whole genome methylation, will result in the acceleration of cancer development by oncogene activation, hot mutation spots formation, transposons abnormal expression and genomic instability. Thus, more and more studies were performed recently to investigate the effects of DNA methylation on the development of cancer.Wnt proteins are secreted signaling factors with multiple functions in development and tumourigenesis. In the previous studies, secreted frizzled-related proteins (SFRPs) have been identified as possible negative modulators of Wnt signaling pathway, in which SFRP2gene at human chromosome4q31.3was claimed as a tumor suppressor gene inactivated by hypermethylation. Disruption of Wnt pathway by downregulation of the SFRP2gene through promoter methylation has been shown in a few human cancers including colon, bladder, oesophagus, lung, and head and neck cancers. However, the role SFRP2gene methylation in smoking and OSCC development remains poorly defined. In the present study, we analyzed the effects of SFRP2gene methylation on the development of OSCC and its molecular mechanisms in vivo and in vitro, respectively. The main thesis consists of the following four parts. Part I:Clinical significance of SFRP2gene methylation status in OSCCObjective:To investigate the methylation status of SFRP2gene in OSCC tissue specimens and its clinical significance.Methods:Between Oct.2010and Aug.2011,49cancer tissue specimens from pathologically diagnosed OSCC patients were collected. Meanwhile,49corresponding tissue specimens were taken from the normal surgical margin of the carcinoma. The methylation status of SFRP2gene was identified by methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP). The SFRP2mRNA in tissue specimen was detected by Real-Time PCR (RT-PCR) with commercial reagents.Results:The incidence of SFRP’2gene methylation in OSCC tissues was75.51%(37/49), which was significantly higher than those in adjacent normal tissues6.12%(3/49). Compared to the corresponding tissue specimens, significantly higher methylation levels of CpG island of SFRP2gene promoter were determined in OSCC specimens (P<0.01). As the results shown, the methylation rates of SFRP2genes in OSCC patients were statistically associated with clinical stages and smoking index, but not with age, gender, tumor histological grade and lymph node metastasis. In addition, the SFRP2mRNAs were detected in100%of adjacent normal tissues but only detected in14.29%of OSCC tissues. Moreover, SFRP2mRNAs were detcted in2of37OSCC samples with SFRP2gene methylation, and detected in7of12OSCC samples without SFRP2gene methylation, respectively.Conclusions:Compared to the adjacent normal tissue, higher incidence of SFRP2gene promoter methylation and significant downregulation of SFRP2mRNA expression were observed in OSCC tissue. The present results suggested that SFRP2gene activation might be inhibited by SFRP2gene methylation. Moreover, the status of SFRP2gene methylation might be affected by smoking history.Part Ⅱ:Effects of5-Aza-dC on Tca8113cells proliferation and SFRP2gene methylationObjective:To investigate the effects of5-Aza-2’-deoxycytidine (5-Aza-dC) on the proliferation and cell cycle distribution of Tca8113cells (OSCC derived cell line) and its SFRP2gene methylation.Methods:Tca8113cells were treated with different concentrations of5-Aza-dC for24h, and the methylation status of SFRP2gene promoter was identified by MSP assay. SFRP2mRNA and SFRP2were detected by RT-PCR and Western Blot, respectively. In the present study, cells proliferations were measured by MTT assay and colony formation assay. Cell cycle distribution and apoptotic cells were detected by flow cytometry assay.Results:As the results shown, hypermethylation status of SFRP2gene promoter was observed in cultured Tca8113cells. Compared to the untreated cells, the levels of SFRP2mRNA and SFRP2were significantly increased in5-Aza-dC treated cells in dose-dependent manner. MTT assay and colony formation assay showed that the viability of Tca8113cells were decreased after5-Aza-dC treatment. Moreover. the cell proliferations of Tca8113were inhibited by5-Aza-dC. After treatment with5-Aza-dC for24h the cell percent of G1phase and apoptotic Tca8113cells were significantly increased in dose-dependent manner.Conclusions:The promoter region of SFRP2gene was highly methylated in Tca8113cells as well as in human OSCC tissues.24hours after treated the Tca8113cells with5-Aza-dc, the methylation of SFRP2gene was significantly decreased, followed by increased level of SFRP2mRNA and SFRP2. By regulating the cell cycle distribution,5-Aza-dC could inhibit cell proliferation, block G1phase and induce cell apoptosis.Part III:Study on the biological function of SFRP2gene in Tca8113cellsObjective:To investigate the biological function SFRP2gene in Tca8113cells and its mechanisms in vitro.Methods:With recombinant SFRP2expression vector pcDNA3.1+/SFRP2and small interfering RNAs (siRNA) for SFRP2, Tca8113cell models with SFRP2overexpression or with SFRP2inhibition were developed, respectively. As described before, the signaling proteins of Wnt pathway were detected by Western Blot assay. The effects of SFRP2on the cells proliferation were characterized by MTT assay and colony formation assay. Cell cycle distribution and apoptotic cells were analyzed by flow cytometry assay.Results:Recombinant plasmid pcDNA3.1+/SFRP2was constructed and confirmed by sequencing. After transinfected the plasmid into Tca8113cells and selected by G418, we obtained Tca8113/SFRP2cell lines which could stable overexpress SFRP2. In addition, after screened three SFRP2siRNAs, we also developed the cell model which could sufficiently suppress SFRP2expression. As the results shown, increased levels of phosphorylated GSK-3β and β-catenin, and decreased level of Cyclin D1expression were observed in the cells with SFRP2overexpression. Meanwhile, decreased levels of phosphorylated GSK-3(3and β-catenin were observed in the cells with SFRP2inhibition. However, SFRP2inhibition has not influence on the expression of Cyclin D1. Compared to parental and mock transfected control cells, SFRP2overexpression significantly inhibited Tca8113cells proliferation and arrested cell cycle in G1phase. On the contrary, SFRP2knockdown increased Tca8113viability and arrested cells in S phase.Conclusions:By partially affecting GSK-3β and β-catenin phosphorylation and Cyclin D1expression in Wnt signaling pathway, SFRP2could regulate the cell cycle distribution and inhibit Tca8113cell proliferation.Part IV:Study on the biological function of SFRP2gene in OSCC xenograft nude mice modelObjective:To investigate the inhibition effects of SFRP2gene on OSCC development in xenograft nude mice model and its molecular mechanism.Methods:20nude mice were selected and divided into two groups randomly (10mice/group). Tca8113/SFRP2cells and Tca8113/pc3.1cells were subcutaneously inoculated on the right forelimp of animals from different group, respectively. The tumor volume was measured according to the formula V=ab2π/6every3days after tumor formation. At day28, all of animals were euthanized sacrificed. The xenografts were dissected, followed by fixation, paraffin-embedded and sectioned. The histopathologic features were characterized by HE staining. In addition, the immunohistochemical assay was used to analyze the expression levels of Ki67, β-catenin and Cyclin D1in the xenografts.Results:As the results shown, Tca8113/SFRP2and Tca8113/pc3.1OSCC xenograft nude mice models were successfully developed in the present study. All animals exibited typical histopathological characteristics of human OSCC. However, the mean tumor volumes of the animals from Tca8113/SFRP2group were significantly smaller than those in the mice from Tca8113/pc3.1group. Additionally, in the animals from Tca8113/SFRP2group, the expression levels of protein Ki67and Cyclin Dlwere dramatically decreased, while the expression levels of β-catenin were significantly increased, in both cell cytoplasm and cell membrane.Conclusions:Our present results indicated that SFRP2overexpression could inhibit the development of OSCC in vivo by regulating the cell cycle distribution and partially suppressing the Wnt signal pathway.