Molecular Mechanism Of DEC1 Expression Effecting On The Chemo-sensitivity Of Alkylating Agent Temozolomide In Human Gliomas

Glioma is the most common malignant tumor in the central nervous system, which accounts for more than 70% of all brain tumors, and the prognosis is very poor. Among them, glioblastoma multiforme(GBM, WHO IV) has the characteristics of low differentiation, high degree of malignancy, and easy tolerance to radiotherapy and chemotherapy. Functions of transcriptional regulation factor Differentiated Embryo Chondrocyte Expressed Gene 1(DEC1) is critical for tumor cell differentiation. This molecule is highly expressed in a variety of human tumors and its expression status is closely related the tumor malignancy. Our previous study found that DEC1 expression was significantly correlated with the pathology grade of gliomas. DEC1 could not only be as an independent prognostic factor, but also predict the response of alkylating agent temozolomide(TMZ) in high-grade gliomas. However, effect of DEC1 expression on the molecular mechanism of temozolomide cytotoxicity is not clear.The interacting molecule of DEC1, the 58-kD microspherule protein(MSP58) is an oncogene and is also highly expressed in many human tumors. DEC1 plays a variety of biological functions through the interaction with MSP58. MLH1 is a key factor in the DNA mismatch repair system(MMR) and the expression status of MLH1 is very important to the cytotoxicity of the alkylating agent. Cell lines with MLH1 mutations or deletions are resistant to temozolomide. As a transcriptional regulation factor, DEC1 decreases MLH1 expression through binding to the E-box motifs in MLH1 promoter region. As the protein molecules which are closely related to the oncogenic functions of DEC1,whether MLH1 and MSP58 can be the key molecules in the influences of DEC1 on the sensitivity of TMZ chemotherapy, is the main content of this research. Therefore, this study mainly consists of the following two aspects in the molecular mechanism of the effect of DEC1 on the sensitivity of TMZ chemotherapy.Part I Expression of MSP58 in human gliomas and its contribution to the sensitivity of temozolomide-chemotherapyObjective: In our previous study, we vertified the oncogenic roles of MSP58 in human gliomas for the first time, and found that DEC1 expression had important effects on the reponse of temozolomide chemotherapy in glioma patients. However, whether MSP58 can be used as a prognostic factor in glioma patients has not been clearly reported. As an interacting molecule of DEC1, MSP58 is closely related to oncogenic functions of DEC1. Effects of MSP58 expression on the response of temozolomide chemotherapy in glioma patients are also unknown.Methods: We mainly used the method of immunohistochemistry to detect MSP58 expression in 158 patients with primary glioma and 63 cases of recurrent GBM, who received postoperative TMZ chemotherapy but had a relapse during the TMZ period. Corelations of MSP58 expression and the clinical data, the prognosis, the response of TMZ chemotherapy of glioma patients were further analyzed. The proliferative ability was evaluated by Ki-67 immunohistochemistry and analyzed between different MSP58 expression levels.Results: The expression levels of MSP58 were significantly correlated with the WHO grade(P < 0.001) and the KPS score(P=0.003) in patients with primary gliomas. Survival analysis showed that with the higher level of MSP58 expression, patients with primary glioma had the worse prognosis(P < 0.001). Multivariate Cox regression model showed that MSP58 expression could be used as an independent prognostic factors in primary glioma patients. In addition, with the increasing level of MSP58 expression, proliferative ability of glioma cell has also increased(P < 0.01). However, there was no significant difference between MSP58 expression and the response to TMZ chemotherapy in recurrent GBM.Conclusion: The significant correlations of MSP58 expression with WHO grades and the proliferative ability of tumor cells in glioma patients indicates that MSP58 plays an important role in carcinogenesis and progression of glioma. However, no significant difference of MSP58 expression levels is found between the primary and recurrent glioma patients, which demonstrates that there is no significant effect of TMZ chemo-therapy on MSP58 expression levels in glioma patients. Furthermore, there is no significant correlation between MSP58 expression and the reponse of TMZ chemotherapy in recurrent GBM patients, which indicated that the molecular mechanisms of DEC1 affecting TMZ cytotoxicity are not through the interaction with MSP58. Moreover, MSP58 might be a novel prognostic marker that could be used as an adjunct to the WHO grade to direct the therapy regimen for individual patients. These results demonstrate the great potential for developing MSP58 as a candidate therapeutic target for glioma.Part II Molecular mechanism of DEC1 expression affecting the chemo-sensitivity of temozolomide in human gliomasObjective: Our previous study found that DEC1 expression plays critical roles in the reponse of TMZ chemotherapy in glioma patients. MMR is the most important mechanism in the DNA damage repair and regulates the sensitivity of alkylating agent chemotherapy. MLH1 is a key molecule in the MMR system. Cell lines with mutations or deletions of MLH1 are resistant to TMZ. However, as the transcriptional regulation factor of MLH1, whether DEC1 can affect the cytotoxicity of TMZ through inhibiting MLH1 expression has yet to be verified.Methods: Firstly, we used the method of immunohistochemistry to detect DEC1 and MLH1 expression in 63 cases of recurrent GBM, who received postoperative TMZ chemotherapy but had a relapse during the TMZ period, and performed TdT-mediated dUTP nick Ending-labeling assay to evalucate the apoptosis status in glioma cells. Secondly, human glioma cell lines U251 and U87 were used as study objects to further establish stable expressing glioma cell lines with DEC1-shRNA via constructing lentiviral expressing vector with specific interference fragment of DEC1. Finally, we analyzed the differences of TMZ cytotoxicity in glioma cell lines with different DEC1 expression background, and detected the expression level of related protein molecules to clarify the molecular mechanisms of DEC1 affecting the sensitivity of TMZ chemotherapy.Results: The expression levels of Dec1 and MLH1 were negatively correlated(r=-0.55, P<0.001), and the apoptosis index of GBM tumor cells increased with the increasing of MLH1 expression level(r=0.29, P < 0.001). In addition, DEC1 specific interference fragment were sequenced. Results show that the lentiviral expression vector psi-LV-DEC1-shRNA was successfully constructed and the stable expressing DEC1-shRNA glioma cell lines U251/U87-DEC1-shRNA were confirmed in fluorescence microscopy. Cytotoxicity analysis showed that MLH1 expression was significantly upregulated and the sensitivity of tumor cells to TMZ was significantly increased in glioma cells with DEC1-shRNA expression; on the contrary, expression of MLH1 was significantly inhibited and the sensitivity to TMZ was significantly decreased in cells of DEC1 over-expressed. Conclusion: The expression level of MLH1 is closely corelated with the sensitivity of TMZ chemotherapy in patients with recurrent GBM. As a transcriptional regulated gene of DEC1, the molecular mechanism of DEC1 affecting TMZ sensitivity in patients with GBM is partly through the regulation of MLH1 expression. These conclusions not only revealed that DEC1 played an important role in the sensitivity of TMZ chemotherapy, but also enriched the molecular mechanism of the resistance to TMZ chemotherapy, and futher provided molecular targets and new therapeutic strategies in TMZ chemotherapy for GBM.