Investigation of Dna Methylation-Influenced Microrna-Gene Networks in Temozolomide-Induced Glioma Cell Death and Chemoresistance

Glioblastoma multiforme (GBM), which belongs to high-grade glioma, is the most common and serious primary glioma in adults. Malignant gliomas are highly mobile, invasive, and difficult to completely resect through surgery. Therefore, radiation and chemotherapy generally follow surgical treatment as adjuvant therapies. Temozolomide (TMZ), an alkylating agent of the imidazotetrazine series, is the first line chemotherapeutic drug in the clinical GBM therapy. However, the drug resistance of TMZ decreases the therapeutic effects on the patients. Moreover, the course of TMZ therapy will last a lifetime which may cause a financial burden. Therefore, to further identify the mechanisms of TMZ-mediated cytotoxicity and chemoresistance may enhance the efficacy and application of TMZ in clinical therapy of GBM. MicroRNAs (miR), endogenous small non-coding RNAs, could control gene expressions by targeting their target genes for degradation and/or translational repression. Several microRNAs have recently been shown to be definitely linked to GBM, like miR-181 and miR-21. Besides, DNA methylation is an important mechanism to influence gene expressions via methylation on CpG dinucleotides. DNA methyltransferases (DNMTs), including DNMT1, 3A and 3B, play the major roles in regulating DNA methylation levels. Except normal physiological development, aberrant DNMTs expressions and DNA methylation changes are highly associated with carcinogenesis by hypermethylation of tumor suppressor genes and hypomethylation of oncogenes. However, no studies reported that TMZ-induced microRNAs could regulated DNA methylation via inhibiting DNMTs expressions in GBM pathogenesis. Moreover, the roles and functions of these methylation-related microRNAs and genes in TMZ-enhanced chemoresistance are still unclear. The overall specific aims are to better characterize the methylation-related microRNAs and genes expressions during GBM development and chemoresistance formation. In this proposal, three major objectives that we expect to achieve including: For Aim 1, we will identify the relations between TMZ-induced microRNAs and DNMTs in U87-MG cells. By ChlP-sequencing, we will explore the DNMTs-influenced gene profiles upon TMZ treatment. Finally, we will evaluate the effects of DNA methylation-related microRNAs and genes in TMZ cytotoxicity. For Aim 2, we will create the TMZ-induced chemoresistant GBM cells (U87R). By MeDIP-seq, we will explore the DNA methylome signatures in TMZ-resistant cells compared with normal GBM cells. Finally, we will evaluate the roles of DNA m ethyl ati on -related microRNAs and genes in TMZ chemoresistance. For Aim 3, we will evaluate the synergistic effects with the TMZ and methylation-related microRNAs combined therapy in vitro and in xenograft animal models with U87-MG and U87R cells. The significance of methylation-related microRNAs and target genes will also be investigated by routine molecular experiments and TCGA database analysis. The present proposal is an innovative study in researching methylation-related microRNAs and their functions in GBM pathogenesis. The results will not only provide a better insight to the roles of methyl ati on-related microRNAs in GBM tumorigenesis and chemoresistance, but also give new strategies for development of new chemotherapeutic combination of microRNAs and TMZ in clinical. We expect these new findings can improve the good prognosis and survival rate of the GBM patients.