Cloning And Characterization Of The Human CCRK Promoter

GBM (Glioblastoma multiform) is the "most common primary brain tumor" and the most aggressive form of brain cancer. Malignant gliomas are a heterogeneous group of tumors that are associated with significant morbidity and mortality. The etiology of glioma is not known; however, most likely, it involves a multi-step process involving activation of oncogenes and inactivation of tumor suppressor genes. The most severe form of glioma is the Glioblastoma (GBM, high grade, WHO grade IV), which is highly malignant, usually resistant to chemotherapy, and lethal within 9-12 months. Statistics show its outbreak rate occupies 40% in the all brain cancer. But the glioblastoma of high relapse rate, high death rate to reside still high, currently, no effective method in this disease. Therefore, to find out a cancer-causing thoroughfare with authenticate new target gene treatment toward malignant glioma with diagnosis there is the very important meaning, now.By subtraction cloning strategy, we have recently identified a novel gene over-expressed in glioblastoma patient tissue samples. Bioinformatics analysisindicates that this gene is a novel Cell Cycle Related Kinase (CCRK) with unknown function. We found that CCRK expression is significantly elevated in seventeen of the nineteen high-grade (grade IV) glioblastoma tissue samples obtained from Hong Kong patients as compared to normal brain tissues. Interestingly, in the seven low-grade (grade I-III) glioma samples, CCRK level appear normal. We have also screened six glioblastoma cell lines and found CCRK level is elevated in the 5 GBM cell lines but not in one glioma cell lines with low growth rate and tumorigenicity.These results suggest that CCRK may play a role in the progression of the low-grade glioma to the malignant GBM and in the cell growth and tumorigenicity.Our objective aims at to characterize CCRK promoter and its regulatory elements. A 1072 bp fragment was isolated from the human genomic total DNA by nest PCR. This fragment three flanking end location is in the first translation start codon ATG (+1) up stream 128 bp.Promoter deletion analysis, 1072 bpusing as the nest PCR template, amplification 951 bp, 564 bp, 313 bp, 111 bprespectively, to construct with pGL3-Basic for the convenience, the artificial design the linker, five flanking carry Kpnl, three flanking carry XhoI.Transient transfection the malignant glioma U373, Dual-luciferase assay revealed that the 564 bp fragment there is the strongest activity, In order to identify the transcriptional start sites of the human CCRK gene, adopting the classical technique of 5'-RACE, the outcome of PCR that get measures the sequence directly, located position(-242) for the transcriptional start sites of the human CCRK gene. Further to start the sub-deletion analysis,-249 bp,-194 bp,-144 bp, we located a region of 74 bp as the core promoter(-441/-367).By the bioinformatics software analysis, we found three very important binding sites within this region, Delta EF-1, NF-kB and SP1.We decided to mutate the three elements with their core sequence. By dual-luciferase assay, the Delta EF-1 activity was higher obviously after mutagenesis, the NF-icB has no change, the SP1 lower obviously. We further confirm in this region by the gel-shift. Two specificity complexes were formed with both Delta EF-1 andNF-kB element. Results from our studies suggest the Delta EF-1 and NF-kB two transcription factors were relative with human CCRK gene expression by site-directed mutagenesis and Electrophoresis mobility shift assay (EMSA).