| Glioma is one of the most common neural epithelial tumors. There are some differentdegrees of improvement in imaging, Minimally Invasive Surgery, chemotherapy, radiotherapyand so on, but the prognosis of tumors has not significantly extended. So how to improve thecomprehensive treatment of glioma has become one of the difficult problems in modernneurosurgery. Recent years, as the research on molecular biology of tumors increasing, howto elaborate molecular mechanism of the glioma and look for specific diagnostic andtherapeutic targets will become the hot spot in the research.Alternative splicing mechanism is a precise and complex regulatory mechanism in genepost-transcription. It is closely linked with human genetic diseases and considered as thenatural source of cancer. So it is very important to study the alternative splicing of tumor forprompting the diagnosis and treatment of cancer.MDM4gene (Murine double minute4) ismy is one of the chemotherapy and prognosis-related gene which was selected from the chipanalysis of biological information by our previous work.MDM4is a key regulator of p53，otherwise its function as oncogene or suppressor gene is still controversial. The p53gene isan important regulatory factor in carcinogenesis, but is an oncogene or tumor suppressor genefunction is still controversial. At present, MDM4splicing variants were considered as thereason for the MDM4gene’s complex function and controversy in existence. Although thefunctional study of MDM4were deeper than ever in glioma, the alternative splicingphenomenon was rarely reported. In the view of the importance of the alternative splicing incancer and MDM4in glioma,our experiments focus on the alternative splicing of glioma.Our research is divided into two parts. First: The discovery, validation, bioinformaticsanalysis of the three new splice variants of MDM4in glioma. Second: The expression of newsplice variants in different kind of tissues and glioma of different grades; and its relationshipwith p53, and clinical significance. Objective: To study on the splicing variants of MDM4in glioma. Validate the new variantsand use the bioinformatics to analyze their structure and function.Methods: To design the appropriate primer according to the MDM4cDNA sequence. Thedifferent kinds of mRNA expression of MDM4in glioma were detected by RT-PCR. Then thespecific of the products were confirmed by Double-PCR, nested PCR, and DNA sequencing.Use the BLAST to make the way of alternative splicing clear. Finally, analyze the structureand function of the three new variants by the use of ExPASy and SWISS-MODEL.Results: There are six bands in the products by RT-PCR. The nonspecific result wasprecluded by the double-PCR and nested PCR. The results of six bands were about800-900bp,750-800bp,700bp,650bp,550bp,450-500bp through the comparison of theMarker. And they are not coincident with the expected five bands, which show the possibleexistence of new variant. By tapping, purifying, and sequencing the PCR products, we foundthe first three bands were consistent with MDM4-FL, MDM4-S, and MDM4-A ever reported.The others were missing exon6,9; exon8,9; exon6,8,9respectively and finally identified asthe novel splice variants of MDM4, which named MDM4-1, MDM4-2, and MDM4-3. Thebioinformatics analysis showed us that the stop codons were ahead by the exon skipping inall new splice variants. This may cause to encode the short isoforms weighted by140aa,172aa,140aa. Both MDM4-1and MDM4-3were missing the exon6, which to lead the stopcodon to advance on exon7.They may encode the same protein as the MDM4-S which hasp53binding domain with the absence of Zn Finger and Ring Finger domain.MDM4-2missedexon8,9and the coding peptide would lost the Acidic domain which helps the MDM4phosphorylation by CK1alpha. Finally, analyze the structure and function of the three newvariants by the use of ExPASy and SWISS-MODEL.Conclusions: There are more than6kinds of splice variants of MDM4in glioma. There ofthem had ever reported, while the other three were novel. All of them were exon skipping,such as exon6,8,9, and named MDM4-1, MDM4-2, MDM4-3respectively. Both MDM4-1 and MDM4-3may encode the same protein weighted140aa, while the MDM4-2encode a172aa one. Finally, we analyze the structure and function of the three new variants by the useof ExPASy and SWISS-MODEL. The three new splice variants all have the p53bindingdomain, which strong illustrate their potential functions of p53precisely regulation. Objective: To investigate the expression of splice variants of MDM4in different kinds oftissues and glioma of different grades. Test the mutation of p53gene, furthermore to analyzethe relationship between the splice variants and clinical significance.Methods: The expressions of splice variants of MDM4were detected by the RT-PCR indifferent kinds of tumors and glioma of different grades. The quantitative expression level ofthe new splice variants were evaluated by Real-time PCR in different grades of glioma. Usethe PCR-SSCP to test the mutation of p53gene in glioma samples, and then analyze thecorrelation between splice variants of MDM4and p53, clinical significance.Results: Three novel splice variants were not specific expressed, but can be detected both intumors of different kinds and normal tissue. Quantitative real time PCR analysisdemonstrated elevated expression ratios of MDM4-1/MDM4-FL in low-grade glioma versushigh-grade glioma(p＜0.05).meanwhile elevated expression ratios of MDM4-S/MDM4-FL inhigh-grade glioma versus low-grade glioma(p＜0.05).There are close correlation in MDM4,MDM2, p53. And the mutation rate of p53was39.2%（29/74）which were detected byPCR-SSCP from the74glioma samples, and most of the mutations site from exon5to exon8as other reported. Glioblastomas and anaplastic astrocytoma got the top high in the mutationrate with44.4%and52.6%, however, we haven’t detected any mutation in the normal brain tissue, pilocytic astrocyoma, and ependymocytoma. There are no enough evidences tosupport the correlation between mutation rate with gender，age，and pathology, but data showthat the elevated rate of p53mutation in high-grade glioma versus low-grade glioma by52.6%vs26.1%. Compare p53mutation with wild type in glioblatoma, it shows that elevatedexpression levels of total mRNA of MDM4and lower levels of MDM4-1in p53wild typeversus mutation,.Higher expression of MDM4-1correlated significantly with poor overallsurvival time in univariate analyses. Meanwhile p53gene mutation correlated significantlywith poor overall survival time in univariate analyses.Conclusions: Three novel splice variants can be examined both in tumors of different kindsand normal tissue. The expression ratios of MDM4-1/MDM4-FL significantly decrease inhigh-grade glioma versus low-grade glioma.The expression ratios of MDM4-S/MDM4-FLincrease in high-grade glioma versus low-grade glioma.There are close correlation in MDM4,MDM2, p53. And the mutation rate of p53in74cases of glioma patients was39.2%（29/74）and most of the mutations site from exon5to exon8as other reported. Glioblastomas andanaplastic astrocytoma got the top high in the mutation rate with44.4%and52.6%, and thedata shows the elevated rate of p53mutation in high-grade glioma versus low-grade gliomaby52.6%vs26.1%. Compare p53mutation with wild type in glioblatoma, it shows thatelevated expression levels of total mRNA of MDM4and the lower levels of MDM4-1in p53wild type versus mutation. MDM4-1expression increased and p53mutation significantly asdisease stage advanced and prognosis worsen, suggesting that MDM4-1and p53mutationmay play important roles as a negative regulator to glioma development andprogression.MDM4-1and p53mutation might be useful molecular marker for predicting theprognosis of high-grade glioma. |
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