Dataset Information

Microarray analysis of aberrant microRNA expression patterns in spinal cord gliomas of different grades.

ABSTRACT: MicroRNAs (miRNAs) are involved in the development of several types of tumor; however, their role in spinal gliomas remains unknown. The present study aimed to identify potentially novel spinal cord gliomas (SCG)-associated miRNAs and to characterize their roles in the development and progression of SCG. miRNA expression levels in low-grade SCG (classed as stage I-II SCG based on the World Health Organization grading system), high-grade SCG (classed as stage IV SCG based on the World Health Organization grading system) and 5 control cases were measured using a miRNA expression microarray. Subsequently, blood samples from the spinal cord of patients with differing grades of SCG were screened for differentially expressed miRNAs (DEmiRNAs). Compared with the control group, 7 upregulated and 36 downregulated miRNAs were identified in the low-grade SCG group and a total of 70 upregulated and 20 downregulated miRNAs were identified in the high-grade SCG group (P?0.05, fold change >2). Gene Ontology analysis revealed that the regulation of cellular metabolic processes, negative regulation of biological processes and axon guidance were primarily involved. Moreover, pathway analysis showed that the target genes of DEmiRNAs were enriched in tumor-related signaling pathways, such as the MAPK and Wnt signaling pathway. The results suggest that DEmiRNAs in peripheral blood may serve as novel target markers with high specificity and sensitivity for the diagnosis of SCG.

SUBMITTER: An T

PROVIDER: S-EPMC7640765 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Microarray analysis of aberrant microRNA expression patterns in spinal cord gliomas of different grades.

An Tian T Zhang Xin-Qing XQ Liu Yu-Fei YF Lian Juan J Wu Yan-Xiang YX Lv Bo-Han BH Liang Cong C Chen Chun-You CY Yu Qi-Shuai QS Ma Meng-Hua MH Wang Yin-Qian YQ Jiang Guang-Jian GJ Fan Tao T

Oncology letters 20201021 6

MicroRNAs (miRNAs) are involved in the development of several types of tumor; however, their role in spinal gliomas remains unknown. The present study aimed to identify potentially novel spinal cord gliomas (SCG)-associated miRNAs and to characterize their roles in the development and progression of SCG. miRNA expression levels in low-grade SCG (classed as stage I-II SCG based on the World Health Organization grading system), high-grade SCG (classed as stage IV SCG based on the World Health Orga ...[more]

PMID: 33154769

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Project description:Intramedullary spinal cord tumors are a rare and understudied cancer with poor treatment options and prognosis. Our prior study used a combination of PDGF-B, HRAS, and p53 knockdown to induce the development of high-grade glioma in the spinal cords of minipigs. In this study, we evaluate the ability of each vector alone and combinations of vectors to produce high-grade spinal cord gliomas. Eight groups of rats (n = 8/group) underwent thoracolumbar laminectomy and injection of lentiviral vector in the lateral white matter of the spinal cord. Each group received a different combination of lentiviral vectors expressing PDGF-B, a constitutively active HRAS mutant, or shRNA targeting p53, or a control vector. All animals were monitored once per week for clinical deficits for 98 days. Tissues were harvested and analyzed using hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining. Rats injected with PDGF-B+HRAS+sh-p53 (triple cocktail) exhibited statistically significant declines in all behavioral measures (Basso Beattie Bresnahan scoring, Tarlov scoring, weight, and survival rate) over time when compared to the control. Histologically, all groups except the control and those injected with sh-p53 displayed the development of tumors at the injection site, although there were differences in the rate of tumor growth and the histopathological features of the lesions between groups. Examination of immunohistochemistry revealed rats receiving triple cocktail displayed the largest and most significant increase in the Ki67 proliferation index and GFAP positivity than any other group. PDGF-B+HRAS also displayed a significant increase in the Ki67 proliferation index. Rats receiving PDGF-B alone and PDGF-B+ sh-p53 displayed more a significant increase in SOX2-positive staining than in any other group. We found that different vector combinations produced differing high-grade glioma models in rodents. The combination of all three vectors produced a model of high-grade glioma more efficiently and aggressively with respect to behavioral, physiological, and histological characteristics than the rest of the vector combinations. Thus, the present rat model of spinal cord glioma may potentially be used to evaluate therapeutic strategies in the future.

Project description:To investigate the regulatory mechanisms governing the malignant signature of different gliomas we analyzed microRNA expression profiles in human tumor samples of world health organization (WHO) grade I (benign tumors), II (low grade tumors) and IV (high grade tumors) and from primary cultures obtained from tumor samples of grade II and IV. Patients This study included tumor samples histologically verified as astrocytic gliomas obtained from patients who had undergone craniotomy for microsurgical tumor removal. According to the revised WHO classification, tumors were diagnosed as: grade I or pilocytic astrocytomas; grade II or diffuse fibrillary astrocytomas; grade IV or glioblastoma multiforme. Primary cell cultures from grade II and grade IV gliomas were also obtained and miRNA expression in these cultures were analyzed RNA extraction Total RNA, including small RNA, was isolated from tissue samples using the mirVanaTM miRNA Isolation Kit (Ambion) following the standard protocol. The quantity and quality of the purified RNA was evaluated by spectrophotometric analysis and electrophoresis on denaturing gel of acrylamide. Multiplex Real-Time Quantitative Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) The miRNAs were first converted to cDNA using Multiplex RT for TaqMan Array Human MicroRNA Panel. The RT Master mix included 100 mM each of dNTPs , 50 U/ml MultiScrabe reverse transcriptase (Applied Biosystems), 20 U/µl RNase inhibitor (Applied Biosystems) and 10X RT Buffer. The 10 µl reactions, including 7 µl of RT master mix, 2 µl of purified microRNA and 1 µl of Multiplex RT Human primer pool (Applied Biosystem), were incubated in ice for 5 min and then in a thermal cycler for 30 min at 16°C, 30 min at 42°C, 5 min at 85°C, and then hold at 4°C. miRNA levels were normalized to the expression of small nucleolar RNAs, RNU44, RNU48 and RNU6B. All reverse transcriptase reactions, including no-template controls and RT controls, were run in duplicate. Real-time PCR was performed using a standard TaqMan PCR kit procedure on an “Real Time Fast 7900 HT” PCR System (Applied Biosystems). The 100 µl PCR included 50 µl RT product (before diluited 1:60) and 50 µl TaqMan Universal PCR Master Mix (2X) (Applied Biosystems). The total volume were loaded into Card TaqMan Low Density Array Human MicroRNA Panel (Applied Biosystem) including a total of 384 human microRNAs publicated on databases www.sanger.ac.uk. The reaction cards was runned at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 97°C for 30s and 59,7°C for 1 min. All reactions were run in triplicate. Analysis of data was performed using the SDS 2.3 software using the 2-∆∆Ct (relative quantitative) method . The ∆Ct of every miRNA was determined in relation to the endogenous control RNA U6 that was invariably expressed in all samples. The ∆∆Ct value was determined in relation to the calibrator, namely the normal brain tissue. Resulting data were grouped according to the tumor grading e selectioned using a cut-off value of 3. Results were expressed as “fold change” over normal brain tissue. We analyzed two samples of grade I, two samples of grade II, two samples of grade IV gliomas. Four samples form norma brain were used as norma control. Primary cell cultures form grade II and grade IV samples were used for the analysis. All reverse transcriptase reactions, including no-template controls and RT controls, were run in triplicate.

Dataset Information

Microarray analysis of aberrant microRNA expression patterns in spinal cord gliomas of different grades.

Publications

Microarray analysis of aberrant microRNA expression patterns in spinal cord gliomas of different grades.

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