5-ethynyl-2--deoxyuridine and Glioma

Mounting literatures have revealed the crucial effects of long noncoding RNA (lncRNA) in various cancers, including glioma. HNF1A-AS1, a novel lncRNA, is reported to modulate tumorigenesis and development of multiple cancers. However, the tumorigenic function of lncRNA HNF1A-AS1 in glioma remains largely unknown. quantitative reverse transcription and polymerase chain reaction and western blot assays were applied to evaluate the expression of relevant mRNAs and proteins. 5-Ethynyl-2'- deoxyuridine, terminal deoxynucleotidyl transferase dUTP nick-end labeling, flow cytometry, and transwell assays were conducted for examining the influence of HNF1A-AS1 on glioma cell functions. The relationship among RNAs was investigated by mechanical experiments. The results demonstrated that HNF1A-AS1 was predominantly highly expressed in glioma cell lines compared with nontumor glial epithelial cell, which was associated with the stimulation of transcription factor myelocytomatosis oncogene. Knockdown of HNF1A-AS1 remarkably inhibited glioma cells proliferation, migration, and invasion, while accelerating cell apoptosis in vitro. Mechanically, HNF1A-AS1 served as a miR-32-5p sponge. Moreover, SOX4 was discovered as a target of miR-32-5p. Inhibited miR-32-5p or upregulated SOX4 could markedly counteract the inhibitory effects of silencing HNF1A-AS1 on glioma malignant biological behaviors. HNF1A-AS1 exerted oncogenic property in glioma progression via upregulating miR-32-5p-mediated SOX4 expression, suggesting potential novel therapeutic target for future glioma treatment.

Topics: Animals; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Deoxyuridine; Disease Progression; DNA Nucleotidylexotransferase; Flow Cytometry; Gene Silencing; Glioma; Humans; In Situ Nick-End Labeling; Male; Mice; Mice, Nude; MicroRNAs; Neoplasm Proteins; Proto-Oncogene Proteins c-myc; RNA, Long Noncoding; SOXC Transcription Factors; Transcription Factors

The characterisation of dividing brain cells is fundamental for studies ranging from developmental and stem cell biology, to brain cancers. Whilst there is extensive anatomical data on these dividing cells, limited gene transcription data is available due to technical constraints.. We focally isolated dividing cells whilst conserving RNA, from culture, primary neural tissue and xenografted glioma tumours, using a thymidine analogue that enables gene transcription analysis.. 5-ethynyl-2-deoxyuridine labels the replicating DNA of dividing cells. Once labelled, cultured cells and tissues were dissociated, fluorescently tagged with a revised click chemistry technique and the dividing cells isolated using fluorescence-assisted cell sorting. RNA was extracted and analysed using real time PCR. Proliferation and maturation related gene expression in neurogenic tissues was demonstrated in acutely and 3 day old labelled cells, respectively. An elevated expression of marker and pathway genes was demonstrated in the dividing cells of xenografted brain tumours, with the non-dividing cells showing relatively low levels of expression.. BrdU "immune-labelling", the most frequently used protocol for detecting cell proliferation, causes complete denaturation of RNA, precluding gene transcription analysis. This EdU labelling technique, maintained cell integrity during dissociation, minimized copper exposure during labelling and used a cell isolation protocol that avoided cell lysis, thus conserving RNA.. The technique conserves RNA, enabling the definition of cell proliferation-related changes in gene transcription of neural and pathological brain cells in cells harvested immediately after division, or following a period of maturation.

Topics: Animals; Brain; Brain Neoplasms; Cells, Cultured; Click Chemistry; Deoxyuridine; Embryonic Stem Cells; Female; Gene Expression Profiling; Glioma; Humans; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Neural Stem Cells; Neurogenesis; Neurons; Olfactory Mucosa; RNA; Single-Cell Analysis