LncRNA PART1 inhibits glioma proliferation and migration via miR-374b/SALL1 axis.

BACKGROUND: Abnormal expression of lncRNA is involved in a diversity of diseases and plays a vital role in targeted therapy. However, few studies have been conducted on lncRNA PART1 in glioma. We aimed to investigate the function and the potential regulatory mechanism of lncRNA PART1/miR-374b/SALL1 axis in glioma. METHODS: qRT-PCR and western blotting detected genes and proteins expression. Dual-luciferase reporter assay was performed to examine the binding relationship of lncRNA PART1, miR-374b, and SALL1. MTT assay and clone formation assay were performed to detect the cell viability and proliferation. Transwell assay detected glioma cell migration. In vivo tumor development experiments detected changes in tumor size, volume, and weight of the tumor after overexpression of lncRNA PART1. Immunohistochemistry was used to detect ki-67, E-cadherin, and N-cadherin expression. RESULTS: The expression of lncRNA PART1 and SALL1 were down-regulated and miR-374b was up-regulated in different glioma cell lines. Overexpression of lncRNA PART1 inhibited glioma cell proliferation, migration, and epithelial mesenchymal transition (EMT). LncRNA PART1 targeted miR-374b to promote SALL1 expression. The knockdown of miR-374b inhibited glioma cell proliferation and migration and EMT by SALL1. What's more, overexpression of miR-374b or knockdown of SALL1 reversed the inhibitory effect of lncRNA PART1 on the proliferation, migration, and EMT of glioma cells. Furthermore, overexpression of lncRNA PART1 inhibited glioma growth in vivo. CONCLUSION: LncRNA PART1 inhibited glioma proliferation and migration via miR-374b/SALL1 axis. These results might provide new insights for comprehending the complex lncRNA-miRNA network in gliomas.

miR-376a inhibits glioma proliferation and angiogenesis by regulating YAP1/VEGF signalling via targeting of SIRT1.

BACKGROUND: Glioma is the most common cancer in the central nervous system. Previous studies have revealed that the miR-376 family is crucial in tumour development; however, its detailed mechanism in glioma is not clear. METHODS: Cellular mRNA or protein levels of miR-376a, SIRT1, VEGF and YAP1 were detected via qRT-PCR or Western blotting. We analysed the proliferation, angiogenesis and migration abilities of glioma cell lines using colony formation, tube formation and Transwell assays. A luciferase assay was performed to determine whether miR-376a could recognize SIRT1 mRNA. Xenograft experiments were performed to analyse the tumorigenesis capacity of glioma cell lines in nude mice. The angiogenesis marker CD31 in xenograft tumours was detected via immunohistochemistry (IHC). RESULTS: miR-376a expression was lower in glioma cells than in normal astrocytes. miR-376a mimic inhibited SIRT1, YAP1, and VEGF expression and suppressed the proliferation, migration and angiogenesis abilities of the glioma cell lines LN229 and A172, whereas miR-376a inhibitor exerted the opposite functions. In a luciferase assay, miR-376a inhibited the luciferase activity of WT-SIRT1. SIRT1 overexpression upregulated YAP1 and VEGF in glioma cells and promoted proliferation, migration and angiogenesis. Xenografts with ectopic miR-376a expression exhibited lower volumes and weights and a slower growth curve. Overexpression of miR-376a inhibited YAP1/VEGF signalling and angiogenesis by inhibiting SIRT1 in xenograft tissues. CONCLUSION: miR-376a directly targets and inhibits SIRT1 in glioma cells. Downregulation of SIRT1 resulted in decreased YAP1 and VEGF signalling, which led to suppression of glioma cell proliferation, migration and angiogenesis.

[Isolation and identification of brain tumor stem cells from human brain neuroepithelial tumors].

OBJECTIVE: To establish a simplified culture system for the isolation of brain tumor stem cells (BTSCs) from the tumors of human neuroepithelial tissue, to observe the growth and differentiation pattern of BTSCs, and to investigate their expression of the specific markers. METHODS: Twenty-six patients with brain neuroepithelial tumors underwent tumor resection. Two pieces of tumor tissues were taken from each tumor to be dissociated, triturated into single cells in sterile DMEM-F12 medium, and then filtered. The tumor cells were seeded at a concentration of 200,000 viable cells per mL into serum-free DMEM-F12 medium simply supplemented with B27, human basic fibroblast growth factor (20 microg/L), human epidermal growth factor (20 microg /L), insulin (4 U/L), L-glutamine, penicillin and streptomycin. After the primary brain tumor spheres (BTSs) were generated, they were triturated again and passed in fresh medium. Limiting dilution assay was performed to observe the monoclone formation. 5-bromodeoxyuridine (BrdU) incorporation test was performed to observe the proliferation of the BTS. The BTSCs were cultured in mitogen-free DMEM-F12 medium supplemented with 10% fetal bovine serum to observe their differentiation. Immunocytochemistry was used to examine the expression of CD133 and nestin, specific markers of BTSC, and the rate of CD133 positive cells. RESULTS: Only a minority of subsets of cells from the tumors of neuroepithelial tissue had the capacity to survive, proliferate, and generate free-floating neurosphere-like BTSs in the simplified serum-free medium. These cells attached to the poly-L-lysine coated coverslips in the serum-supplemented medium and differentiated. The BTSCs were CD133 and nestin positive. The rate of CD133 positive cells in the tumor specimens was (21 +/- 6.2)% - (38 +/- 7.0)%. CONCLUSION: A new simplified culture system for the isolation of BTSCs is established. The tumors of human neuroepithelial tissue contain CD133 and nestin positive tumor stem cells which can be isolated, proliferate and differentiate in vitro and give rise to brain tumor spheres. This tumorigenic subset may provide both a platform for brain tumor research and a target for clinical treatment.

[Correlation research between cancer stem cells and the pathological grades of neuroepithelial tumors].

OBJECTIVE: To explore the methods of isolation, culture and identification of brain tumor stem cells (BTSCs) in neuroepithelial tumor tissues in vitro, and to study the correlation between BTSCs and the patholorical grades of neuroepithelial tumors. METHODS: Tumor cells from patients undergoing neuroepithelial tumors excision were acutely dissociated, triturated into single cells, and then seeded into serum-free medium. After the primary brain tumor spheres (BTSs) were generated, they were triturated again and passaged in fresh medium. The expression of Nestin and CD133 of BTSs was detected by immunocytochemistry staining, and the expression of CD133 of tumor specimen sections was detected by immunohistochemistry staining . The expression of CD133 of 46 brain tumors and 5 normal brain tissues were analysed by SABC immunohistochemical staining, and the correlation between the expression and pathological grade of the tumors was analysed. RESULTS: BTSCs from neuroepithelial tumors could be isolated and cultured, and could be generated and passaged in vitro. The expression of Nestin and CD133 could be detected in BTSCs. CD133 could be detected in neuroepithelial tumor tissues, but not in normal brain tissues. There was significant difference between the expression of CD133 and the different grades of tumors (P < 0.01), and there was a positive correlation between the expression of CD133 and the histologic grading of tumors (P < 0.01). CONCLUSION: A small proportion of stem cells have the ability to self-renew in human neuroepithelial tumors, and there is a positive correlation between the expression of CD133 and histologic grading of tumors.

[Isolation and characterization of brain tumor stem cells in human medulloblastoma].

BACKGROUND & OBJECTIVE: Tumor stem cells have been isolated from several kinds of solid tumors, including primary brain tumors such as glioma and medulloblastoma. This investigation was to establish a simplified culture system to isolate and passage brain tumor stem cells (BTSCs) from human medulloblastoma, observe their proliferation and differentiation, and determine the expression of normal neural stem cell antigens, CD133 and Nestin, in BTSCs. METHODS: Eleven specimens of medulloblastoma were acutely dissociated and triturated into single cells without trypsin digestion. The tumor cells were seeded into serum-free DMEM/F12 medium (200,000 viable cells/ml) containing B27 (1:50), basic fibroblast growth factor (bFGF, 20 microg/L), epidermal growth factor (EGF, 20 microg /L), insulin (4 u/L), L-glutamine, and antibiotics. After generation, the primary brain tumor spheres (BTSs) were mechanically dissociated and passaged in the above serum-free medium. The monoclonal formation experiment was performed to determine the proportion of BTSCs in medulloblastoma and to observe the formation of BTSs. The differentiation of BTSCs was induced in mitogen-free DMEM/F12 medium supplemented with 10% fetal bovine serum. The expression of CD133 and Nestin in BTSs was observed with immunofluorescence staining; the distribution of CD133-positive cells in tumor sections was assessed by immunohistochemistry. RESULTS: In each of the 11 specimens, only a minority of medulloblastoma cells showed the capacity of self-renew and proliferation. These BTSCs generated free-floating neurosphere-like BTSs in the simplified serum-free medium. The proportion of BTSCs with monoclonal formation capacity in primary tumor cells was (31.18+/-6.18)%. The BTSCs attached to poly-L-lysine-coated coverslips and differentiated when the serum-supplemented medium was added. The expression of CD133 and Nestin was detected in BTSCs. CD133-positive cells scattered or formed nest-like aggregations in tumor masses, and accounted for (33.06+/-8.57)% of all tumor cells. CONCLUSIONS: BTSCs, with the capacity of self-renew and proliferation and express CD133 and Nestin, are exist in human medulloblastoma. They could be isolated and cultured in the simplified serum-free medium, and their differentiation could be induced in serum-supplemented medium.