miR-497 promotes the migration and invasion of human medulloblast Daoy cell line through targeted regulation of ARHGDIA in vitro.

OBJECTIVES: To further investigate the effects of miR-497 on the biological behavior of human medulloblastoma cell line in vitro. METHODS: Human medulloblastoma cell lines, Daoy and D341, were used in this study, and the miR-497 expression in the cells was measured by Quantitative PCR with fluorescence. The Daoy cells were divided into the mimics group (Daoy cells treated with mimics), inhibitor group (Daoy cells treated with inhibitor), normal Daoy cells, ARHGDIA siRNA group (Daoy cells transfected with ARHGDIA siRNA), ARHGDIA control group (Daoy cells did not receive any treatment), and negative control group (normal cells transfected with ARHGDIA siRNA). The expression of miR-497 and ARHGDIA mRNA was measured by Quantitative PCR with fluorescence, while the level of ARHGDIA protein was measured by Western blot. The binding capability of ARHGDIA and miR-497 was assessed by luciferase assay, the migration of cells was assessed by wound healing assay, and the invasion of cells was assessed by Transwell assay. RESULTS: Compared to D341 cells, the miR-497 level was significantly higher in the Daoy cells (P < 0.01). The dual-luciferase reporter assay showed that miR-497 targets ARHGDIA. Transfecting the normal Daoy cells with miR-497 mimics significantly reduced the expression of ARHGDIA protein (P < 0.05), while transfecting normal Daoy cells with miR-497 inhibitor significantly increased the expression of ARHGDIA protein (P < 0.05). Consequently, the migration and invasion capability of cells increased significantly after transfection with miR-497 mimic (P < 0.05), and decreased significantly after transfection with miR-497 inhibitor (P < 0.05). In addition, the migration and invasion capabilities of the cells also increased significantly after transfection with ARHGDIA siRNA (P < 0.05). CONCLUSIONS: miR-497/ARHGDIA axis participates in the in vitro migration and invasion of human medulloblastoma cell lines.

The 90-day prognostic value of serum cyclophilin A in traumatic brain injury.

BACKGROUND: Cyclophilin A is involved in many inflammatory diseases and its expression is up-regulated after brain injury. We determined if serum cyclophilin A could be used as a marker for severity and 90-day outcome in patients with traumatic brain injury (TBI). METHODS: Serum cyclophilin A concentrations were quantified in 105 severe TBI patients and 105 healthy individuals. Its association with Glasgow Coma Scale (GCS) score, 90-day mortality and 90-day poor outcome (Glasgow Outcome Scale score of 1-3) were investigated. RESULTS: Serum cyclophilin A concentrations were significantly higher in TBI patients than in healthy individuals. Cyclophilin A concentrations had a close relation to GCS scores and showed a high discriminatory ability for 90-day mortality and poor outcome according to area under receiver operating characteristic curve (AUC). Its AUC was in the range of GCS scores. Moreover, its combination with GCS scores significantly improved the predictive performance of GCS scores alone. In addition, serum cyclophilin A emerged as an independent predictor for 90-day mortality, overall survival and poor outcome. CONCLUSIONS: Increased serum cyclophilin A concentrations could reflect trauma severity and unfavorable outcome after head trauma, substantializing cyclophilin A as a potential biomarker for prognostic prediction of TBI.

[Effect of long noncoding RNA SPRY4-IT1 on proliferation and metastasis of medulloblastoma].

OBJECTIVE: To investigate the effects of long non-coding RNA SPRY4-IT1 (LncRNA) on proliferation and metastasis of medul-loblastoma cells. METHODS: SPRY4-IT1siRNA and control fluorescence siRNA were transfected into medulloblastoma cell line Daoy with Lipo-fe ctamine 2000 and were divided into control group and si-SPRY4-IT1 group. Relative expression of SPRY4-IT1 mRNA were detected by real-time quantitative PCR. The change of cell proliferation were examined using CCK-8 kit and clone forming experiment. The change of cell inva-sion and metastasis were examined by matrigel invasion assay and cell metastasis experiment respectively, The expression of matrix metallopro-teinase MMP-2 and MMP-9 were detected by Western blot. RESULTS: In si-SPRY4-IT1 group,the SPRY4-IT1 mRNA expression level, cell pro-liferation in vitro,cell invasion and migration ability, MMP-2 protein expression were significantly lower than those in the control group. CONCLUSIONS: Interference SPRY4-IT1 expression has prominent inhibitory effect on the cell proliferation、invasion and metastasis of medulloblastoma cell line Daoy.

[Effect of matrine on cell apoptosis and proliferation and the apoptosis related proteins of human medulloblastoma D341 cells in vitro].

OBJECTIVE: To investigate the apoptosis and proliferation effect of matrine on human medulloblastoma cell line D341 in vitro and the effect of the expression of the related caspase 3 and caspase 9 proteins. METHODS: The D341 cells were cultivated successfully in vitro. Then the cells were divided into 5 groups according to the concentration of matrine (0.5 mg/mI group, 1.0 mg/ml group, 1.5 mg/ml group, 2.0 mg/ml group and the control group was 0 mg/ml). All the experiments were repeated three times. The cell morphologic and structure change was observed with the optical microscope and the transmission electron microscope. The proliferation of D341 cell was analyzed using Cell Counting Kit-8 assay. Apoptosis was detected by Annexin V-FITC/PI double staining. The expression of Caspase3 and Caspase9 was detected by Western blot. RESULTS: With the effect of matrine, the proliferation inhibition rate gradually increased with drug concentrations increasing, and there was a significant difference (P < 0.01). The inhibitory effect of matrine on cell proliferation was different with the different treatment time, there was a significant difference between the 24 h to 72 h groups (P < 0.01). The apoptotic rate increased with matrine concentrations increasing. There were significant differences between the group of 0.5 mg/mI or 1.0 mg/mI to the group of 1.5 mg/mI or 2.0 mg/mI (P < 0.05). The apoptotic rate increased with the prolonged treatment time. There were significant differences between the group of 24 h or 48 h to the group of 72 h ( P < 0.05). With the increase of matrine concentration, the expression of Caspase 3 and Caspase 9 increased (P < 0.01). CONCLUSION: Matrine induces the apoptosis, and inhibits the proliferation of human medulloblastoma D341 cells in vitro by up-regulation of the expression level of Caspase3, Caspase9.

[The relationship between hypermethylation of Syk gene promoter and medulloblastoma cell invasion and metastasis].

OBJECTIVE: To investigate the effect of demethylation of Syk gene promoter by the methylation transferase inhibitor 5-aza-CdR on the invasion and metastasis of medulloblastoma cell line Daoy. METHODS: Medulloblastoma cell line Daoy was treated with 5-aza-CdR in vitro. Methylation-specific PCR, real time-PCR and Western blot were used to detect Syk gene promoter methylation status, Syk mRNA and protein expression respectively. Transwell was employed to study the invasion and metastasis of medulloblastoma cell line Daoyby counting the cells that had invaded through Matrigel and migrated to the undersurface of the membrane before and after treatment of 5-aza-CdR. RESULTS: In comparison to control group, Syk gene promoter of 5-aza-CdR-treated groups was demethylated and expression of Syk mRNA and protein was significantly up-regulated by 3.40±0.24 folds (P<0.01) and 3.23±0.19 folds (P<0.01) respectively. The invasiveness and metastasis of medulloblastoma cell line Daoy was decreased(P<0.05). CONCLUSIONS: Hypermethylation of Syk gene promoter is responsible for the down-regulation of Syk gene expression in medulloblastoma cell line Daoy, which may be one of the mechanisms that enhanced cell invasion and metastasis. While 5-aza-CdR can reverse the hypermethylation of Syk gene promoter and restore Syk gene expression and thus suppresses invasiveness and metastasis of tumor cells.