| Background Astrocytomas are the most common neoplasm of the central nervous system in human. Although progress has been made, the survival rate of astrocytoma patients is still poor. Thus it is important to study the molecular mechanisms in order to find new therapeutic targets. p38y is one of the four subtypes of p38mitogen-activated protein kinases (MAPK), only expressed in skeletal muscle tissue under normal circumstances. p38γ has been reported to invovle in cell proliferation, cell cycle or appotosis in tumorigenesis, suggesting that it might be involved in the occurrence and development of cancer. However, the molecular mechanism of p38y has not been fully uncovered. To date, there is no correlative report on its expression and mechanism in human astrocytoma.Objectives The present study determined the expression of p38γ in normal brain tissues and WHO Ⅰ-Ⅳ grade astrocytoma tissues, to initially explore the relationship between p38γ and malignant degree of astrocytoma. We then applied RNA interference technology to downregulate the expression of p38γ in astrocytoma cell line U251, to further investigated the biological characteristics (proliferation, apoptosis, migration and invasion capacitis) of U251cell after p38y was downregulated, as well as the involved molecular mechanisms.Methods1. Western Blot and immunohistochemistry were used to detect the protein expression levels of p38γ and hTERT in normal brain tissues and WHO Ⅰ-Ⅳ grade astrocytoma tissues.2. p38y specific siRNA was used to downregulate the p38γ expression in astrocytoma cell line U251, then real time-PCR and Western Blot were performed to detect the interference effect.3. CCK-8Kit was used to examine the proliferation ability of astrocytoma cell line U251after p38γ was downregulated.4. Flow cytometry assay was performed to examine the apoptosis level in astrocytoma cell line U251after p38γ was downregulated.5. Transwell was used to determine the migration and invasion ability of astrocytoma cell line U251after p38γ was downregulated.6. After downregulation of p38γ in astrocytoma cell line U251by siRNA, Western Blot was applied to determine expression levels of hTERT, MMP-2and MMP-9. TRAP-PCR was applied to determine telomerase activity. Caspase-3/-9activities were detected by spectrophotometry and cytoskeletal morphology was detected by immunofluorescence staining.Results1. The protein expression level of p38y in astrocytoma tissues were significantly increased when compared with those in normal brain tissues, which were lower in low-grade astrocytomas compared with high-grade ones (P<0.05). The protein expression pattern of hTERT were similar to p38y, and its expression were positively correlated with p38y (P<0.05). p38y protein level had no correlation with age, gender and tumor size (P>0.05).2. After transfection with three p38γ specific siRNAs, both the mRNA and protein expression of p38γ decreased in in U251cell lines. SiRNA-3(target site1168) was the most efficient one. The mRNA expression level of p38γ (siRNA-3) in astrocytoma cell line U251was decreased by (92.7±1.12)%, and the protein expression level was decreased by (62.2±1.29)%(P<0.01), suggesting that the RNA interference was successful.3. After transfection with p38y specific siRNAs, the cell proliferation rate of U251was(47.0±2.3)%. The cell apoptosis level was(11.97±0.41)%. The differences were statistically significant when compared with control groups (P<0.01).4. After transfection with p38y specific siRNAs, hTERT protein level were decreased, telomerase activity was repressed, and caspase-3/-9activity was upregulated. The differences were statistically significant when compared with control groups (P<0.01).5. The migration and invasion ability of U251was impaired after the expression of p38y was decreased (P<0.01). The numbers of migration and invasiveness cells in p38y RNAi group was (44.0±1.0) and (30.6±0.9), respectively.6. After p38y knockdown, the actin cytoskeleton was obviously disorganized and sparse, with a reduction in the beam structure in U251cells observed by immunofluorescence microscopy. Protein levels of MMP-2and MMP-9were significantly decreased when compared with control groups (P<0.01).Conclusion1. The expression of p38γ in astrocytomas is not only significantly increased but is also positively correlated with the pathologic stage.2. When p38y in U251cells is down-regulated by siRNA, the expression of hTERT is inhibited, thereby reducing telomerase activity, thus the proliferation of U251cells is decreased, and apoptosis is induced.3. When p38γ in U251cells is down-regulated by siRNA, it inhibits U251cells invasiveness by disordering cytoskeleton structure of actin and downregulating of the MMP-2and MMP-9.4. p38y might be a useful oncogene biomarker for the diagnosis and treatment of astrocytomas. |
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