Influence Of Induced MSP58 Overexpression On Biological Behavior In Human Brain Glioma Cells

Glioma is the most common malignant tumor in the centrol nervous system and accounts for 30%~50% of all brain tumors. Although comprihensive treatments, which comprise surgical removal, radiotherapy and chemotherapy, has been applied in clinical management, the outcome is not promising. This type of tumor is characterized by progressive overgrowth, diffuse and relentless invasion of glial tissue, even with treatment, a poor prognosis due to universal recurrence. These features make effectiveness of surgical therapy intensively impaired. Therefore, it needs to reveal the molecular biolodical mechanism involved in occurrence and development of glioma , and to elucidate the genes influencing the malignant biological behavior of glioma. This will be very helpful and significant for us to overcome this hard disease.MSP58(58-kDa microspherule protein) was picked up as the binding partner of NDRG2 (N-myc down-stream regulated gene 2 ), a candidate gene for anti-oncogene , as we performed yeast two-hybrid screening of an adult brain cDNA library using NDRG2 as a bait protein. Protein of MSP58 consists of 462 amino acids. This molecule contains a coiled-coil domain and a FHA(forkhead associated domain). Now in the current study, the expression of MSP58 messenger RNA and protein were closely related to the proliferation ability of cells. MSP58 is a cell cycle-dependent transcription factor. MSP58 can promote the cells colony forming ability and malignant transformation and MSP58 is named as protooncogene interacting with the important molecules relating to trumorigenesis and tumor hyperplasia.Our previous study showed that both messenger RNA and protein of MSP58 expressed in brain glioma tissue. There were specially significant differences (p<0.01 for both) respectively between the positive expression rate of MSP58 mRNA and protein in benign brain glioma and those in malignant brain glioma and among those in different pathological grades of glioma. The results suggested that MSP58 may associate closely with initiation and progress of brain glioma. This research observed the effects of induced MSP58 overexpression on the proliferation, invasion and apoptosis in glioma cells. The results would be profitable for us to understand the role of MSP58 in malignant progress of brain glioma. The experiments what we did as follows.1. MSP58 eukaryotic expression vector transfecting glioma cells in vitroOur previous study investigated expression of mRNA and protein of MSP58 by reverse transcription polymerase chain reaction and Westernblot respectively in glioma cells. The results showed that, both mRNA and protein of MSP58 were expressed in U251, U87, BT325 and SHG44 cells. But There was no significant difference among the cell lines. MSP58 eukaryotic expression vector(pCI-neo-MSP58) and empty vector(pCI-neo) were identified by double-enzymed digestion and sequencing. These two vectors were transfected respectively into human brain glioblastoma U251 cells by lipofectin 2000 medium and the permanent transfectants U251-P msp58 and U251-P neo were established through selecting with G418. RT-PCR results showed that mRNA expression of MSP58 was increased markedly, while the results of Western blot indicated that protein expression of MSP58 was also increased significantly in U251-P msp58 cells.Based on the above results, the MSP58 mRNA and protein were successfully induced overexpression, which may be excellent cell models that could be used to exploit the effect of MSP58 on the biological features of brain glioma cells.2. Impact of induced MSP58 overexpression on the proliferation, invasion and apoptosis in glioma cellsThe cell growth curve drawn by MTT test showed that U251-P msp58 cells grew significantly fast than U251 and U251-P neo cells (p <0.01). The results of plate colony formation and Soft-Agar Assay showed that the colony number of U251-P msp58 cells markedly increased than those of U251 and U251-P neo cells (p <0.01). The cell cycle analysis by flow cytometry (FCM) showed that the G1 phase cells significantly decreased, whereas S phase cells markedly increased in U251-P msp58 cells, as compared with those in U251 and U251-P neo cells.These data suggested that the pCI-neo-MSP58 can increase significantly expression of mRNA and protein of MSP58, thereby markedly promote growth and invasion of human brain glioma cells in vitro.The invasiveness of cells was measured by using the monolayer wound healing assay and transwell invasion assay. The migration of U251-P msp58 cells increased notably notably. There were no obvious differences in migration among U251 and U251-P neo cells. Matrigel invasion assay showed that MSP58 significantly increased the invasiveness of U251 cells. The number of invaded cells increased markedly in U251-P msp58 cells. No obvious difference in invasion were observed among U251 and U251-P neo cells. These data indicated that MSP58 increases cell migration and invasion.The above results suggested that the overexpression of MSP58 was closely related with proliferation, migration and invasion in human brain glioma cells. In summary, MSP58 may be a key gene in initiation and progress of human brain glioma, and its mechanism may involve multiple aspects such as promoting proliferation and invasion of brain glioma cells. MSP58 may be not only a valuable biomarker of brain glioma, but also a promising candidate tagert for gene therapy aiming at malignant glioma.