The Role And Mechanism Of MSP58 Gene In The Proliferation And Invasion Of Human Glioma

Glioma is the most common malignant tumor in the centrol nervous system. Approximately 40 percent are diagnosed as glioma in intracranial tumors. Patients with the most malignant glioblastoma, carry the worst prognosis, with an average survival time of less than 1 year, despite surgical treatment combined with radiotherapy and/or chemotherapy. About 20000 new cases are diagnosed as glioma each year in American. Among these diagnosed cases, nearly half are high-grade gliomas. Half of those with high-grade glioma die within the first year. The researches indicate that the glioma morbility in young people is rising in mainland, Taiwan and Hongkong of China. 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, there is a need for a deeper understanding of the molecular mechanisms which are involved in high proliferation and invasion of glioma to allow for the development of an adjuvant to current therapy.NDRG2 ,which was first identified and cloned from normal human whole brain cDNA library in our lab in 1999[GenBank registration number AF159092], belongs to a new family of differentiation-related genes, the NDRG family. Human NDRG2 locates at chromosome 14q11.2 and encodes a protein about 41kDa which is composed of 357 amino acids. Since human NDRG2 shows about 60% homology to NDRG1, NDRG2 was also named as a N-myc down-stream regulated gene.Bioinformation analysis do not indicate any known motif or domain in NDRG2 and other members of NDRG family. The precise molecular and cellular functions of NDRG2 is still unknow so far. Some preliminary researches suggest that NDRG2 may play a role in growth arrest and cell differentiation, and it may be regulated by cellular stress. So, identification of NDRG2 interaction proteins may provide important informations for its function research. For this purpose, we performed yeast two-hybrid screening of an adult brain cDNA library using NDRG2 as a bait protein. A cell cycle-dependent transcription factor MSP58(58-kDa microspherule protein) was picked up as the binding partner of NDRG2. Sequence analysis revealed that MSP58 contains a conserved FHA domain and a coiled-coil domain. GST-pull down and co-immunoprecipitation furtherly showed that NDRG2 and MSP58 possessed physical interactions both in vitro and in vivo.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 MSP58 could interact with NDRG2, a protein intimately involved in regulation of proliferation and differentiation, and the protein–protein interaction between them might play an important role in controlling cell cycle. In addition, ectopic expression of NDRG2 had been reported to contribute to inhibition of glioma cell proliferation.At present, research on the relationship between MSP58 and brain glioma is little, and the role of MSP58 in malignant proliferation and invasion of brain glioma is not completely clear. To observe the mechanism of MSP58 in the proliferation and invasion, we performed some experiments as follows.Four human brain glioma cell lines (U251, U87, BT325 and SHG-44), 41 cases of human brain glioma tissue were investigated by reverse transcription polymerase chain reaction (RT-PCR) for expression of mRNA of MSP58 and by Westernblot for expression of protein of MSP58. The results showed that, both mRNA and protein of MSP58 were expressed in U251, U87, BT325 and SHG44 cells. There was no significant difference among the cell lines. There were specially significant difference (P<0.01) and significant difference (P<0.05) respectively between the positive expression rate of mRNA and protein of MSP58 in brain glioma tissues and those in normal brain tissues. 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 mRNA and protein are highly expressed in brain glioma, and which may associate closely with initiation and progress of brain glioma.RNAi-mediated inhibition of MSP58 leads to decreased tumor growth, migration and invasion in glioma. According to MSP58 cDNA sequence, the specific RNAi fragments targeting MSP58 were designed and synthesized, which were cloned into pSilencer3.1-H1neo vector, and the eukaryotic expression vector pSilencer3.1-MSP58 of MSP58 shRNA was constructed. The pSilencer3.1-MSP58 vector, blank pSilencer3.1-H1neo and pSilencer3.1-NC were transfected respectively into human brain glioblastoma U251 cells by lipofectin 2000 medium, and the permanent transfectants U251-S, U251-H1neo, and U251-NC cells were established through selecting with G418. RT-PCR results showed that mRNA expression of MSP58 was inhibited markedly, while the results of Western blot indicated that protein expression of MSP58 was also suppressed significantly in U251-S cells. The cell growth curve drawn by MTT test showed that U251-S cells grew significantly slow than U251, U251-H1neo and U251-NC cells (P<0.01). The results of plate colony formation and Soft-Agar Assay showed that the colony number of U251-S cells markedly decreased than those of U251, U251-H1neo and U251-NC cells (P<0.01). The cell cycle analysis by flow cytometry (FCM) showed that the G1 phase cells significantly increased, whereas S phase cells markedly decreased in U251-S cells, as compared with those in U251, U251-H1neo and U251-NC cells. The invasiveness of cells was measured by using the monolayer wound healing assay and transwell invasion assay. The migration of U251-S cells declined notably (P<0.05). There were no obvious differences in migration among U251, U251-H1 neo, and U251-NC cells. Matrigel invasion assay showed that MSP58 targeting shRNA significantly suppressed the invasiveness of U251 cells. The number of the invaded cells declined notably in U251-S cells (P<0.01) with contrast of the control groups. No obvious difference in invasion were observed among U251, U251-H1 neo, and U251-NC cells (P>0.05). These data indicated that MSP58 inhibition suppresses cell migration and invasion.The above results suggested that the MSP58 shRNA can suppress significantly expression of mRNA and protein of MSP58, thereby markedly inhibit growth and invasion of human brain glioma cells in vitro.U251, U251-H1neo, U251-NC and U251-S cells were inoculated respectively in flank subcutaneous tissue of nude mice to establish xenograft models of human brain glioma. The tumor growth status was observed and tumor volume was measured termly, and the tumor growth curve was drawn. The animals were killed and the tumor weight was investigated 24 days after inoculation. The results showed that the tumorigenesis time delayed, tumor grew slow, both tumor volume and tumor weight decreased significantly (P<0.01) in U251-S group as compared with those in U251, U251-H1neo and U251-NC cells groups. The above results suggested that RNAi targeting MSP58 can suppress significantly tumorigenesis and growth of human brain glioma in nude mice, which may be associated with marked inhibition of proliferation and invasion of human brain glioma cells.Total RNA was extracted from U251-NC and the U251-S transfectants using TRIzol reagent, and two micrograms of total RNA were reverse-transcribed to generate cDNA probes. cDNA microarray analysis was performed to analyze the apoptosis-specific signaling pathway by GEArray according to a manufacturer's instructions, and mRNA levels were quantitated by GEArrayAnalyzer software. The relative mRNA expression of cell cycle-associated genes was normalized to the signals derived from four different housekeeping genes on the same membrane and expressed in arbitrary units. For the cDNA array expression analysis, a twofold change in gene expression between control and treated cultures was considered significant. MSP58 inhibition induced up-regulation of tumor suppression genes such as BRCA1 and p53, and, conversely, downregulation of proliferation-related genes such as Ki-67 and the transcription factor E2F2. Marked changes were also observed in cell cycle-associated genes, including ATM, ATR, Cyclin G1, Cyclin G2, Cullin 4A and Cullin 5, which are involved intimately in the G1-S phase transition. These alterations were consistent with MSP58 shRNA mediated inhibition of the proliferation of U251 cells. Moreover, we found that inhibition of MSP58 concomitantly up-regulates a set of genes essential for the G2/M phase transition, including anaphase-promoting complex subunit 2 (ANAPC2), ANAPC4, and ANAPC5, which is consistent with decreased cell numbers in the G2/M phase in U251-S cells. In summary, MSP58 may be a key gene in initiation and progress of human brain glioma, its mechanism may involve multiple aspects such as promoting proliferation and invasion of brain glioma cells. MSP58 may act as a valuable biomarker for molecular diagnosis of glioma and a potential target for gene therapy of glioma, meanwhile, RNAi technology may provide a novel and important means for gene therapy of glioma.