Cloning Of Human Brevican CDNA And The Expression Of Its MRNA In Human Glioma

Glioma is the most common among the malignant primary tumors in the central nervous system. The highly invasive behavior of giloma, which makes it particularly difficult to completely remove them by surgery, is an important cause for the high rate of recurrence. More and more studies focus on the mechanism of invasion of glioma in recent years. Former data showed that the extracelluar matrix (ECM) plays important roles in the aggressive invasion of glioma. During brain development, the neural ECM tends to be more liquid and is permissive for the moving of neural cells and the extension of axons. In adult brain, ECM becomes more insoluble and the moving of cells is restricted to maintain the morphology and function of brain. Changes of ECM in glioina are similar to that in brain development, which make it easier for tumor cells to move and invasion. Regulation of chondroitin sulfate proteoglycans (CSPGs) as the main elements of ECM in the nervous system is correlated with glioma. Brevican/BEHAB (Brain Enriched Hyaluronan Binding), which was found in 1994, is a new member of the lectican family of CSPGs. It is one of the most enriched extracelluar matrix molecules in brain. Brevican has both secreted and glycisyphosphatidylinsitol (GP1)- anchored isoforms and is expressed by neurons and glial cells. The expression of brevican is remarkably restricted to the CNS and can'S be found in peripheral tissues. First detected at embryonic days, brevican increases with neural development and maintains low levels of expression in adult brain. In brain injury, the expression of brevican is upgraded. Interestingly, brevican is increased in glioma. Both in tow and in mtm studies show that the expression of brevican in glioma needs intracranial brain environment and correlates with glioma invasion. The roles of brevican in giloma and its invasive growth are not completely understood now. In this study, we cloned the full cDNA of human brevican secreted isoform from a human astrocytoma by RT-PCR and detected the levels of brevican mRNA in glioma and nonglial brain tumors by in situ hybridization and semiquantitative RT-PCR. 5 The results are as follows: 1. cDNA cloning of human brevican secreted isoform. Surgical sample of a human astrocytoma was fresh-frozen in liquid nitrogen. Total RNA was prepared by guandimium isothiocyanate. First strand cDNA was synthesized by RT using oligo-dT. A fragment of [3-actin 476-924 was amplified by PCR to test the quality of cDNA. Two pairs of primers were designed according to secreted brevican mRNA from GeneBank search results: EHI/EH2 (corresponding to brevican 48-1637) and SHI/SH2 (corresponding to brevican 1605-2812). A 1.6kb fragment (EH-PCR) and another 1.2kb fragment (SH-PCR) were expected, with a unique Hind III restriction site existed in the overlapping part. The PCR products were cloned into pBluescnpt SK+ vectors and then ligated into a complete one, which included the whole open reading frame of brevican and was inserted into pBluescript SK+ vector between EcoR I and Sal I sites. Tow point mutations were found: T~ to C and A to T. Expression of brevican mRNA in human glioma. In situ hybridization was carried with a digoxin-labelled brevican cRNA probe (corresponding to brevican 1605-2812). Under microscope, positive tumor cells with blue...