MicroRNA-148a Promotes Human Glioma Cell Metastasis By Targeting DLGAP1

Objective: mir-148 a is an important micro RNA(mi RNA) which expression level has a close connection with genesis and progression of multiple malignant neoplasms. Through mining of large-sample genomic data, we established a novel glioblastoma multiform(GBM) molecular prognostic model. We found there was a reverse correlation between mir-148 a and DLGAP1(discs large homolog associated protein 1) in expression level in GBM which affect patient’s survival. Bioinformatics prediction discovered DLGAP1 is a potential target gene for mir-148 a.This study was to 1) invesigate mir-148a’s expression level in human glioma tissue samples and glioma cell lines; 2) construct mir-148 a overexpression stable cell lines and mir-148 a knock down stable cell lines;to 3) verify the relationship between DLGAP1 and mir-148a; 4)investigate the role of mir-148 a in cell proliferation, apoptosis, migration and invasion. This will help to clarify the molecular mechanism of the development of GBM and apply mir-148 a as a new target for GBM diagnosis and treatment.Method: 1. We utilized in-situ hybridization and q RT-PCR to detect expression level of mir-148 a in human glioma tissue samples and glioma cell lines.2. We synthesized precursor mir-148 a and reverse complement sequence of mir-148 a based on human mir-148 a sequence, and we constructed mir-148 a overexpression and mir-148 a anti-expression lentivirus and use them to infect glioma cell lines. Then we obtain mir-148 a overexpression and anti-expression stable cell lines throughpuromycin screening and q RT-PCR verification of the expression of mir-148a3. We evaluated mir-148a’s influence to the proliferation of glioma cells through Ed U, CCK8, plate clone formation assays, mir-148a’s influence to the migration and invasion of glioma cells through scratch,tanswell migration and invasion assays, mir-148a’s influence to the apoptosis of glioma cells through flow cytometry.4. We utilized immunohistochemical method to detect the expression of DLGAP1 in glioma tissue samples and control normal brain tissues in different grades and utilized q RT-PCR, Western Blot method to detect the expression of DLGAP1 in human glioma cell lines.5. We verified whether DLGAP1 is a target gene for mir-148 a through luciferase assay, and the expression of DLGAP1 m RNA and protein level in cell lines which has been infected by mir-148 a overexpression or suppression expression lentivirus. We investigated the role of mir-148a-DLGAP1 in the migration and invasion of glioma cells through transwell migration and invasion assays.Result: 1. Mir-148 a expressed in both glioma tissue samples and normal brain tissues. Comparing with which in normal brain tissues, the expression of mir-148 a up-regulated in glioma of different grades. The difference within had statistical significance(P<0.05). Similar to the detection result of expression level mir-148 a in tissues, mir-148 a also expressed in normal glial cell HA1800 and glioma cell lines U87, U251,BT325, T98 G, SWO38-C2. Comparing with which in HA1800, the expression of mir-148 a up-regulated remarkably in U87, U251, BT325,T98 G, SWO38-C2. The difference within has statistical significance(P<0.05).2. We constructed the overexpression and suppression expressionmir-148 a lentivirus vectors successfully and obtained the stable overexpression and suppression expression mir-148 a stable cell lines U87,U251, BT325, T98 G are successfully obtained through puromycin screening and q PCR detecting.3. We found that the overexpression mir-148 a promoted the proliferation of U87, U251, BT325, T98 G and The down-regulation expression of mir-148 a suppressed the proliferation of U87, U251, BT325,T98 G by Ed U, CCK8 and plate clone formation assays. We also certified that the overexpression of mir-148 a promoted the migration and invasion of U87, U251, BT325; and the down-regulation expression of mir-148 a suppressed the migration and invasion of U87, U251, BT325 by scratch experiment, transwell migration and invasion assays. The result of Annexin V PE/7-AAD staining detection of apoptosis discovered the overexpression of mir-148 a promotes the apoptosis of U87, BT325, T98G;and the down-regulation expression of mir-148 a suppressed the apoptosis of U87, BT325.4. DLGAP1 expressesd in both glioma tissues and cells. Comparing to which in normal glial cells, the expression of DGLAP1 down-regulated in glioma cells, including m RNA and protein level; The result of immunohistochemical assay detection showed the positive rate of DLGAP1 lowered along with the growth of malignant degree of gliomas.The difference between each group has statistical significance(P<0.05);Bioinformatic predicted DLGAP1 was one of the targets of mir-148 a.Through cotransfecting 293 T cells with mir-148 a over expression lentivirus, we found mir-148 a can directly act on DLGAP1-3’UTR target sequence thus down-regulating the expression of DLGAP1; q PCR,Western Blot verification indicated when mir-148 a over expressed,mir-148 a suppressed the gene and protein level of DLGAP1; when theexpression of mir-148 a is down-regulated, the gene and protein level of DLGAP1 was up-regulated. The result of transwell migration and invasion assay vertified that when DLGAP1 suppressed the migration and invasion in U87, U251, BT325 cell lines was promoted.Conclusion: 1. In glioma, the high expression of mir-148 a and the low expression of DLGAP1 are negatively correlated.2. The mir-148 a overexpression stable cell lines and mir-148 a knock down stable cell lines were successfully constructed.3. Mir-148 a promoted the proliferation, migration and invasion and suppresses the apoptosis of glioma cells.4.Mir-148 a promoted the migration and invasion of glioma cells through target regulating of DLGAP1.