Study On The Suppressive Effect Of MiR-451 On Human Glioma Cell Growth In Vitro And Vivo

Glioma is the most common primary malignant tumor in the central nervous system. Owing to the feature of its invasive growth, fast cell proliferation and high recurrence rate. The molecular development of glioma is a complex process which involved the activation of proto-oncogenes and inactivation of tumor suppressor genes and the abnormal expression of mutiple cytokines in the cellular message transmission pathway. Glioma is hard to be removed completely by the surgical resection. The postoperative radiotherapy, chemotherapy and immunotherapy can improve the part of life quality and overall survival of the patients. Therefore, the gene therapy for correcting the aberration of genetic events in glioma may be the best method, which has been the hot spots in the study of glioma.MicroRNAs (miRNAs) are small, non-coding RNAs about 22 nucleotides in length that negatively regulate gene expression at the post-transcriptional and/or translational level by binding loosely complimentary sequences in the 3'-untranslated regions (UTRs) of target mRNAs. MicroRNAs are predicted to regulate the expression of approximately one-third of all human genes. Some miRNA can target hundreds of genes, and some genes may be targeted by multiple miRNAs, suggesting that miRNAs play important roles in coordinating many cellular processes. As such, miRNA-mediated gene regulation is now considered an important role in biologic processes of human cells.MiR-451 is located on chromosome 17q11.2, a region known to be amplified in certain types of cancers, and is in close proximity to HER2 (17q12). In recent years, studies have found that abnormal expression of miR-451 in breast cancer, ovarian cancer, gastric cancer, colorectal cancer and glioma has been reported. Gal reported that transfection of glioblastoma (GBM) cells with the mature miR-451 could inhibited neurosphere formation, and inhibited GBM cell growth. Furthermore, transfection of miR-451 combined with Imatinib mesylate treatment had a cooperative effect in dispersal of GBM neurospheres, however, the regulatory mechanism of miR-451 is unclear. Another report showed that miR-451, down-regulated in migrating GBM cells, could regulate LKB1/AMPK signaling by directly regulating CAB39 expression in GBM cells. Noticeably, it showed that miR-451 could reduce GBM cell migration but pomote its proliferation, which were partly contrary to Gal H's results. In breast cancer, Olga showed that miR-451 could regulated the·expression of multidrug resistance 1 gene. Transfection of the MCF-7/DOX-resistant cells with miR-451 resulted in the increased sensitivity of cells to DOX. In ovarian cancer, expressions of miR-27a and miR-451 were up-regulated in multidrug resistant (MDR) cancer cell lines A2780DX5 and KB-V1, as compared with their parental lines A2780 and KB-3-1. Treatment of A2780DX5 cells with the antagomirs of miR-27a or miR-451 decreased the expression of P-glycoprotein and MDR1 mRNA. In contrast, the mimics of miR-27a and miR-451 increased MDR1 expression in the parental cells A2780. The sensitivity to and intracellular accumulation of cytotoxic drugs that are transported by P-glycoprotein were enhanced by the treatment with the antagomirs of miR-27a or miR-451. In gastrointestinal cancer Cells, down-regulation of miR-451 was associated with worse prognosis. MiR-451 was decreased expression in gastric and colorectal cancer versus nontumoral tissues. Overexpression of miR-451 in gastric and colorectal cancer cells reduced cell proliferation and increased sensitivity to radiotherapy. Microarray and bioinformatic analysis identified the novel oncogene macrophage migration inhibitory factor (MIF) as a potential target of miR-451. In fact, overexpression of miR-451 down-regulated mRNA and protein levels of MIF and decreased expression of reporter genes with MIF target sequences.In our previous studies, we profiled miRNA expression in five GBM cell lines, one astrocytoma cell line, and normal brain tissue. Our data revealed that the miRNA most significantly decreased in GBM compared to normal brain tissue in these studies was miR-451. In the present study, we used 2'-O-methyl-oligonucleotides to overexpress miR-451 in the human GBM cell lines, then we observed whether there were jointly or collaborative enhancement to suppress the proliferation, invasion and apoptosis of tumor, and then explored the possible mechanisms with a view to provide a new direction for the comprehensive treatment of glioma.The present study was divided into two parts.In the first part of this study, we used 2'-O-methyl-oligonucleotides to overexpress miR-451 in A172, LN229 and U251 GBM cell lines, then miR-451 mRNA was quantified by real time PCR. MTT method was used to evaluate cell proliferation rate. Flow cytometry and the invasion and migration ability was detected by Transwell analysis and Scarification test. Annexin V-FITC were used for cell cycle apoptosis analysis respectively. Western blot was used to evaluate the expression of proteins. The result showed that miR-451 was upregulation following in miR-451 mimics transfection of A172, LN229 and U251 cells. The cell multiplication rate in miR-451 mimics group presented decreasing trend since the second day in culture (P<0.05), as compared with control and scramble group. The number of cells inhibited at G0/G1 phase in miR-451 mimics group was more than any other group (P<0.05). Transwell and Scarification test indicated that the invasion and migration ability of the cells in miR-451 mimics group were decreased obviously (P<0.05). The apoptosis rate in miR-451 mimics group was higher than that in the other two groups (P0.05). The protein expression of AKT1, Cyclin D1, MMP2, MMP9 and bcl-2 were lower in miR-451 mimics group, while p27 was significantly higher than those in the other two groups (P<0.05).In the second part, in vivo study was carried out to further investigate the miR-451 and its concrete mechanism in glioma. Eighteen athymic mice were randomly divided into 3 groups (control, scramble and miR-451 mimics group), and were treated respectively. Athymic mice xenogeneic transplant model was established by inoculation (sc) with LN229 glioma cells. Body mass (BM) and diameter of tumor mass were measured. Furthermore, The protein expressions of AKT1, CyclinD1, p27, MMP2, MMP9 and Bcl-2 in tumor tissues were analyzed with immunohistochemistry. The results show that the tumor-inhibiting rate of miR-451 mimics was significantly higher than the control (P<0.05) and scramble (P<0.05). The protein expression of Akt1, Cyclin D1, MMP2, MMP9 and bcl-2 were lower in miR-451 mimics group, while p27 was significantly higher than those in the other two groups (P<0.05).Conclusion:The suppressive effect of miR-451 mimics group is better than control and scramble group in vitro and vivo, which indicate that miR-451 may function as tumor suppressor in human gliomas. Its molecular mechanism may be as followed:the tumor suppressor activity of miR-451 may be regulated by the PI3K/AKT pathway to inhibit cell proliferation and invasion and induce cell apoptosis in GBM.