Study On The MiR-451 For Treating Human Gliomas Through Regulating AMPK Pathway Mediated By Bone Mesenchymal Stem Cells

The molecular development of glioma is a complex process which involves multi-genes changes and their relating signal pathways abnormalities. All the signal pathways comprise a tight manipulation net to regulate the abnormal pathological process of cell cycle, development and differentiation, apoptosis and death, signal transduction and metabolism of the tumor cells.It is reported that more than 100 miRNAs are in correlation with glioma.Abnormal expression of miR-451, which locates on chromosome 17q11.2, has been reported in many malignant tumors, such as glioma, breast cancer, ovarian cancer, etc.In our previous research, it was found that miR-451 was low expression in human brain glioma tissues, and its’ expression level was negatively correlated with the pathological grade of gliomas. By up-regulating the miR-451 level in the glioma cell lines through the vehicle of Lipofectamine 2000, and by means of the experiments of cell cycle test, MTT, invasion test and apoptosis test, the suppressive function of miR-451 as a suppressor gene was verified. Through bio-infomatics analysis and Luciferase reporter gene assay, it was found that the target gene of miR-451 was CAB39 and the miR-451 acted as a suppressor gene through the pathway of LKB1(CAB39)-AMPK-PI3K-AKT in vitro and vivo experiments. But the signal pathway of LKB1( CAB39)- AMPK-PI3K-AKT is just a part and the shared part of signal pathways of malignant tumors, so whether miR-451 could regulate other pathways through AMPK and its specific mechanism is not yet completely clear.The fast growth of solid tumors can lead to hypoxia condition and then the AMPK and HIF-1α will be activated and the expression of VEGF will be enhanced, and eventually, the tumors’ angiogenesis will be promoted and the ability of proliferation and metastasis will be boosted up. It was reported that the expression level of miR-23 b was high in glioblastoma which acted as a oncogene in essence, and the HIF-1α—VEGF signal pathway would be inhibited by the down-regulation of miR-23 b. Because the function of miR-451 was just on the opposite to miR-23 b, so the problem of whether the pathway of AMPK-HIF-1αcould be inhibited by up-regulating miR-451 had attracted our great attention.Nowadays, With the development of molecular biology, gene therapy becomes the most promise measure in the treatment of malignant gliomas. Selecting appropriate drug / gene carriers for gene therapy is the first and foremostconsideration. Cell-based carriers are emerging as a hopeful therapeutic choice in the field of cancer treatment. Because mesenchymal stem cells(MSCs) have the great capability of self-renewal, relative ease of isolation and culturing, powerful ability of expansion in vitro, good tropism and homing to tumors, they will be promising cell therapy vehicles for delivering treatment agents into tumor cells.The main topic of this research is to investigate how the miR-451 regulate the biological characteristics of glioma by targeting CAB39 through AMPKHIF-1α-VEGF signal pathway after the level of miR-451 in the tumor cells was up-regulated by hBMSCs which severed as cellular vehicles to deliver miR-451 into glioma cell lines. There are 3 correlative parts in this essay.The first part of this article is about the isolation, culture and identification of hBMSCs and the literature review study of which act as cellular vehicle for gene therapy of glioma. The bone marrow tissue was taken from an adult male femoral shaft fracture patient and the hBMSCs were isolated by density gradient centrifugation combined with adherence screening method. After the hBMSCs were acquired, through conventional subculture for 3-5 generations, the non-adherent and various mixed cells could be eliminated gradually, and as a result, the highly purified, stable hBMSC cell lines were established. By means of flow cytometry, the surface antigens of CD29, CD44, CD71 were positive and HLA-DR, CD34, CD45 were negative. Then the ability of hBMSCs differentiating into adipocytes and osteoblasts was tested, and the results were in line with the test requirements. In terms of literature review and analysis, we found that hBMSCs showed a clear tumor tropism ability and could migrate into and fuse with the tumor tissue, and thus could be used as a promising cellular vector carrying the therapeutic genes and modified cytokines for malignant glioma gene therapy.In the second part of this thesis, firstly, the lentivirus was used as vehicle to deliver the miR-451 into hBMSCs, and RT-PCR was performed to detect the miR-451 expression level in the transfected hBMSCs. Then the supernatant of the culture medium was collected and used as culture medium to cultivate the U87, U251,LN229 human glioma cell lines to make the miR-451 level up-regulated. The expression level of miR-451 in the tumor cells was verified by RT-PCR. In the following experiments, the nude mice were divided into control group, hBMSCs group and LV-miR-451-hBMSCs group according to different treatment measures.The inhibiting function of miR-451 which acted as a tumor suppressor gene in the glioma was certified by a series of experiments, including MTT assay,plate colonyformation assay, cell cycle analyzing by flow cytometry, transwell migration assay,wound healing study, Annexin V test, and Western blot. All these results revealed the promising cellular carrier function of hBMSCs, and the miR-451 regulate the AMPK-HIF1α-VEGF pathway by targeting CAB39.The third part is mainly about the experiments in vivo by creating the heterogenous intracranial glioma model in nude mice. After the construction of U87 glioma cells with over-expressed luciferase, the intracranial glioma model in-situ was established by the application of stereotactic injection. 7 days after the establishment of the model, all the nude mice were divided into 3 groups randomly:LV-miR-451-hBMSCs group, hBMSCs group and control group. Then the real-time bioluminescence imaging for tumor tissue was performed and treatment measures were given. The data of the body weight, the fluorescence intensity and the death time of dead mice were recorded and analyzed. The survival curves were depicted and survival analysis was carried out using Kaplan-Meier method. The brain tissue of the dead mice was dealt with by H-E and immunohistochemical staining. Results showed a down-expression trend of the proteins of CAB39, LKB1, AMPK, HIF-1αand VEGF in the LV-miR-451-hBMSCs group, which are important members of the AMPK-HIF-1α-VEGF signal pathway. All these results indicated that the miR-451 inhibit glioma through this pathway and act as a tumor suppressor gene.