The Research On Mechanism Of MiRNA-451 Regulation Of Human Brain Glioma Cells Energy Metabolism Of Glucose And Biological Behavior

Malignant glioma is the most common primary brain in the human central nervous system tumors. The median survival time is about 14 months. It is easy to relapse, unlimited proliferation, and invasive growth, and adds great difficulty to clinical treatment. The traditional neurosurgery, radiation and chemotherapy method is still hard to cure malignant glioma. Glioma is found more abnormal gene expression of tumor, including the anomaly over expression of protooncogene and deletion or mutation of tumor suppressor genes, leading tumor cells to escape regulation mechanism of the normal growth. With the rapid development of genetic and molecular biology in recent years, to explore the molecular mechanism of the occurrence and development of glioma and find new treatments becomes a field of research at home and abroad.Micrornas is a kind of length is about 19-25 nucleotide sequence of non-codingRNAs, and the 3 ’end of the target genes of transcription area part or all of the specific binding, and degradation of the target mRNA or inhibition of its protein translation process, so as to realize to adjust and control of gene transcription. So far, the thousands of miRNAs were found in the human tissues, and about a third of protein-coding genes through the miRNA regulation, which own highly specificity of time, space and tissue. The same miRNA can targeting multiple mRNA genes at the same time, and a target mRNA gene can also be controled by a number of different micrornas. Therefore, the role of micrornas was called "one-to-many or many-for-one". Interact with target genes, they not only involved in the cell and tissue growth, metabolism, new blood vessels formation, but slso mediated cell proliferation,apoptosis and invasion biology process. And they play an important role in regulation of energy metabolism in the process of the occurrence and development of tumor.Therefore, It will provide a new research direction for cancer research field including tumor markers and tumor targeting therapy.Numerous studies have demonstrated the miRNA-451 locates on chromosome17q11.2 position, and is adjacent to HER2 17q11.2-21 area. It was abnormal expression in many malignant tumors, especially significantly reduced in malignantglioma stem cells.Godlewski thinked the miRNA-451 could inhibit the migration of glioma cells, and the RT-polymerase chain reaction(PCR) to prove that the miRNA451 expression of migration glioma cells appeared to reduce. Godlewski also proposed the miRNA-451 under certain conditions could control the switch of energy metabolism, the metabolism of tumor cells could still change adaptively in a terrible environment pressure. Kim Y testified the role of mutual antagonism between miRNA-451 and AMPK existed in the process of malignant glioma cell proliferation and invasion. And the miRNA-451 overexpression or CAB39/AMPK pathway by blocking could significantly inhibit malignant glioma cell invasive ability. David Carling thought AMPK was a kind of natural energy receptor, mediating the energy balance of eukaryotes. Christine K thought that inhibition of PI3K/AKT pathway can prevent tumor cells from glycolysis metabolism glucose for energy. Warburg confirmed tumor cells both in aerobic and anaerobic conditions are through glycolysis reaction glucose to provide energy(ATP). And glucose into energy metabolism within the tumor cells must depend on the carrier protein on the cell membrane(glucose transporters, GLUT). And GLUT1 is most widely expressed in mammalian transporter, especially in the more common parenchymal cells. And GLUT1 as the typical representative of the family of GLUTs, significantly higher expression in a number of cancers, especially the expression of malignant glioma increased obviously.Our pilot study has elaborated the miRNA-451 could inhibit the growth of malignant glioma cells, but the inhibition mechanism is unclear. So the purpose of this study was that to explore the miRNA-451 was likely to regulate and control glucose transporter in malignant glioma cell, thus affected the efficiency of energy metabolism of glucose and eventually impacted on biological behavior of malignant glioma cells.This topic will study from the following two aspects:At first, the miRNA-451 influenced on glucose energy metabolism of brain malignant glioma cell line. Routine cultivating human brain glioma cell line LN229 and U87 two cell lines, and lentivirus vector carrying oligonucleotide miRNA-451 and miRNA-NC to transfect. Experiments were divided into Blank control group,Lentivirus micrornas negative control(LV-miRNA-NC)and Lentivirus miRNA-451group(LV-miRNA-451); Real-time quantitative polymerase chain reaction(RT-PCR)results showed the miRNA-451 transfection efficiency and GLUT1 mRNA expression level from malignant glioma cell line in different groups; cell immunofluorescence and Western Blot experiments verified GLUT1 protein expression level in each group;STRING computer software and preliminary research builded the miRNA-451 regulation signaling pathways of GLUT1 gene; Western Blot experiments further verified the miRNA-451 control the expression of GLUT1 gene related to protein in the signaling pathway; Automatic microplate reader analyzed glucose uptake, lactic acid production and ATP in malignant glioma cell line. The result showed: compared with Blank control and LV-miRNA-NC group, RT-PCR experiments proved that the miRNA-451 expression quantity increased obviously in LV-miRNA-451 group, and GLUT1 mRNA expression level decreased obviously;Cell immunofluorescence and Western Blot experiments proved that GLUT1 protein expression quantity significantly reduced in LV-miRNA-451 group; Signal path diagram showed the miRNA-451 targeted CAB39 through LKB1/ AMPK/PI3K/AKT pathway regulating the expression of GLUT1 gene; Western blot experiments results proved that the miRNA-451 regulatory GLUT1 gene related purpose protein LKB1,AMPK, PI3 K, AKT and GLUT1 expression quantity were significantly reduced in LV-miRNA-451 group; Automatic microplate reader detection results proved that intracellular glucose uptake, lactic acid secretion and ATP product significantly reduced in LV-miRNA-451 group, differences were statistically significant(P<0.05).The second aspect: the MiRNA-451 affects brain malignant glioma cell biology behavior. according to the above methods transfection and grouping, quantitative polymerase chain reaction(RT-PCR) research results show miRNA-451 transfection efficiency from malignant glioma cell line in different groups; CCK-8 method and cell plate clone formation experiment tested glioma cells proliferation activity ability from different treatment group; Transwell experiment tested glioma cell invasion ability; wound healing assay tested glioma cell migration ability; Western blot experiments detected malignant glioma cell biology behavior related to protein expression. The results showed: compared with Blank control and LV-miRNA-NC group, RT-PCR experiments proved that miRNA-451 expression quantity increasedobviously in LV-miRNA-451 group; CCK-8 method and cell plate cloning experiments detected glioma cell proliferation activity ability significantly reduced in LV-miRNA-451 group; Transwell experiments results proved that glioma cells invasion ability decreased significantly in LV-miRNA-451 group; wound healing assay results showed that glioma cell migration ability was restrained obviously in LV-miRNA-451 group. Western blot experiments results showed that malignant glioma cell biology behavior related protein CyclinD1, MMP-2 and MMP-9, PCNA and E-Cadherin protein expression quantity decreased in LV-miRNA-451 group,differences were statistically significant(P<0.05).Conclusion: the miRNA-451 targets CAB39 through LKB1/AMPK and PI3K/Akt pathway obvious inhibition GLUT1 gene expression in the glioma cell line,reducing the rate of glucose transporter, and decreacing malignant glioma cells glucose uptake and discharge energy; Eventually the miRNA-451 significantly inhibits malignant glioma cell proliferation, invasion and migration ability.