Clinical Relevance And Underlying Mechanism Of Abnormal Expression Of GLUT3 In Glioma

Part Ⅰ The expression and functional role of GLUT3 in gliomaBackgroundGlioma is one of the most common tumors in our brain. It makes up about 30% of all brain and central nervous system tumors and 80% of all malignant brain tumors [2]. Because of the diffuse invasive growth pattern and its important location, it is difficult to be completely removed by surgery. Because of the existence of the blood-brain barrier (BBB), many chemotherapy drugs are difficult to reach the tumor and the chemotherapy effectiveness is limited [3]. Because the tumors contain many different types of cells, although some cells may respond well to certain therapies, while others may not be affected at all. So, treatment of glioma remains one of the most challenging tasks. Even with standard treatment (Surgical resection+ chemotherapy+radiotherapy), median survival for adults with an anaplastic astrocytoma is about two to three years. For adults with more aggressive glioblastoma, treated with concurrent temozolamide and radiation therapy, median survival is about 14.6 months and five-year survival is less than 6%.It is necessary to do more research in glioma, and make good understanding of the molecurlar mechanism of glioma development, and to find some new therapy targets. Glucose is a vital metabolic fuel for all mammalian cells. However, glucose moleculers can not diffuse the phospholipids’plasma membrane freely[7]. Glucose is transported into the cell via facilitative glucose transporters (GLUT) protein family. The GLUT protein family has 14 members [8]. According to their sequence similarity, these fourteen GLUT proteins can be categorized into 3 classes:Class 1 (GLUTs 1-4, 14); Class 2 (GLUTs 5,7,9, and 11); and Class 3 (GLUTs 6,8,10,12, and HMIT)[9] 10]. Glucose transporter family members can be divided into two categories as well: sodium-dependent and non-dependent sodium ions. Sodium-independent glucose transporters (facilitated transport; GLUT) and sodium-dependent glucose transporters (secondary active transport; SGLT), each with different kinetic property[11, 12] Most cells express a variety of glucose transporters and the pattern of expression in different tissues is related to specific metabolic requirements. In the brain, more glycosylation GLUT1 is mainly expressed in the vessel wall and in charge of transferring glucose across the blood-brain barrier, less glycosylation GLUT1 mainly expressed in astrocytes[11]. GLUT2 is mainly expressed in the hypothalamus neurons [13,14] GLUT3 is most abundant in the brain, and is mainly expressed on neurons. The glucose transport capacity of GLUT3 is about five times more than that of GLUT1 [15]. However the expression pattern can be changed under pathological condition. Under physical condition, GLUT3 is hard to be detected in glia cells [33]. But in glioma, the expression of GLUT3 increased gradually with the increase of pathological grade of glioma, respectively[20]. Why does GLUT3 express in glioma? Does it play some important roles in the development of glioma? In this study, we want to investigate the abnormal expression of GLUT3 on glioma and what roles of GLUT3 play in the development of glioma.Objective:Detect the expression level of GLUT3 and Ki-67 in glioma specimens by immunohistochemistry, and identify the relationship between the amount of GLUT3 and the proliferation of glioma cells. Then we inhibit the expression of GLUT3 by virus-mediated RNA interference, and observe the influence of GLUT3 silencing on glioma cells’ proliferation, invasion and migration capability and apoptosis of glioma.(1) Changes of the expression of GLUT3 and ki67 in glioma during its developmentMethods:1) 20 glioma specimans (including 5 specimans of each grade) were collected.2) Immunohistochemical studies were performed for the expression of GLUT3 and ki-67 in these specimans.3) Statistic analysis:all data were analyzed by SPSS 17.0 software, with t-test or one way ANOVA, p< 0.05 was considered as significance.Results:1) The expression of GLUT3 in the glioma specimens are increasing as the pathological grade increasing (grade Ⅰ:0.900±0.909%, gradeⅡ:13.420±3.653%, gradeⅢ:62.280±8.094%, grade Ⅳ:93.340±5.212%, Tamhane’s, P<0.001).2) The expression of Ki-67 in the glioma specimens are increasing as the pathological grade increasing (grade Ⅰ:0.600±0.833%, gradeⅡ:11.140±4.291%, gradeⅢ:20.560±3.759%, grade Ⅳ:31.500±4.896%, Tamhane’s, P<0.001).3) The expression of GLUT3 and Ki-67 in the fast growing region are greater than that in the low growing region of same glioma specimen (GLUT3: 93.340±5.212%/53.267±6.341%,t=-23.963, P<0.001; Ki-67:32.333 ±6.562%/ 21.333±5.158%, t=-7.224, P<0.001)4) High expression of GLUT3 is positive related with high expression of ki-67(Spearman, rs=0.928, P<0.001).Conclusion:As glioma developing, the pathology grade was increasing, the tumor cells have growing faster and faster and the expression of GLUT3 was increasing accordingly.(2) The influence of GLUT3 silencing on glioma cells.Methods1) Constructed lentiviral vectors: Lv-GLUT3 (expressing GLUT3 shRNAs) and Lv-NC (expressing non-specific shRNAs)2) Infected the U251 glioma cells respectively;3) Detected and compared the expression of GLUT3 in each group;4) Detected and compared the proliferation, invasion capability, migration capability and apoptosis in each group;5) Statistic analysis:all data were analyzed by SPSS 17.0 software, with t-test or one way ANOVA, p< 0.05 was considered as significance.Results1) Compared with Lv-NC, Lv-GLUT3 can effectively inhibit the relative expression of GLUT3-mRNA (0.0000615±0.0000202/0.000647±0.000237,t=7.383, P<0.001), inhibit the expression of GLUT3-protein (0.108±0.0159/0.364±0.0444, t=16.267, P <0.001)2) Compared with Lv-NC, Lv-GLUT3 infection can inhibit glioma cell uptaking glucose (OD505:0.532±0.0308/0.449±0.0323, t=-5.619,P<0.001)3) Compared with infected Lv-NC, U251 cells infected with Lv-GLUT3 have lower proliferation in 48 h(OD490:0.780±0.0257/0.859±0.0157),72 h(OD490: 1.018±0.0068/1.206±0.0165),96 h(OD490:0.9294±0.0115/1.022±0.00387) after infection.4) Compared with infected Lv-NC, U251 cells infected with Lv-GLUT3 have lower invasion capability and the number of cells passed though the matrigel are: 14.400±3.851/67.267±10.053, t=19.019, P<0.001.5) Compared with infected Lv-NC, U251 cells infected with Lv-GLUT3 have lower migration capability and the percentage of unhealed wound area are:12 h(0.779±0.088/0.515±0.072, t=-9.802, P <0.001),24 h(0.566±0.121/0.211 ± 0.0305, t=-13.991, P<0.001),36 h(0.186±0.0744/0.0826 ± 0.150,t=-2.612, P=0.013).6) Compared with infected Lv-NC, U251 cells infected with Lv-GLUT3 have higher apoptosis rate (0.133±0.00290/0.0777±0.00630,t=2.757, P<0.001)Conclusion:GLUT3 silencing can inhibit glioma cells’glucose uptake and slow down glioma cells’ proliferation, invasion capability,migration capability and promote apoptosis.Part Ⅱ The study on the involvement of SP1 in the regulation of GLUT3 expression in gliomaBackgroundIn the glioma specimens, both 4.2 and 2.7 kb GLUT3/actin mRNA ratios showed a linear correlation with the glioma grade.It suggests that the transcription of GLUT3 gene is up-regulated. In eukaryotes, regulation of transcription is a result of the combined effects of DNA structural properties and the interactions of proteins called transcription factors.Transcription factors whose function is to activate (or inhibit) transcription of DNA by binding to specific highly conserved DNA sequences . These basic components required for eukaryotic gene transcription have been highly conserved in evolution.With the development of bioinformatics science, we can find these conserved DNA sequences by bioinformatics sofwares.Specificity protein 1 (SP1) is an important transcription factor. It is a zinc finger transcription factor that binds to GC-rich motifs (5’-(G/T)GGGCGG(G/A) (G/A)(C/T)-3’)of many promoters. SP1 is invoLved in many cellular processes, including cell differentiation, cell growth, apoptosis, immune responses, response to DNA damage, and chromatin remodeling.After analyzing the 5’ un-translated region of human, rat and mouse GLUT3 gene by TFsearch, we find that they all have three SP1 putative binding sites. Rosario A. Rajakumar, etc. reported that Spl down-regulated the expression of GLUT3 in N2A cells and the suppressive effect of Spl is dependent on the presence of putative trans-acting factors interacting with the DNA sequence between bp-203 and-177.Copland JA, etc. reported that in L6 cells Sp1 can increased the expression of Glut3, and Sp1 specifically bound to only one of three Sp1 binding sites . Ganguly A reported that calories restriction (CR) in C57BL6 pregnant mice from gestational days 10 to 19 observed a 50% diminution in placental Glut3 expression. The mechanism under this phenomenon is that CR enhanced DNA methylation of a CpG island in the promoter region of GLUT3 gene, enhanced MeCP2 binding to the CpG island of the glut3 gene, but interfered with Sp1 binding.These results suggest that SP1 invoLved in the regulation of GLUT3 expression though binding to certain sites, and this regulation has tissue specific. Sp1 is upregulated in human glioma, high Spl expression was correlated strongly with the WHO grading [32]. Here we wonder that what role of SP1 plays in the transcriptional regulation of GLUT3 gene in glioma.Objective:To investigate what role of SP1 playing in the transcriptional regulation of GLUT3 gene in glioma.SP1 bind to the promoter of GLUT3 gene and up-regulate the expression of GLUT3 in gliomaMethods:1) Construct recombinant vectors pIRES2-ZsGreenl-SP1 expressing Spl, the firefly luciferase reporter gene recombinant vectors pGL3-GLUT3-wt and Spl binding site mutunt pGL3-GLUT3-mut.2) Culture U251 cells, and transfect pIRES2-ZsGreenl-SP1, pIRES2-ZsGreenl, respectively, detect the relative expression of SP1 and GLUT3 by RT-PCR and western blot, analyze the relation between the expression of SP1 and GLUT33) Culture U251 cells, and cotransfect pGL3-GLUT3-wt+pRL-TK+ pIRES2-ZsGreen1-SP1,pGL3-GLUT3-mut+pRL-TK+pIRES2-ZsGreenl-SPl and pGL3-GLUT3-wt+pRL-TK+pRL-TK+pIRES2-ZsGreenl respectively, detect the relative luciferase activity by Dual-Luciferase(?) Reporter Assay System, analyze the influence of SP1 on the promoter activity of GLUT3 gene.4) Culture U251 cells, do CHiP assay to test whether SP1 bind to the promoter of GLUT3 gene.5) Statistic analysis:The data was analyzed with one-way analysis of variance or t-test using SPSS version 17.0. In all statistical analyses, a P<0.05 was considered as statistically significance.Results:1) The sequencing results show that the sequences of pIRES2-ZsGreen1-SP 1, pGL3-GLUT3-wt and pGL3-GLUT3-mut are correct.2) Compared with control group which transferred with pIRES2-ZsGreen1, the expression of SP1 and GLUT3 increased in the group transferred with pIRES2-ZsGreenl-SP1(SP1-mRNA:0.573±0.0900/0.0844±0.00919,t=-16.204, P<0.001; GLUT3 mRNA:0.513±0.0656/0.0392±0.0104,t=-16.204,P<0.001; SPl-protein:0.414±0.0573/0.212±0.0394, t=-8.585, P<0.001; GLUT3-protein: 0.337±0.0557/0.114±0.0400,/=-9.721, P<0.001)3) Compared with control group which transferred with pGL3-basic+ pRL-TK, the relative luciferase acitivity in the group transferred with pGL3-GLUT3-wt+ pRL-TK increased(37.477±4.870/2.367±0.760,t=21.370,p<0.001).4) The relative luciferase acitivity in the group transferred with pGL3-GLUT3-wt+ pRL-TK+ pIRES2-ZsGreenl-SP1 is higher than that in the groups transferred with pGL3-GLUT3-mut+pRL-TK+pIRES2-ZsGreenl-SP1 or pGL3-GLUT3-wt + pRL-TK + pIRES2-ZsGreen1 (56.561±11.012/27.212±5.920, LSD, p<0.001; 56.561±11.012/25.841±4.726, LSD.<0.001).There is no significantly relative luciferase acitivity’s difference between the groups cotransferred pGL3-GLUT3-wt + pRL-TK + pIRES2-ZsGreenl and pGL3-GLUT3-mut + pRL-TK + pIRES2-ZsGreenl-SP1(27.212±5.920/25.841±4.726, LSD, P=0.710).5) The CHiP assay thows that a 182 bp DNA fragment corresponding to putative Spl binding site within the promoter of GLUT3 gene (nt-381/-200) was amplified from U251 cells when chromatin was immunoprecipitated with Sp1 antibody. That is to say, SP1 binded to the promoter of GLUT3 gene in glioma cells.Conclusion:1) Increase the expression of SP1 will upregulate the expression of GLUT3 in glioma cells2) SP1 binded to the 5’-UTR of GLUT3 gene, and promote the transcription of GLUT3 gene.