MicroRNA-517c Inhibits Autophagy And Epithelial-to-mesenchymal(Like)Transition Phenotype In Human Glioblastoma Via Disturbing P53Nuclear Translocation Conducted By KPNA2

Epithelial-mesenchymal transition (EMT) represents the molecular reprogramming and phenotypic changes involved in the conversion of polarised immotile epithelial cells to motile mesenchymal cells. This process allows the remodeling of tissues during embryonic development and is also implicated in the promotion of tumor invasion and metastasis.Autophagy, also called Type Ⅱ programed cell-death,refers to the process that cell degrade and recycle itself cytoplasmic cargo be transported to the lysosome. Resently, some new studies imply the close relationship pertinent autophagy to EMT in various epithelial carcinoma. Autophagy plays important role in benign to malignant transformation of tumor and in the initiation stage of tumor metastasis.Nevertheless, the precise contribution of autophagy to glioblastoma invasion and EMT is still needing more research to explain.Glioma represents the most common malignant tumor type in the central nervous system, which accounts for41-44.6percent of intracranial tumors. This type of tumor is characteristic of high mortality and morbidity and high recurrence rate. Patients diagnosed with the most malignant subtype, glioblastoma multiforme(GBM), only have a median survival time of no more than14months. Although remarkable progresses have been made on the available treatments for gliomas including surgery, chemotherapy and radiotherapy, the prognosis of patients is still dismal, which is mainly attributed to two major reasons. Firstly, the resistance to the chemo-and radiotherapy attenuates the treatment for GBM. And secondly, the intrinsic capacity of isolated tumor cells to invade the surround parenchyma makes that it is impossible for totally surgical resection in nearly all cases. Therefore, there is an urgent need for identification of key molecular targets in tumor migration and invasion to cure this disease.The role of microRNA (miRNA)in the tumorigenesis has been extensively studied and it sheds light on the potential application as an important adjuvant treatment since the beginning of this century. In our previous work, the chr19q13.41microRNA (miRNA) cluster (C19MC) that encodes54microRNAs was discovered with high DNA copy number in the11/45cases of supratentorial primitive neuroectodermal tumors (sPNET). Expression profiles demonstrate significant enrichment of self-renewal and survival genes in C19MC amplified tumors. Among the microRNAs, miR-512-3p,517a,517c,549a, and520g has significantly higher expression in sPNET samples with C19MC amplification. Moreover, these miRs expression data in two neural stem cell, different parts of fetal brain and Adult brain hinted miR-517c and519a might play important biological function in embryonic development. In order to test if the miRs also play some biological function in GBM cells, six cell lines of GBM were selected to undergo the Realtime-qPCR. Results indicated that the miR-517c and519a have higher expression level in two cell lines The above data promoted us to do further investigation.In this study, we sought to comprehensive analyze the precise biological function of miR-517c in GBM. Whether the function of miR-517c is related to autophagy and EMT? Whether the biological function is dependent on p53phenotype in GBM cells in vitro and in vivo? Aim to find a new strategy for treating GBM.Chapter1Autophagy promotes U87(p53wt)cell migration but not U251(p53mt)cellPurpose:To investigate the role of autophagy in glioma cell migration, whether its role is related to glioma cell p53phenotype.Methods:Temozolomide (TMZ) and low glucose medium culture were used to induce cell autophagy,3-MA and Chloroquine(CQ) were used as autophagy inhibitor. Cell migration ability of p53wild type U87cell and p53mutant type U251was verified under circumstances mentioned above by wound healing assay and Transwell assay. Western-blot and Immunofluorescence assay were used to confirm the autophagy activity in U87and U251cell treated with these reagents by detecting the autophagy marker protein LC3B.Results:After treatment of TMZ (150μM)24h, morphologic change were find in U87cell (p53wt) but not in U251(p53mt) cell,compared to non-treatment group. When used in combination with autophagy inhibitor3-MA or CQ, the morphologic change was disappeared. Both of the wound healing assay and Transwell assay showed TMZ enhanced U87cell migration (F=12.890, P=0.002), which can also be diminished by3-MA and CQ, the P value of TMZ group and normal culture TMZ+3-MA、TMZ+CQ wer0.002,0.007,<0.001respectivley.However, the migration enhancement effect of TMZ was not find in U251cell (p53mu) even the concentration of TMZ was increased to300μM. Western-blot and Immunofluorescence assay results showed that TMZ induced an increasing of the lapidated form of LC3B (LC3B-Ⅱ), which is a generally accepted marker of autophagy activity in U87cell but not in U251cell, and this increasing effect can be decreased by3-MA. Besides similar results were observed when U87cells were starved by low glucose culture, which also enhanced cell migration and infiltration through autophagy induction (F=25.629, P=0.001), the P value of LG vs normal culture,3-MA+LG, CQ+LG were0.017,<0.001,<0.001respectivley,but in U251cell low glucose culture had no these effects.Conclusion:TMZ (150μmol) and low glucose culture promotes U87(p53wt) cell migration through autophagy induction. In U251(p53mt) cell, TMZ (150μmol) and glucose culture cannot induce autophagy and had no effect in cell migration. These clues leads to close relationship between autophagy and glioma migration and this relationship maybe different in various p53phenotype glioma cells.Chapter2Section1In vitro, miR-517c inhibits U87cell migration, autophagy and EMT but not in U251cellPurpose:To investigate the biological function of miR-517c in U87and U251cell.Methods:Transfect chemical synthetized RNA oligonuclotide miR-517c mimics, Negative control (NC),miR-517c inhibitor (inhibitor) in U87cell. Cell counting kit (cck-8) assay was used to test cell viability. Annexin V-PI flowcytometry was used to test cell apoptosis. Cell cycle distribution were detected by PI-flowcytometry. Protein level of caspase-3, caspase-9and cleavage caspase-3were detected by Western-blot. Lentivirus as vehicle carrying a sequence of miR-517c, negative control (NC), miR-517c inhibitor(inhibitor) were used to established stable cells in U251and U87cell (U87-miR-517c, U87-NC, U87-inhibitor,U251-miR-517c, U251-NC, U251-inhibitor).Expression level of miR-517c in each type of cell were verified by qRT-PCR.Then we further used wound-healing assay and Transwell assay to measure cellular migration regulation by miR-517c on U87and U251cells. Western-blot were used to detect autophagy and EMT related protein in each kind of stable cells. LC3B level were also verified in each stable cell by Immunofluorescence assay. Transmission Electron Microscopy examination were carried out in each stables cell.Results:CCK-8data showed that miR-517c had no role in regulating U87cell viability when treated with TMZ, there were no significant differences of cell viability at6h(F=1.019P=0.416),24h(F=0.677,P=0.543),48h(F=0.112,P=0.896),96h(F=1.019,P=0.257).The cell cycle distribution had no differences between three groups (F=0.063,P=0.680).Cell apoptosis were examined by using flowcytometric analysis with Annexin V/FITC staining,results showed no significant differences between each groups. Western-blot showed the Caspase3and Caspase-9protein level had no significant differences between these three groups. After puromycin selection, we find that there were morphologic differences between U87miR-517c, NC and inhibitor stable cell. But in U251cell, the miR-517c, NC,517c group had no such morphologic discrepancy. Wound healing migration and Transwell results showed, no matter under normal medium(F=45.473, P<0.001) or TMZ application(F=36.055, P<0.001), under normal culture the P value of miR-517c VS NC and miR-517c VS inhibitor were0.003,<0.001, and under TMZ treatment, the P value of miR-517c VS NC and miR-517c VS inhibitor were0.005,<0.001respectively,which hinted miR-517c inhibits U87cell migration induced by TMZ (150μM). But in U251, the three groups had no such difference in migration capability. As the Western-blot results indicated, in miR-517c group, autophagy key proteins level, such as, Beclin1, Atg3, Atg7, Atg5, and Atg12were down-regulated in p53wild type U87cell. The decreasing of LC3B convert (LC3B-Ⅱ/Ⅰ) is also observed, which indicated the inhibition of autophagy status by miR-517c. Meanwhile, in U87-miR-517c group, the epithelial marker protein level, such as T-cadherin, Claudin were higher than NC and inhibitor group, but the mesenchymal marker protein as N-cadherin, β-catenin, Vimentin were significantly down-regulated. Similarly, the expression of EMT-related regulators, such as ZEB1, slug and Snail were down regulated in miR-517cgroup. The fluorescence intensity indicated a significant decreased numbers of LC3B punctate in U87miR-517c group. However in U251cell, these autophagy and EMT-related proteins had no significant different level. Transmission Electron Microscopy examination results showed, among the three groups of U87cell, miR-517c cells had less autophagic vacuoles which is the direct evidence of autophagy process. But in U251cell, each groups almost had similar number of autophagic vesicles, which indicated miR-517c had no such functions in U251.Conculsion:In vitro, miR-517c had no significant role in cell viability, cell cycle, cell apoptosis of U87cell treated with TMZ, but miR-517c inhibits changes cell morphology appearance,inhibits cell migration, autophagy and EMT. In U251cell, such effect of miR-517c were not found.Chapter2Section2In vivo, miR-517c inhibits U87cell migration, autophagy and EMTPurpose:To confirm whether miR-517c also inhibits U87cell migration, autophagy and EMT in vivoMethods:To further testify the speculation that miR-517c reduces the tumor invasion and inhibits autophagy and EMT phenotype in vivo. Subcutaneous transplantation tumor in nude mice were carried out.10nude mice were divided into two groups, each contains5.5×106U87miR-517c(left), U87NC(right) or U87inhibitor(left),U87NC(right) cells were subcutaneously injected into the flank of nude mice. Every four days, the tumor volume was measured, then after24days, mice were sacrificed, tumor formation was confirmed by KODAKA2000system. The tumor and periphery tissue were meticulously dissected and photographed. Tumor tissue slid were under immunofluorescence observation or H&E stain. EMT related protein expression and localization was analyzed by immunochemistry. Autophagy related vacuoles in tumor tissue was verified by Transmission electronic microscopy.ResuIts:Tumor formation was succeed in all10mice, the tumor volume of miR-517c group was large than NC group (F=2.155, P=0.216), and the tumor volume of inhibitor group was large than NC group with significant differnces (F=36.685,P=0.004). As indicated in H-E and immunoflurocence photos of tumor tissue, the miR-517c tumors had less invasive characteristics, compared to NC and inhibitor tumors. Immunehistochemical staining showed in U87-miR-517c group, the epithelial marker protein level, such as T-cadherin, Claudin were higher than NC and inhibitor group, but the mesenchymal marker protein as N-cadherin, β-catenin, Vimentin were significantly down-regulated. Similarly, the expression of EMT-related regulators, such as ZEB1, slug and Snail were down regulated in miR-517c group. By transmission electron microscopy examination, the number of autophagic vacuoles were different in three types of tumor, miR-517c tumor with less autophagic vacuoles, NC and inhibitor tumor have more. Conclusion:In vivo study also confirmed miR-517c’s function in U87cell,which inhibits tumor invasion and also inhibits autophagy and EMT.Chapter3Section1KPNA2is the target of miR-517c and exert its function by disturbing p53nuclear cytoplasmic transportPurpose:Two-dimensional electrophoresis and MALDI-TOF MS combined with biological database searching to find out target of miR-517c. To confirm the target by biological experiment; To explore the mechanism of target’s function.Methods:Two-dimensional electrophoresis analysis of U87transfected with miR-517c mimics and NC were done. The protein spots of interest were successfully identified by MALDI-TOF MS and by subsequent comparative sequence search in the Mascot database, which indicate the KPNA2gene is the possible target of miR-517c. Western-blot were used to detect the KPNA2protein level in U87and U251stable cells.luciferase reporter plasmids were constructed by inserting wild-type and mutant3’-UTR of KPNA2mRNA bearing miR-517c putative binding sites into pGL3-control vector immediately downstream of the luciferase coding sequence. Effect of plasmid pGL3-KPNA2and pGL3-KPNA2-mutant was verified by RT-PCR. Luciferase report assay was used to verify the KPNA2-3’UTR region’s luciferase activity. P53localization in U87and U251cell after transfected with miR-517c,NC,inhibitor were confirmed by Confocal immunofluorescence.Results:Two-dimensional electrophoresis analysis of U87transfected with miR-517c mimics and NC were done.The protein spots of interest were successfully identified by MALDI-TOF MS and by subsequent comparative sequence search in the Mascot database, which indicate the KPNA2gene is the possible target of miR-517c. Although we did not find KPNA2as the target gene of miR-517c in TargetScan and micron database, but we find only two nucleotides were different between miR-517c and miR-519a and as chance would have KPNA2is the target gene of miR-519a, so we still believe KPNA2is possible the target of miR-517c.Further repeatedly Western-blot validation showed in both U87and U251cell, KPNA2protein level is down-regulated in miR-517c group compared to NC, and inhibitor groups Effect of plasmid pGL3-KPNA2and pGL3-KPNA2-mutant was verified by RT-PCR. Co-transfected luciferase reporter plasmids with the miR-517c mimics, mimics NC, miR-517c inhibitor, inhibitor NC into U87cells to perform luciferase reporter assays. MiR-517c significantly inhibited the luciferase activity in the reporter bearing the wild type KPNA23’-UTR, but not in the plasmid with a mutated KPNA23’-UTR. In contrast, inhibitor did significant increase the luciferase activity of the reporter containing the wild-type KPNA23’-UTR(F=61.113, P<0.001; both of the P value of miR-517cVS mimicsNC InhibitorVS Inhibitor NC were less than0.0010), there were no such differneces in pGLV-3vector (F=0.074,P=0.653) and pGL3-KPNA2-3’UTR-mutant vector(F=0.973, F=0.590), which suggesting KPNA2is one direct target of miR-517c. Confocal immunofluorescence result showed in U87miR-517c cell, more p53protein was located to the cytoplasm compared to NC and inhibitor groups. However, in p53mu U251cells, there was no significant difference in different groups.Conclusion:KPNA2is the target of miR-517c, miR-517c has function in disturbing p53nuclear cytoplasmic translocation, which hints miR-517c exert its autophagy and EMT inhibitory function through disturbing p53nuclear cytoplasmic translocation by KPNA2. Chapter3Section2miR-517c exert its function through KPNA2and is p53dependentPurpose:To explore the relation of KPNA2expression level and autophagy activity; To explore whether miR-517c’s function is p53dependent.Methods:Plasmid pCDNA-KPNA2and pSilencer-KPNA2-shRNA to achieve KPNA2overexpression and knock-down in U87and U251cell. Autophagy related protein were analyzed in U87-KPNA2-oe, U87-KPNA2-shRNA, U87-NC, U251-KPNA2-oe, U251-KPNA2-shRNA,U251-NC cell by Western-blot and immunofluorescence assay. Plasmid psilencer-p53-shRNA was used to knockdown p53level in U87cell, then Western-blot were used to test autophagy related protein.To further validate if the miR-517c’s function is through KPNA2and wide-type p53dependent, the human P53+/+and P53-/-HCT116cell were used for Western-blot.Results:Western-blot and immunofluorescence assay showed, lower autophagy related protein level were observed in U87-KPNA2shRNA cell compared to U87-KPNA2oe and U87-NC cell, but there was no such differences in U251-KPNA2oe,U251-KPNA2shRNA, U251-NC cell. Result of Western-blot showed that after p53knockdown in U87cell, miR-517c cannot down regulation autophagy related protein. In P53+/+HCT116cell KPNA2and autophagy related proteins such as Beclin1,Atg3,Atg5,Atg7,Atg12level in miR-517c mimics group were significantly down-regulated as the same way in U87glioma cell. The convert of LC3B-Ⅰ to Ⅱ also decreased. But in p53-/-cell, though in miR-517c mimics group KPNA2protein level was down-regulated, but the autophagy related protein level has no changes between groups. Conclusion:Expression level of KPNA2was positive related to autophagy activity in U87cell but not in U251cell. MiR-517c exert its autophagy inhibition function through KPNA2and is p53dependent, the miR-517c/KPNA2/p53pathway plays important role in regulation of autophagy and EMT in glioma cell.Chapter4Expression level of miR-517c relates to outcomes of GBM patientsPurpose:To analysis the expression level of m miR-517c and prognosis of GBM patientsMethods:Expression levels of miR-517c and miR-519a were measured in46glioma samples using real-time quantitative PCR. Patient clinical data was reviewed and analyzed. Relation between patient outcomes and miR-517c were analyzed by Kaplan-Meier analysis.Results:In17cases (36.9%), the miRs level is higher than average mean, being thought as high expression as miR-517c (+). The other29cases are thought as low expression as miR-517c (-). By Kaplan-Meier analysis, the tumor-free survival curve showed better prognosis for miR-517c (+) patients compare to miR-517c (-) patients(χ2=1.403, P=0.001).Futhermore, the overall survival cure also showed better prognosis for miR-517c(+) patients compare to miR-517c(-) patient(x2=6.619, P=0.01).Conclusion:lower expression level of miR-517c related to poorer outcomes in GBM patient.