Study On The Roles Of The Abnormal Decrease Of MiR-135a-5p In Gliomagenesis And Progression As Well As The Mechanisms

Objectives:The abnormal expression of miR-135a-5p exerts opposite roles in different tumors.Previous studies have found that miR-135a-5p is abnormally decreased in gliomas,thus we use bioinformatics to predict that the TRAF5 is a potential target gene for miR-135a-5p.As a signal transduction protein,the expression of TRAF5 is abnormal increased in several kinds of tumors.However,whether miR-135a-5p targets at TRAF5 in glioma cells and the relationships between their expression levels and glioma cell proliferation or patients' survival still need further study.We design the study for: 1.To examine the expression levels of miR-135a-5p and TRAF5 in the different grades of gliomas.Analyze the correlation between the expressions of miR-135a-5p and TRAF5,and the relationships between their expressions and the proliferation of tumor cells or the prognoses of glioma patients.2.Determine whether TRAF5 is a potential target gene of miR-135a-5p in the glioma cells.3.To observe the effects of miR-135a-5p and TRAF5 on cell cycle and proliferation of glioma cells,and further explore the molecular pathways by which miR-135a-5p and TRAF5 affect the biological behavior of glioma cell proliferation.4.To further confirm the effects of miR-135a-5p and TRAF5 on glioma growth and cell proliferation,and observe the influences of miR-135a-5p on the weight and survival time of mice by inhibiting TRAF5 in vivo.Methods:1.ISH with the LNA-modified probe and IHC were used to detect the endogenous expression levels of miR-135a-5p,TRAF5 and Ki-67 in 120 gliomas and20 non-tumoral brain tissues(tissue microarray),and we also observed the differences between the expressions of the three and glioma grades.According to the labeling indexes of miR-135a-5p,TRAF5 and Ki-67,pearson correlation analysis was used to explore the correlation between two of the three.The relationships between the expression levels of miR-135a-5p and TRAF5 and patients' survival were analyzed by Kaplan-Meiers.The Cox's proportional hazards regression model was applied to screen out independent survival predictors for glioma patients.2.Bioinformatics prediction showed the potential targets of miR-135a-5p.The dual-luciferase assay was adopted to verify the silencing effect of miR-135a-5p on TRAF5.3.The qRT-PCR and Western blot assays were used to confirm the expression efficiencies of the exogenous genes after infecting with the miR-135a-5p or TRAF5 overexpressing lentivirus.The glioblastoma cell lines of U251 and U87 MG were infected with the corresponding lentivirus(es)to construct the stable sub-cell lines U251/U87MG-miR-135a-5p(infected with miR-135a-5p lentivirus only),U251/U87MG-miR-135a-5p+TRAF5(infected with miR-135a-5p and TRAF5lentiviruses)and U251/U87MG-Con(infected with the control lentivirus).4.qRT-PCR and Western blot were used to detect the mRNA and protein expression levels of TRAF5 in Con or miR-135a-5p overexpression sub-cell lines.Examine the silencing efficiency of miR-135a-5p on TRAF5 and explore its molecular mechanisms.5.Flow cytometry(FCM)cell cycle detection,EdU and MTS assays were used to reflect the cell cycle distribution and proligeration activity of U251 and U87 MG sub-cell lines.Observe the effects of miR-135a-5p on proliferation of glioma cells and whether TRAF5 could reverse the anti-proliferation effect of miR-135a-5p.6.qRT-PCR and Western blot were used to detect the endogenous TRAF5 silencing efficiency of U251 and U87 MG,which were transfected with the specific siRNA of TRAF5(siTRAF5#1 ? siTRAF5#2).FCM,EdU and MTS assays were performed to detect whether siTRAF5 s could mimic the effects of miR-135a-5p on glioma cell cycle and cell proliferation.7.Athymic nude mice were orthotopically implanted with U87 MG stable sub-cell lines(Con,miR-135a-5p,miR-135a-5p+TRAF5).Mice weight and survival were recorded and the bioluminescence imaging was used to detect the tumor growth regularly.HE staining was performed to observe the xenograft samples and IHC was used to detect TRAF5 and Ki-67 expression levels in the xenograft samples.8.To unveil the michanism by which miR-135a-5p effected on glioma cell cycle and cell proliferation,Western blot was applied to detect the expression levelsof TRAF5,p-AKT,AKT,c-Myc and cyclin D1 using U251 and U87 MG stable sub-cell lines(Con,miR-135a-5p,miR-135a-5p+TRAF5)or transfected with siTRAF5(siTRAF5#1?siTRAF5#2).Results:1.ISH results showed that the expression level of miR-135a-5p was significantly lower in gliomas than in non-tumoral brain tissues,and decreased with the elevation of glioma grades(P<0.001).While IHC indicated that TRAF5 and Ki-67 expression levels were both higher in gliomas than in the control,and accompanied with the elevation of glioma grades(P<0.001).Pearson correlation analysis showed that miR-135a-5p LI% was inversely correlated with TRAF5 LI%(r=-0.831;P<0.001)and Ki-67 LI%(r=-0.782;P<0.001),while TRAF5 LI%positively correlated with Ki-67 LI%(r=0.946;P<0.001).These results suggest the negative regulation between miR-135a-5p and TRAF5 in glioma tissues,and they are negative and positive regulators for tumor cell proliferation respectively.2.Kaplan-Meier analyses proved that glioma patients with higher miR-135a-5p level or lower TRAF5 level enjoyed longer disease-free survival(DFS)and overall survival(OS)whether in 120 glioma specimens or in patients with the same grade,same IDH status,similar ages and KPS groups(P<0.01~0.0001).Cox's proportional hazards regression showed that miR-135a-5p and TRAF5 were independent survival predictors for DFS and OS of glioma patients.3.Bioinformatics prediction showed that the nucleotides 422-428 in the 3'UTR region of TRAF5 mRNA are complementary to the seed sequence of miR-135a-5p,suggesting that TRAF5 is a potential target gene for miR-135a-5p.Dual-luciferase reporter assay demonstrated that miR-135a-5p could effectively suppress the luciferase activity by binding with the wild type 3'-UTR of TRAF5 mRNA,while the mutant type,without the binding site,failed to suppress the above luciferase activity.qRT-PCR and Western blot further confirmed that the expression levels of TRAF5 mRNA and protein in miR-135a-5p overexpressing sub-cell lines(U251/U87MG-miR-135a-5p)were significantly lower than the Con(U251/U87MG-Con,P<0.5~0.01).The above results demonstrate that TRAF5 is adirect target of miR-135a-5p in glioma cells,which could inhibit the expression of its encoded protein by inducing the degradation of TRAF5 mRNA.4.FCM showed that the percentage of G0/G1 cells in miR-135a-5p overexpressing sub-cell lines were significantly higher than that in the Con and miR-135a-5p+TRAF5 sub-cell lines(P<0.01).EdU and MTS assays showed that the cell proliferation ability of the miR-135a-5p sub-cell lines were significantly lower than that in the Con and miR-135a-5p+TRAF5 sub-cell lines(P<0.01~0.001).These results demonstrate that miR-135a-5p could effectively inhibit the proliferation of glioma cells by inducing G1 arrest,while exogenous TRAF5 reversed the effect of miR-135a-5p in glioma cell proliferation.5.qRT-PCR and Western blot affirmed that siTRAF5 significantly suppressed the expression level of TRAF5 mRNA and protein in U251 and U87 MG cells.FCM demonstrated that the proportion of G0/G1 cells in the siTRAF5 transfected groups were higher than than in Scr group(P<0.001).EdU and MTS assays showed that siTRAF5 could inhibit the proliferation ability of glioma cells(P<0.05~0.001).These results confirm that specific siRNA of TRAF5 could mimics the inhibitory effect of miR-1335a-5p on proliferation of glioma cells.6.The in vivo assay showed that the xenografted tumors overexpressing miR-135a-5p showed lower growth rate,mice weight loss rate and longer mice survival time comparing with Con and miR-135a-5p+TRAF5 groups.The pathological results showed that the tumor cell density and Ki-67 labeling index in the miR-135a-5p group were significantly lower than the other two groups(P<0.05~0.001).The above results demonstrated that miR-135a-5p can also inhibit the proliferation activity of xenograft tumor cells by knocking down the expression of TRAF5 in vivo.7.Western blot results indicated that miR-135a-5p could significantly inhibit AKT phosphorylation as well as the expression of c-Myc and cyclin D1,while overexpression of TRAF5 restored the above protein levels inhibited by miR-135a-5p(P<0.05~0.001),except for total AKT remained constant(P>0.05).Furthermore,siTRAF5 could mimic the effect of miR-135a-5p with above detections.Conclusions:1.The aberrant decrease of miR-135a-5p and increase of TRAF5 are common feature of human gliomas.The miR-135a-5p level decreases whist the TRAF5 level increases with the elevation of glioma grade and the proliferation activity,hence the expression levels of miR-135a-5p and TRAF5 could be used as biomarkers to assess the malignant degree of gliomas and evaluate tumor cell proliferation activity.2.The expression levels of miR-135a-5p and TRAF5 are closely related to the DFS and OS of glioma patients.Their LI% are the protective factor or risk factor for patients' survival respectively as well as the independent survival predictors for glioma patients.3.TRAF5 is a direct target of miR-135a-5p in glioma cell lines,which inhibits TRAF5 expression via inducing the degradation of its mRNA and then suppressing its expression at the post-transcriptional level.The downexpression of miR-135a-5p is common in glioma cells,and is responsible for the abnormal increase of TRAF5.4.The abnormal expressions of miR-135a-5p and TRAF5 are the important causes leading to the disorder of cell cycle and infinitely proliferation of glioma cells,and play important role in glioma progression.5.Exogenous miR-135a-5p can inhibit AKT phosphorylation and block AKT pathway by targeting TRAF5,thereby suppressing the expression of c-Myc and cyclin D1,and inducing G1 arrest and to inhibit the proliferation of glioma cells in vitro and in vivo.6.This study demonstrates that miR-135a-5p is a tumor suppressor,which shows the anti-proliferation effect in gliomas cells and TRAF5 is selected as a direct target of miR-135a-5p to mediate the effect.miR-135a-5p inhibits glioma cell proliferation by targeting TRAF5.Our study suggests that TRAF5 could serve as an important candidate target for molecular therapy,and miR-135a-5p could be used as tumor suppressor for gene therapyof malignant gliomas.