Study On The Inhibition Roles Of MiR-146b-5p And MiR-361-5p In Glioma

?Objective?The expression of miR-146b-5p in various human tissues with different levels and in the lung,thymus and spleen specificity high.miR-146b-5p was originally found as one of the important regulatory factor of inflammation and have an important role in the process of innate immunity.Studies have found that the expression of miR-146b-5p was abnormally decreased in malignant tumors,such as malignant melanoma,breast cancer and pancreatic cancer,confirmed that miR-146b-5p was an important tumor suppressor miRNA of those tumors.The coding gene of miR-146b-5p location on chromosome 10q24-26,our previous studies have demonstrated that 10q24-26 is a hotspot of glioma genomic DNA loss extents,so miR-146b-5p is characteristic and highly suggestive of may also glioma suppressor miRNA.After that,we continued our study and found that overexpression of miR-146b-5p in glioma cell lines can be targeted to reduce the expression of MMP-16 and significantly reduce the invasion and migration ability of glioma cells.However,miR-146b-5p for glioma cell proliferation,the effects of apoptosis and its related target pathways remain to be further research.If there have any abnormal expression of miR-146b-5p in glioma cells and if the abnormal expression of miR-146b-5p related on the grade of glioma,the glioma cell's biology characteristics and the prognosis of glioma patients also remain unclear.Our previous studies have confirmed that SND1 is abnormally high expression in glioma tissues and the high expression of SND1 predicts the worse prognosis of glioma patients.As a transcription coactivator,SND1 can increase the transcriptional activity of MMP2 and Rho A,thus increasing their expression,is one of the important factors that lead to tumor cell abnormal biology behavior.But why SND1 abnormally high expression in glioma cells was waited to discuss.miR-361-5p has been demonstrated abnormally low expression in a variety of tumors,and the high expression of it's target genes that caused by it's low expression are some of the most important factors in tumor occurrence and development.There is one article reported that miR-361-5p can inhibits colorectal and gastric cancer growth and metastasis by targeting SND1.Based on our laboratory's previous study about SND1 in glioma and some research papers about miR-361-5p in other tumors,we consider whether the expression of miR-361-5p in glioma also abnormally low.Then explore the relationship between the expression level of miR-361-5p and SND1.Further explore miR-361-5p impact on the biological behavior of glioma cells,explore other related target genes and regulatory pathways.This research adopts the human glioma histological specimens and glioma cell line of the above question has carried on the deep discussion.Through the study of the above problems in-depth system that can further understand the molecular mechanism of glioma occurrence,development,for patients with gliomas molecular pathology,diagnosis and prognosis evaluation of screening and reliable molecular markers and molecular target therapy for malignant glioma gene intervention screening of tumor suppression factor and intervention targets and to establish a corresponding intervention strategies to provide the theoretical basis of the objective,therefore this study has important theoretical significance and practical value.?Method?1.By in situ hybridization method to detect the expression of miR-146b-5p and miR-361-5p in 147 cases of different grade glioma tissues and 20 cases of nontumor brain tissues(control).Using immunohistochemistry method to detect the expression of TRAF6,SND1,Fox M1 and Ki-67 in the same tissue samples that above described.Combined with TCGA database data,we analysed the relationship between miR-146b-5p and TRAF6 and the relationship between miR-361-5p and SND1 or Fox M1.Combined with clinical follow-up data,we analysed the relationship between miR-146b-5p or TRAF6 expression level with the overall survival or disease-free survival in patients.2.Through to the glioma cell line transfection with the miR-146b-5p or miR-361-5p mimics and the corresponding nonsense controlled sequence(scramble),with transfection scramble cells as a control group.The q RT-PCR was used to detect the transfection efficiency of miRNAs mimics,the q RT-PCR and western blot were used to detect the expression of miRNAs' corresponding target genes that the targetscan predicted.Using CCK-8 experiment,the clone formation experiment and Ed U cell proliferation experiment detect cell proliferation activity.Using alkaline single cell electrophoresis experiment and flowcytometry(FCM)detect cell apoptosis.Using transwell and scratch experiment to test cell migration and invadion ability.We adopted these above experiments to make clear different levels of miR-146b-5p or miR-361-5p expression affect the biological behavior of glioma cells.3.Using bioinformatics to predict miR-146b-5p and miR-361-5p potential target genes,use q RT-PCR and western blot detect the expression of TRAF6,SND1 and Fox M1 that the bioinformatics predicted.Dual luciferase reporter gene experiments have established that miR-146b-5p can be combined with TRAF6 3 'UTR region to inhibit its expression and miR-361-5p can combine with SND1 and Fox M1 3' UTR region to suppress their gene expression.Chromatin precipitation(Ch IP)experiment to verify whether SND1 in U87 MG and U251 can improve the expression of MMP2 and Fox M1 can improve the expression of ?-catenin by way of transcriptional activation.4.Through to the glioma cell line transfection with the TRAF6 specificity small interference RNA(TRAF6-si RNA)or its controlled sequence(scramble),with transfection scramble cells as a control group.The q RT-PCR and western blot were used to detect the expression of TRAF6.Using CCK-8 experiment detect cell proliferation activity.Using alkaline single cell electrophoresis experiment and flowcytometry(FCM)detect cell apoptosis.The experiments that described above provided circumstantial evidence that the abilities of glioma cell proliferation and apoptosis is exerted by miR-146b-5p target on TRAF6.5.Through to the glioma cell line co-transfection with miR-361-5p and SND1 eukaryotic expression plasmid(p SG5-SND1),co-transfection with miR-361-5p and control plasmid(p SG5)or co-transfection with nonsense controlled sequence(scramble)and control plasmid(p SG5),with the group that co-transfection scramble+p SG5 as control group;co-transfection with miR-361-5p and Fox M1 eukaryotic expression plasmid(pc DNA3.0(+)-Fox M1),co-transfection with miR-361-5p and control plasmid(pc DNA3.0(+))or co-transfection with nonsense controlled sequence(scramble)and control plasmid(pc DNA3.0(+)),with the group that co-transfection scramble+ pc DNA3.0(+)as control group.Using CCK-8 experiment,the clone formation experiment and Ed U cell proliferation experiment detect cell proliferation activity.Using alkaline single cell electrophoresis experiment and flowcytometry(FCM)detect cell apoptosis.Using transwell and scratch experiment to test cell migration and invadion ability.The rescue experiments that described above provided solid evidence that the abilities of glioma cell biological behavial is at least patialy exerted by miR-361-5p target on SND1 or Fox M1.6.Using western blot to detect the expression of phosphorylation TAK1,TAK1,phosphorylation I?B? and I?B? after transfection of miR-146b-5p mimics or TRAF6 si RNA;to detect the expression of SND1,MMP2,Fox M1,?-catenin,cyclin D1,and c-myc after transfection of miR-361-5p mimics.Our purpose is to make clear of the molecular mechanism that miR-146b-5p targeted TRAF6 to reduce glioma cell proliferation and to inhibit apoptosis and miR-361-5p targeted SND1 to reduce glioma cell invasion and migration or targeted Fox M1 to reduce glioma cell proliferation and to inhibit apoptosis.? Results ?1.According to the result of ISH,we found that miR-146b-5p and miR-361-5p expression in gliomas were lower than that in their correspongding control and that their expression were significantly decreased with the elevation of glioma grades and was the lowest in glioblastoma.Immunohistochemical results show that the expressions of TRAF6,SND1,Fox M1 and Ki-67 in gliomas were higher than that in the control and that their expression were significantly increased with the elevation of glioma grades and were the highest in glioblastomaglioma group.Moreover,miR-146b-5p expression in gliomas was negatively correlated with their TRAF6 and Ki-67 expressions.The inverse relationship between the expressions of TRAF6 and miR-146b-5p was further validated by the glioblastoma data from TCGA database.Kaplan-Meier analyses showed that the patients with higher level of miR-146b-5p had longer disease-free survival(DFS)and overall survival(OS)and demonsteated that the high level of TRAF6 expression predicted a short-term DFS and OS in patients with gliomas.Cox regression analysis showed that both low expression of miR-146b-5p and high expression of TRAF6 are the risk factors influencing the patients' survival.Both multivariate and univariate analyses showed that miR-146b-5p was an independent predictor for DFS and OS of patients with gliomas.2.The results of q RT-PCR showed that after transfection with miR-146b-5p mimics or miR-361-5p mimics in glioma cells,the corresponding miRNA expression leves rise obviously,that prove transfection success.q RT-PCR and western blot results showed that TRAF6 m RNA and protein expression levels were significantly reduced after transfection of miR-146b-5p mimics and Fox M1 m RNA and protein expression levels were significantly reduced after transfection of miR-361-5p mimics.All above results confirmed that high expression of miR-146b-5p can decrease TRAF6 protein levels by degrade its m RNA and high expression of miR-361-5p can decrease SND1 and Fox M1 protein levels by degrade their m RNAs.The dual-luciferase assay have established that miR-146b-5p could specific binding TRAF6 m RNA 3'UTR and miR-361-5p could specific binding SND1 m RNA 3'UTR or Fox M1 m RNA 3 'UTR region and exert their inhibitory effect on the corresponding target genes.3.CCK-8 assay and Ki-67 western blot test results showed that the glioma cell proliferation ability significantly reduced after transfection of miR-146b-5p mimics or TRAF6 si RNA.Alkaline single cell electrophoresis experiment and flow cytometry(FCM)showed that the glioma cell apoptosis ability increased after transfection of miR-146b-5p mimics.These results demonstrated that overexpression of miR-146b-5p exerted its function by lowering the expression of TRAF6 and play a role of inhibition of glioma cell proliferation and promote apoptosis.Transwell and scratch experiment results confirmed that the migration and invasion ability of glioma cells significantly decreased after transfection of miR-361-5p mimics,and co-transfection of SND1 expression plasmid and miR-361-5p mimics can partially reverse the inhibition of migration and invasion that miR-361-5p caused on glioma cells,confirmed that miR-361-5p can effectively restrain glioma cells migration by targeting SND1.CCK-8 experiment,the clone formation experiment and Ed U cell proliferation experiment results showed that the glioma cells proliferation ability significantly reduced after transfection of miR-361-5p mimics.Alkaline single cell electrophoresis experiment and flow cytometry(FCM)showed that the glioma cells apoptosis increased significantly after transfection of miR-361-5p mimics.And cotransfection of Fox M1 expression plasmid and miR-361-5p mimics can partially reverse the inhibition of proliferation and the promotion of apoptosis that miR-361-5p caused on glioma cells,confirmed that miR-361-5p can inhibit glioma cells proliferation and promote apoptosis by targetting Fox M1.4.According to the results of western blot experiments in glioma cells,overexpression of SND1 can effectively increase the expression of MMP2 and Ch IP experiment results showed that SND1 can specificity combine with the promoter region of MMP2 genes encoding,confirmed that SND1 as a coactivator can induce transcription of MMP2 m RNA and then increase its protein expression level.Similarly,western blot experiment results showed that overexpression of Fox M1 in glioma cells can effectively increase the expression level of ?-catenin and Ch IP experiment results showed that Fox M1 can specificity combine with the promoter region of CTNNB1 genes encoding,confirmed that Fox M1 as a coactivator can induce transcription of CTNNB1 m RNA and then increase its protein expression level.5.According to the results of western blot experiments in glioma cells,transfection of miR-146b-5p or TRAF6 si RNA both can effectively inhibit the phosphorylation of TAK1 and I?B?,and the phosphorylation of I?B? directly influences the transloction of NF?B into nuclear,hence miR-146b-5p can blocking the TRAF6-TAK1 pathway and effectively inhibition of the transloction of NF?B into nuclear to exert its inhibitory effect of glioma cells proliferation and promote their apoptosis by targeting TRAF6.In glioma cells that transfection of miR-361-5p mimics,the results of western blot showed that the expression of MMP2 decreased significantly.MMP2 expression levels began to recover after cotransfection of miR-361-5p mimics and SND1 overexpression plasmid,confirmed that miR-361-5p reduce the expression of MMP2 levels by targeting SND1,and thus inhibit the migration and invasion of glioma cells.Likewise,in glioma cells that transfection of miR-361-5p mimics,western blot showed that the expression of ?-catenin,cyclin D1 and c-myc levels decreased significantly,confirmed that miR-361-5p can decrease the expression of ?-catenin by targeting Fox M1 and then inhibition of Wnt-?-catenin pathway,thus inhibit glioma cells proliferation and promote their apoptosis.?Conclusions ?1.The tumor cells in human glioma tissue generally have an abnormally decreased expression of miR-146b-5p and miR-361-5p,but have an abnormally increased expression of TRAF6,SND1 and Fox M1.The expression of miR-146b-5p and the expression of TRAF6 both show a good correlation with glioma grade and the proliferation activity of glioma cells.Therefore,miR-146b-5p and TRAF6 both can be used as biomarker that evaluation of the grade of glioma and the biological behavior of glioma cells.2.miR-146b-5p and TRAF6 were negative and positive control factor respectively of glioma cells proliferation,miR-361-5p and SND1 were negative and positive control factor respectively of glioma cells migration and invasion,miR-361-5p and Fox M1 were also negative and positive control factor respectively of glioma cells proliferation.So the abnormal expression of miR-146b-5p and miR-361-5p both could be the important reasons for glioma cells active proliferation and strong ability of migration and invasion,and play an important role in the occurrence and development of glioma.In glioma cells,SND1 is one of the important regulatory factors that can increase the expression of MMP2 by activation of MMP2 transcription and Fox M1 is one of the important regulatory factors that can increase the expression of ?-catenin by activation of CTNNB1 transcription.3.The expression of miR-146b-5p and TRAF6 are both closely associated with the prognosis of patients with gliomas,the low expression of miR-146b-5p and the high expression of TRAF6 are all risk factors for glioma patients survival,their expression level have certain reference value for prediction of disease-free survival and overall survival in glioma patients.miR-146b-5p LI% is an independent predictor for gliomas prognosis that in line with the Ki-67 LI%.Therefore,miR-146b-5p can be used as an important reference indicator of the prognosis of patients with glioma assessment.4.In glioma cells,TRAF6 m RNA is a target m RNA of miR-146b-5p,miR-146b-5p can combine with the seed sequence of TRAF6 m RNA 3'UTR.SND1 and Fox M1 are both target m RNAs of miR-361-5p,miR-361-5p can combine with the seed sequence of SND1 and Fox M1 m RNA 3'UTR.By inducing TRAF6 m RNA,SND1 m RNA and Fox M1 m RNA respectively,miR-146b-5p and miR-361-5p can prevent their translation in the transcription level and effectively restrain their protein expression.5.In glioma cells,TRAF6 is an important activating factor in NF?B pathway,it can induce phosphorylation of TAK1 and I?B? and then promote the release of active compounds p65:p50,thus promote the glioma cells proliferation and inhibit apoptosis.Overexpression of TRAF6 generally exists in glioma cells is an important molecular mechanism of glioma cells unlimited proliferation capacity and evade apoptosis.The abnormally high expression of SND1 in glioma cells elevated the expression of MMP2 is one of the important molecular mechanism of glioma cells strong migration and invasion ability.?-catenin is a key protein in Wnt-?-catenin pathway.Fox M1 as a coactivator can promote the transcription activity of ?-catenin and thus increase its expression level.On the other hand,by targeting Fox M1 to degrade can inhibit the transcription activity of ?-catenin and decrease its expression,inhibition of Wnt-?-catenin pathway,thus inhibiting the proliferation and promoting apoptosis of glioma cells.Targetting TRAF6,SND1 or Fox M1 to degrade all could become important strategies for effective intervention of malignant glioma.6.Overexpression of TRAF6 in glioma cells caused by the abnormally low expression of miR-146b-5p and overexpression of SND1 and Fox M1 caused by the abnormallu low expression of miR-361-5p are important molecular mechanisms of glioma cells biology behavior regulation disorder,and play important roles in occurence and development of gliomas.Overexpression of miR-146b-5p or miR-361-5p and knocking down TRAF6,SND1 or Fox M1 with some methods,all could play roles of effective glioma tumor suppression,are likely to be effective targeted therapy of malignant glioma and molecular genetic intervention strategies.