MicroRNA-664 Regulates Cell Invasion And Migration And Epithelial-mesenchymal Transition By Targeting TGF-β1 Signal In Glioblastoma

Background:Glioma is one of the most common malignant tumor of the central nervous system,the incidence of which increased in recent years.Glioblastoma(Glioblastoma,GBM),accounting for intracranial tumor 15%,often originate from normal brain cells or low grade astrocytoma,an annual incidence rate of 3/10 million.The treatment of Glioblastoma is still a great problem,generally speaking,after patients diagnosis with GBM,the survival time is about 12-18 months,only 3-5% patients live for more than5 years.Now,many treatments were developed for the GBM,including surgery,radiation therapy,chemotherapy and gene therapy.However,most of them had not meet further expectations.The mechanisms about cells proliferation,migration,invasion and epithelial to mesenchymal transformation(Epithelial-mesenchymal transition,EMT)is a hot topic for the tumor growth.But there is still no breakthrough in the current research..Transforming growth factors(Transforming growth factor-β,TGF-β)is one of the members of multiple functional growth factors superfamily for regulating cell growth and differentiation.Transforming growth factor Beta 1(TGF-β1)is a negative growth regulator in the tumor.Previous research has mostly focused on the inflammation,tissue repair,but recent studies showed that TGF-β1 related to cell differentiation,growth and development,cell proliferation and migration,angiogenesis,tumor immunity.TGF-β1 participated in the negative regulation of tumor,but for the glioma,mechanism of TGF-β1 is still unclear.Relationship between TGF-β1 expression and degree of malignant glioma is still controversial.TGF-β1 showed no biological activity before activated by receptors in all types of cells.In vitro,the cell proliferation of many tumors(such as intestinal,liver cancer,lung cancer,prostate cancer)were inhibited with the up-regulattion of TGF-β1,however,in vivo,there was no such phenomenon.its molecular mechanism is still unclear.The function of TGF-β1 is directly related to the different receptors on the cell membrane.There are type Ⅰ 、Ⅱ 、Ⅲ TGFBR(Transforming growth factor beta receptor,TGFBR)on the surface of cells membrane,of which the type Ⅰ 、Ⅱis the main receptors.Many studies showed the relationships between their levels of expression in glioma and their mechanism of action is unclear.Activin receptor-like kinase 1(Activin receptor-like kinase 1,ALK1)is type Ⅰ receptor.TGF-β1involved in the regulation of cell proliferation,differentiation,cell apoptosis,and other biological processes.It is reported that the abnormal expression or mutations of TGFBR2 may lead to kidney cancer,colon and gastric cancer.It is still unclear that how the TGFBR3 without protein kinase activity involved in the signal transduction.TGF-β1 combined with the type Ⅱ receptor firstly,and then combined with type I receptor to be binary compounds.The binary complexes band to TGF-β1,eventually via phosphorylated to start the intracellular TGF-β1 signal transduction.TGF-β1 receptor may be key points in TGF-β1 signaling pathway.Therefore,via regulation gene expression of TGF-β1 receptor and changing the TGF-β1 signaling transduction is a hot research topic.mi RNAs is a kind of endogenous small,non-coding RNAs,about 22 nucleotides in size.It binded to the target gene m RNA 3’-UTR base pair,to suppress m RNA or facilitating the process of translation m RNA degradation.mi RNAs regulate target gene function by the post-transcriptional inhibition of target gene expression.The biological function of mi RNA is an controversial problem.Current research mainly focused on the organism development,cell differentiation,apoptosis,tumor formation,and so on.Studies have found that mi RNAs as oncogenes or tumor suppressor genes play a key role in the development and progression of tumor process.In recent years many studies have confirmed that mi RNAs play an important role in regulating expression of TGF-β1 in glioblastoma.mi R-664 expression was reported which related to bone sarcomas,breast cancer and cervical cancer,tumor formation,however,mi R-664 plays a role in glioblastoma,and the relationship between glioblastoma and TGF-β1 is still unclear.Therefore,this subject was up to study the the TGFBR and the mi R-664 expression in glioma cells,and explore cell migration,invasion and expression of endothelial-derived mesenchymal transformation after transfeceted with mi R-664 in glioma.and validation the effect of mi R-664 and TGF-β1.In addition,our studies are to clarify the development of the underlying molecular mechanisms of mi R-664 in glioblastoma.Charpter 1 The research of clinico-pathological correlation analysis between the expression of TGF-β1 and its receptor I,Ⅱ(ALK1,TGFBR2)in Human Brain GliomaObjectives:TGF-β1 is a regulatory factor of tissues and cells,via combining with different receptors to regulate the growth of cells.TGF-β1 and the TGFBR expression in glioma tissues of different levels has been reported in many literatures,the relationship with histological grade is not fully clear.After collect the samples of malignant glioma,we apply RT-PCR,Western Blot and immunohistochemistry assay to detect expression of TGF-β1,ALK1 and TGFBR2 m RNA,and analyse the change and correlation between deferent grade glial tumor.Methods:1.Collection of data and specimens: tissue samples were obtained from patients with glioma,immediately stored in liquid nitrogen.All patient information and informed consent through approval of the first affiliated hospital of Nanchang university Ethics Committee.2.Pathological grading and grouping: surgical pathology specimens were confirmed by two pathologists,in accordance with WHO classification.Divided into:Ⅰ-level groups,Ⅱ-level groups,Ⅲ-level groups and Ⅳ-level Group.Normal control groups and glioma groups were acquired from donors.3.Total RNA extraction and RT-PCR detection : brain glioma specimens and normal tissues were stored in access sterile conditions tanks in liquid nitrogen frozen custody for the extraction of total RNA,using parallel RT-PCR to detect the expression levels of TGF-β1,ALK1 and TGFBR2 m RNA in different groups.4.Total protein extraction and Western blot detection: fresh specimens of brain glioma and normal tissues were prepared for extraction of total protein,via Western blot analysis,detect expression of TGF-β1,ALK1 and TGFBR2 in different groups.5.HE staining and Immunohistochemistry assay: follow immunohistochemical SP Kit step,dewaxing,formalin-fixed,endogenous peroxidase and serum after sealing of glioma specimens in each group,via HE stain and immunohistochemical to detect expression of TGF-β1,ALK1 and TGFBR2.The number of positive cells were messured by the double-blind method.6.Data processing and statistical analysis: all data using SPSS 13.0 statistical software for the analysis,the experimental group for homogeneity of variance analysis and means were compared with the two-sample t test,Spearman nonparametric correlation correlation analysis.Results:1.Specimens of different pathological grading in the TGF-β1 m RNA and protein expressions in trend: compared with normal group,m RNA expression of TGF-β1inⅠ,Ⅱ and Ⅲ Group are up-regulated,but the difference of which has no statistical significant(P>0.05);Otherwise,Ⅳ-level group increased significantly,and it has statistical significant(P<0.05).The expression of TGF-β1 in normal and I-level Group is very low,Ⅱ,Ⅲ and Ⅳ group expressed extremly high,the difference has statistical significant(P<0.05),Different pathological grade showed inconsistency about the expression of TGF-β1 m RNA and protein.2.Specimens of different pathological grading ALK1 and TGFBR2 m RNA and protein expression of trends: The m RNA and protein expression of ALK1 and TGFBR2 of glioma specimens in each group were significantly high(P<0.05),compared with normal control group,Ⅰ,Ⅱ,Ⅲ and Ⅳ level group have statistically significant difference(P<0.01),The researchers found a positive correlation between protein expression and pathological grade of the glioma.3.Immunohistochemistry analysis: TGF-β1 expressed in a low level in normal group,but high in ALK1 and TGFBR2.The expression level of TGF-β1 in I-level group and Ⅱ Group is low,expression level of Ⅲ-Ⅳ groups are significantly high(P<0.05).Different groups are present in ALK1 and TGFBR2 staining cells,but rare positive cells in the control group.Compared with the control group,the difference of ALK1 in all level groups of positive cells ratio has statistical significance(P<0.05).TGFBR2 has obviously statistical significance(P<0.01).4.The Spearman rank correlation analysis showed that ALK1 and TGFBR2 protein expression were positively correlated with tumor pathological grade,for ALK1(r=0.297,P<0.05)and TGFBR2(r=0.371,P<0.05).The expression of TGF-β1 is inconsistent with pathological grading of glioma.Conclusions:1.There are some expression of TGF-beta-1 m RNA in normal and low grade glioma groups,but low levels of protein expression.With glioma grade increases,protein expression levels of TGF-β 1 m RNA are gradually increased,but there are some inconsistencies,for the expression of TGF-beta 1 is associated with malignancy of glioma.2.The m RNA expression and protein of ALK1 and TGFBR2 in groups of brain glioma are up-regulated and relevant with pathologic grade of glioma.3.The expression of TGFBR2 in brain glioma group was significantly higher than the ALK1,while its high expression has a strong correlation with pathologic grade of glioma,and it may participate in malignant change of glioma.Charpter 2 The research of micro RNA-664 inhibits migration/invasion and endothelial-mesenchymal transformation(EMT)of glioma cells by TGF-β1signal pathway Objectives:It was reported that abnormal mi RNAs expression may influence tumor cell proliferation,invasion and migration,which is one of the possible TGF-β signaling pathways function.The abnormal expression of mi R-664 was related with the formation of bone sarcomas,breast cancer and cervical cancer.The relationship between mi R-664 and development of glioblastoma and TGF-β1 is still unknown.This experiment apply q RT-PCR and western blot assay to detect the expression levels of mi R-664 in glioma tissues and cell lines,and the influence of mi R-664 on migration or invasion of glioma cells and endothelial-mesenchymal transformationrelated protein(N-cadherin,E-cadherin and Vimentin).Next,this experiment intended to find the targeted of mi R-664 and the relationships of mi R-664,TGF-β1and TGFBR2 receptor.Methods:1.Collection of data and specimens: the proven cases of glioma patients in our hospital.The diagnosis of glioblastoma was pathologically defined according to World Health Organization(WHO)classification and each case was confirmed by two pathologists.Glioblastoma tissue and the normal brain tissue samples were obtained from the enrolled cases.All the patients were informed with the consent and this experiment was approved by our hospital’s protection of human ethics committee.2.Cell culture: glioma cell lines U87-MG,T98 G and normal astrocytes HEB acquired from the European Collection of Cell Cultures(Wiltshire,UK).Resuscitation of cells containing 10% FBS DMEM culture fluid of 5% CO2 at 37°C constant temperature incubator and it is adherent growth.3.Chemical synthesis of mi R-664 mimic mi R-664 scramble and TGFBR2-si RNA: login mirbase.org library to find the latest mi R-664 sequence of the chemical compounds mi R-664 mimic,mi R-664 scramble and TGFBR2-si RNA.4.Transfer: Lipofectamine 2000 Manual on glioma cell line U87-MG,and T98 G normal astrocytes and HEB transfection.5.q RT-PCR and western blot detection : The total RNA and total protein are extracted from normal brain tissue,brain glioma and glioma cell lines(U87-MG and T98 G HEB),using q RT-PCR and western blot technology to detect the protein expression levels of Micro RNA-664 and TGFBR2 m RNA in these cells or tissues.HEB,U87-MG,and T98 G cell transfection,respectively mi R-664 mimic,mi R-664 scramble and TGFBR2-si RNA training 48 h extracted total RNA and protein to detect EMT Related proteins(N-cadherin,E-cadherin and vimentin)m RNA and protein expression levels change in each group.6.Transwell detection: Preparation of matrigel Transwell Chamber,glioma cell line(U87-MG,and T98 G HEB),respectively transfect mi R-664 mimic,mi R-664 scramble TGFBR2-si RNA and serum-free culture 24 h,prepared cell suspension,inoculated in the cab and detected number of cells in different groups cross the PET membrane after the transfection,assess migration and invasion of cells.7.Detection of luciferase report gene: Vectors of TGFBR2-3-UTR was synthesized by Sangon Biotech(Shanghai,China).The dual luciferase reporter plasmids TGFBR2-WT(containing the wild-type TGFBR2 putative 3’-UTR-binding site)and TGFBR2-Mut(containing the mutant TGFBR2 3’-UTR)were constructed.Luciferase activities were measured by using the dual-luciferase reporter assay system(Promega)for 48 h after cell transfection.The relative reporter activity was normalized by r-luc activity.8.All data in this study were expressed as mean ± standard error of mean(SEM).Independent sample t-test was used to calculate the difference between two groups using the graph prism 5.0 software(Graph Pad Prism,San Diego,CA).Analysis of variance(ANOVA)was used to calculate the difference for more than three groups.P< 0.05 was defined as statistically significant.Results:1.Expression of mi R-664 in glioblastoma tissue and cell lines The relative expression levels of mi R-664 and TGFBR2 in glioblastoma and the normal brain tissue tissues,as well as in glioblastoma cells and normal human astrocytes were using q RT-PCR and western blot analyses.The relative m RNA levels of mi R-664 were significantly decreased in glioblastoma tissues and cells compared with the normal tissues and cells(P < 0.01).Additionally,m RNA and protein expression levels of TGFBR2 in glioblastoma tissues and cells were significantly higher than that in normal tissues and cells(P < 0.05).2.mi R-664 overexpression inhibited cell migration and invasion The role of mi R-664 in invasion and migration of glioblastoma cells was assayed by Transwell.The results showed that the number of migrated glioblastoma cells decreased significantly in mi R-664 mimic group compared with mi R-664 scramble and control groups(P <0.05).Similarly,the number of invaded glioblastoma cells in mi R-664 mimic group was significantly less than that in mi R-664 scramble and control groups(P < 0.05).3.mi R-664 overexpression regulated the EMT-related protein expression In order to further explore the mechanisms of mi R-664 inhibiting glioblastoma cell invasion and migration,the expressions of EMT-related proteins,N-cadherin,E-cadherin and vimentin were detected.When mi R-664 was overexpressed,the expressions of E-cadherin increased significantly in both U87-MG and T98 G cells(P< 0.05).On the contrary,the expressions of N-cadherin and vimentin decreased significantly in two cell lines in mi R-664 mimic group than in the other two groups(P< 0.05).4.TGFBR2 was the target for mi R-664 The relative luciferase activity of the reporter that contained the wild-type3’′-UTR of TGFBR2 was significantly decreased in mi R-664-mimic-transfected cells compared to control cells in both U87-MG and T98 G cells(P < 0.05).However,mutation of the predicted binding site of mi R-664 on the TGFBR2 3’′-UTR rescued the luciferase activity.Moreover,the relative TGFBR2 expression was significantly decreased in mi R-664 mimic group compared with in mi R-664 scramble and control groups(P < 0.05).In addition,the relative m RNA and protein expression of TGFBR2 was down-regulated in cells transfected with silenced TGFBR2.These results suggested that TGFBR2 was negatively regulated by mi R-664.5.mi R-664 overexpression regulated cell invasion and migration via TGF-βsignal.Further investigation of the relationships between mi R-664 inhibiting glioblastoma cell invasion and migration and TGF-β signal revealed that both TGFBR2 inhibition and mi R-664 overexpression could significantly inhibit the invasion and migration of glioblastoma cells(P < 0.05).Additionally,when mi R-664 mimic combined with si-TGFBR2,the inhibitory effect was optimal(P < 0.01)(Figure 5A-5D).Furthermore,both mi R-664 mimic and si-TGFBR2 could increase the expression of E-cadherin,and decrease the expressions of N-cadherin and vimentin significantly(P < 0.05),especially when mi R-664 mimic combined with si-TGFBR2(P < 0.01).The results indicated that mi R-664 overexpression inhibiting glioblastoma cell invasion and migration and EMT may be achieved by regulating TGF-β signal.Conclusions:1.Endogenous mi R-664 m RNA and protein expression levels in the glioblastoma tumor tissue and cell lines is low,which may be correlated with the development and progression of gliomas.2.Over expression of mi R-664 can effectively inhibit cell migration,invasion of glioma and change their trend of endothelial-mesenchymal transformation.3.Mi R-664 via TGF-β signaling pathway to inhibit invasion and migration of tumor,regulate the endothelial-mesenchymal transformation.TGFBR2 is probably the direct targets as mi R-664 affect TGF-β signaling pathways.