MicroRNA-16Inhibits Glioma Cell Growth And Invasion Through Suppression Of BCL2and The Nuclear Factor-κB1MMP9Signaling Pathway

Background and Objective:Glioma is one of the most common types of primary brain tumors in adults, andrepresents one of the most aggressive and lethal human cancer types. Over the past decades,an increasing amount of evidences have showed that microRNAs (miRNAs), a class ofnon-coding RNAs, play a pivotal role in glioma. They can act as both tumor suppressorsand oncogenes by negatively regulating their mRNA targets through degradation ortranslational repression. Through utilising the microRNA.org database and the Database onPredicted and Published MicroRNAs(miRWalk), we previously identified miRNA16(miR-16) is most strongly correlated with malignancy in nearly all analyzed human tumors.Other authors have demonstrated the down-regulation of miR-16in a wide range ofcancers, including breast, prostate, and lung cancers, as well as in chronic lymphocyticleukemia. These findings suggest that miR-16is a possible tumor suppressor that act in avariety of cancers.The prognosis of human glioma is poor, and the highly invasive nature of the diseaserepresents a major impediment to current therapeutic modalities. At the molecular level,tumor cell invasion is mediated by a set of factors that initiate or promote cell motility,matrix destruction, angiogenesis and other biological events. NF-κB mediates cellproliferation, cell migration, and angiogenesis. Aberrant activation of NF-κB has beenobserved in many types of human cancer, including glioma. Nuclear localization of p50, anindicator of NF-κB activation, has also been demonstrated in clinical specimens ofglioblastoma multiform (GBM). NF-κB signaling orchestrates several key biological processes during the development and progression of cancer by inducing the transcriptionof a variety of target genes regulating cell apoptosis, cell cycle, and invasion. AmongNF-κB-regulated genes, matrix metallo-proteinases (MMPs) are closely associated withtumour invasion. In particular, matrix metallo-proteinase9(MMP9) and2(MMP2) levelsincrease with tumor progression in gliomas, and are thus known as key enzymes forinvasion. By analyzing the homology between miR-16and the NF-κB1mRNA sequenceson the microRNA.orgdatabase, we found that fifteen nucleotides are complementary.Therefore, miR-16may inhibit the expression of NF-κB1and MMPs proteins, and mayreduce glioma invasiveness. BCL2is an oncogene, an anti-apoptotic protein residing in themitochondria, and is also involved in gliomas development.The aim of the present study was to explore miR-16expression in human braingliomas and in three malignant glioma cell lines (SHG44, U87and U373), as well as thecorrelation between miR-16and NF-κB1expression, cell apoptosis and invasiveness. Thisstudy also aimed to lay the foundation for future in-depth study of the mechanisms ofaction of miR-16in human glioma.Materials and Methods1. Through utilising the microRNA.org database and the Database on Predicted andPublished MicroRNAs(miRWalk), we previously identified miRNA16(miR-16) is moststrongly correlated with malignancy in nearly all analyzed human tumors.2. Chemical synthesis miR-16oligonucleotide random sequence was transfected intothe malignant glioma SHG44、U87and U373cell lines by the Lipofectamine2000liposome.3. To achieve stable cell lines that over express miR-16, U87cells were transfectedwith the pcDNA6.2-GW/EmGFP-miR plasmid that contained computer-designedoligonucleotide sequences expressing the pre-miR-16. The entire transfection process wascompleted using lipofectamine20004. The transfection rates in human glioma cell lines SHG44, U87and U373weredetermined by flow cytometry and Green fluorescence. Real-time quantitative RT–PCR (qRT–PCR) was used to determine the expression of miR-16and NF-κB1in noncancerousbrain tissue and Human glioma tissue samples respectively.Cells were allowed to transfectwith the mimics, inhibitors of miR-16or the negative control oligonucleotide. Theproportion of cells in early-stage apoptosis was detected using the Annexin V PE ApoptosisDetection Kit PE and flow cytometry. Scratch migration assay and Transwell assay wereused to detect invasive effect of miR-16in human glioma SHG44、U87and U373.5. The expression of NF-κB1, BCL2, MMP2and MMP9proteins were detected bywestern blot.6. To observing miR-16has effect with glioma invasion and growth in vivo, we hadestablished intracranial and subcutaneous nude mouse model respectively in U87cellstransfected with miR-16and control oligonucleotide U87cells. HE Staining andImmunofluorescence were used to detect a role for miR-16in glioma invasion and growth.Immunohistochemistry was used to detect the effect of miR-16,NF-κB1, MMP9andKi-67. Luciferase reporter assay demonstrate that NF-κB1and Bcl2are a direct target ofmiR-167. Statistical analyses were performed using SPSS version13.0, and significancedetermined with two-tailed Student’s t test. Data were considered statistically significant atp <0.05.Results1. Expression of miR-16and NF-κB1were negatively correlated in glioma.qRT–PCR showed that miR-16expression in non-cancerous brain tissues was lowerthan in human brain glioma tissues (p<0.01) and decreased with the increasing degree ofmalignancy in glioma (low-grade gliomas VS high-grade gliomas p<0.05). In addition, wefound that the expression of miR-16in glioma cell lines SHG44, U87and U373was lowerthan in non-cancerous brain tissues (p<0.01). However, the expression of NF-κB1increased with the increasing degree of malignancy in glioma (low-grade gliomas VShigh-grade gliomas p<0.01).2. miR-16directly targets NF-κB1in human glioma cells. By analyzing the homology between miR-16and NF-κB1mRNA sequences, weobserved that the fifteen nucleotides were complementary. A Dual-Luciferase ReporterAssay System was utilized to validate whether miR-16directly recognizes the3’UTRregion of NF-κB1mRNA. As shown in Fig.2B, the relative luciferase activities of thepre-miR-16groups transfected with NF-κB13’UTR constructs were significantlydecreased, compared to those transfected with pMIR-NF-κB1constructs, for293T cellline, implying that NF-κB1is a direct target of miR-16. To further demonstrate thecorrelation between miR-16and NF-κB1, mRNA and protein expression of NF-κB1weredetected by qRT-PCR and Western blottings. Data showed that miR-16up-regulationdramatically reduced the protein expression of NF-κB1. Meanwhile, we observed thatNF-κB1expression was increased by down-regulating miR-16.3. Upregulation of miR-16induces apoptosis in human glioma cells by directlydown-regulating BCL2expression.The microRNA.org database shows that BCL2is a target of posttranscriptionalrepression by miR-16. Our data showed that miR-16expression was inversely correlatedwith BCL2in the same tissue samples of human glioma. A Dual-Luciferase Reporter AssaySystem was utilized to validate whether miR-16directly recognizes the3’UTR region ofBCL2mRNA.Our data implys that BCL2is a direct target of miR-16. Flow cytometry dataindicated that increasing miR-16expression induced early apoptosis, compared with thenegative control oligonucleotide and the mock, and that the proportion of early apoptoticcells in the miR-16treatment group was markedly increased (P<0.01, n=3). BCL2wasdetected by Western blot. Data showed that the up-regulation of miR-16led to obviousdown-regulation of BCL2expression.4. Upregulation of miR-16reduces invasiveness ofglioma cells in vitro.To assess the effects of miR-16on the invasiveness of glioma cells, we used aTranswell invasion system. The numbers of invasive cells with miR-16mimics weresignificantly reduced compared with those with the negative control oligonucleotide andmock. 3. Upregulation of miR-16leads to down-regulation of MMP9expression.The transwell invasion system clearly showed that the up-regulation of miR-16obviously reduced the invasiveness of glioma cells. Western blot Data showed that MMP9expression was obviously inhibited by the up-regulation of miR-16and that the oppositeresult was observed by inhibiting miR-16expression. However, miR-16had little effect onMMP2expression.6. Upregulation of miR-16reduces glioma growth and invasiveness in an in vivomouse model.As shown in our experiments, all tumors from U87cells transfected with plasmidconnected pre-miR-16were efficiently suppressed compared with negative control. Theintracranial tumors were removed, and sectioned. HE staining showed that miR-16reducedglioma growth and invasion in the encephalic nude mouse model. Immunofluorescenceshowed that miR-16reduced glioma invasion and MMP9expression in the nudeencephalic mouse model. Sections were stained for Ki-67, MMP9and NF-κB1.Immunohistochemistry showed that tumors with U87cells transfected with miR-16hadreduced Ki-67, MMP9and NF-κB1stainings, compared with negative controloligonucleotide. Thus, the data indicated that miR-16up-regulation led to the inhibition ofglioma cell proliferation and invasion in tumor xenografts, and that the up-regulation ofmiR-16reduced glioma growth in the mouse model in vivo.Conclusions:we identified miR-16as a negative regulator of tumor growth and invasion in vitroand in vivo in glioma. These in vitro and in vivo findings were further validated in patientsamples, where miR-16expression was inversely correlated with the grade of glioma.Mechanistically, the tumor-suppressive role of miR-16can be attributed to inhibition of theBCL2and NF-kappaB1/MMP-9signaling pathway. In conclusion, our results add amechanistic insight regarding the role of miR-16in the growth and aggressiveness ofglioma. As glioma presents a therapeutic challenge due to the lack of molecular targets, atherapeutic miRNA approach, where oncogenic pathways are directly targeted, might provide new insights into the development of therapeutic strategies against cancer viamiR-16.