Study On The Roles Of The Abnormal Decrease Of MiR-320a In Gliomagenesis And Progression Of Gliomas As Well As The Mechanisms

Objectives:miR-320 a is a tumor suppressor in various malignancies. The deregulation of this miRNA plays a pivotal role in tumorigenesis and malignant progression. However, the expression pattern and prognostic significance of miR-320 a in gliomas remain to be delineated. Staphylococcal nuclease domain-containing 1(SND1) and β-catenin, potential targets of miR-320 predicted by bioinformatics, are overexpressed with the elevation of glioma grades. It’s imperative to validate whether they are bona fide targets of miR-320 a in gliomas. Moreover, the functional roles of miR-320 a in gliomas, the underlying mechanisms that SND1 and β-catenin participate in and the downstream effectors involved in this regulatory network remain to be elucidated. In the present study, we set out to investigate these issues thoroughly and systematically using glioma brain tissues of various pathological grades, as well as human glioblastoma cell lines. Methods:1. The endogenous miR-320 a levels were detected in 120 gliomas and 20 non-tumoral control brain tissues by ISH with the LNA-modified and DIG-labeled miR-320 a oligonucleotide probe. Dichotomized by miR-320 a LI%, patients’ survival was analyzed with Kaplan-Meier method. Stem-loop q RT-PCR was employed to detect miR-320 a levels in 7 human GBM cell lines and the human astrocyte cell line UC2.2. To examine effects of miR-320 a in vitro, we performed gain-of-function analysis with transient transfection of miR-320 a. To confirm the efficiency of transfection, miR-320 a levels were quantified by stem-loop q RT-PCR. The CCK8 assay, colony formation assay, transwell assay and wound healing assay were performed to reflect the effect of miR-320 a on GBM cells in U87 MG and U251 cells.3. The potential targets of miR-320 a were predicted by Target Scan and miRanda. The dual-luciferase reporter assay was performed to verify the functional binding of miR-320 a with SND1 or β-catenin 3’UTR. Western blot and q RT-PCR analysis were performed to monitor endogenous changes of SND1 and β-catenin with miR-320 a reintroduction.4. The expression levels of SND1, β-catenin and Ki-67 were detected immunohistochemically in the same batch of tissue samples and comparison was made among pathological grades. Dichotomized by SND1 or β-catenin LI%, patients’ survival was analyzed with Kaplan-Meier method. The Cox’s proportional hazards regression model was applied for univariate and multivariate survival analyses to screen out independent predictors for gliomas.5. The CCK8 assay, Ed U assay and flow cytometry were performed to reflect the effect of miR-320 a on proliferation and cell cycle progression of GBM cells. To determine whether SND1 and β-catenin were functional mediators for miR-320 a in glioma cells, we performed a series of rescue experiments by restoring the decreased β-catenin and SND1 levels in cells transfected with the miR-320 a. To unveil the mechanism by which miR-320 a suppressed the proliferation of GBM cells, Western blot was adopted to detect the expression levels of p21WAF1 and cyclin D1.6. The migration and invasion capacities of GBM cells transfected with miR-320 a were gauged by the transwell assays. To ascertain the roles of SND1 and β-catenin in the suppressive effect of miR-320 a on GBM cells, the rescue experiments were conducted with SND1 and β-catenin restoration. The MMP2 and MMP7 enzymatic activities in the U87 MG and U251 media were evaluated by SDS-PAGE zymography. Western blot and q RT-PCR were adopted to detect the intracellular levels of MMP2 and MMP7.7. To evaluate the effects of SND1 on glioma invasiveness, we established the SND1-knockdown(SND1-SH) and control(SND1-HK) sub-cell lines of U87 MG and U251 with infection of the recombinant lentiviruses. Transwell assays were carried out to assess the migration and invasion capabilities of GBM sub-cells. q RT-PCR analyses were performed to detect m RNA levels of T-Smad2, Smad4 and MMP2 in those sub-cells. Western blot were adopted to detect the expression levels of T-Smad2, P-Smad2, Smad4 and MMP2 in the U87 MG sub-cells with or without TGFβ1 treatment. Results:1. The ISH result showed that miR-320 a expression in gliomas was lower than that in the control and that its expression was significantly decreased with the elevation of glioma grades and was the lowest in GBM(P<0.001). The Kaplan-Meier analyses showed that patients with higher miR-320 a levels had a longer disease-free survival(DFS) and overall survival(OS), which was also the case when comparisons were performed within the same malignancy grade(P<0.0001). The stem-loop q RT-PCR detection showed that miR-320 a expression in 7 human GBM cell lines were also significantly reduced in comparison with human astrocyte cell line UC2(P<0.01~0.001).2. The CCK8 and colony formation assays showed that miR-320 a could effectively inhibit the proliferation(P>0.01~0.001) of U87 MG and U251 cells and potently lower the colony formation efficiency(P<0.01~0.001) of U251 cells. In addition, transwell assays and wound healing assays showed that the migration and invasion capacities were remarkably impaired in the miR-320 a groups(P<0.01~0.001).3. In the list of candidates predicted by Target Scan and miRanda, SND1 and β-catenin were identified as potential targets due to their desired ranking scores and tight connections with the malignant phenotypes of various cancers. The luciferase assays showed that the wild type 3’-UTR fragments of SND1 or β-catenin could effectively mediate the silencing effect of miR-320 a on reporter gene expression, whilst the mutant ones, with the binding sites depleted, failed to exert a similar effect. Moreover, q RT-PCR and Western blot captured a dramatic decrease(P<0.05~0.001) in the m RNA and protein levels of SND1 and β-catenin after miR-320 a reintroduction.4. The IHC results showed that the expression of SND1, β-catenin and Ki-67 in the gliomas were higher than those in the control, and that their expression increased with the elevation of the malignancy grade and were the highest in GBM(P<0.001). Moreover, the expression of SND1 or β-catenin was inversely correlated with that of miR-320a(for SND1, r=- 0.981 and for β-catenin, r=- 0.975) and positively correlated with that of Ki-67(for SND1, r= 0.984 and for β-catenin, r=0.975). The Pearson correlation analysis also showed that the expression of miR-320 a was inversely correlated with that of Ki-67(r=- 0.976). The Kaplan-Meier analyses demonstrated that a high level of SND1 or β-catenin was associated with a poor prognosis in the glioma patients(P<0.0001). Furthermore, multivariate and univariate analyses ascertained miR-320 a and SND1 as independent predictors and β-catenin as an auxiliary predictor for DFS and OS of glioma patients.5. The CCK8 and Ed U assays showed that miR-320 a transfection largely inhibited the proliferative activity of U87 MG and U251 cells, whilst the replenishment of β-catenin or SND1 substantially reversed the anti-proliferative effect of miR-320 a. The FCM results showed that miR-320 a upregulation increased the G0/G1-phase cells and reduced the S-phase cells of these cells, whilst the overexpression of β-catenin or SND1 abrogated the G0/G1-phase arrest induced by miR-320 a. Western blot results showed that miR-320 a transfection significantly reduced cyclin D1 and increased p21WAF1 through downregulating β-catenin and SND1 in GBM cells, and the effects of miR-320 a on cyclin D1 and p21WAF1 could be reversed by β-catenin or SND1 overexpression, respectively.6. The transwell assay results showed that the migratory and invasive capacities of the GBM cells were largely impaired by miR-320 a transfection and partially rescued by SND1 or β-catenin overexpression. Gelatin and casein zymography assays indicated that MMP2 and MMP7 activities were significantly lowered in the culture medium of GBM cells transfected with miR-320 a. The q RT-PCR results also showed significantly lower levels of MMP2 and MMP7 in miR-320 a groups. Western blot results verified that miR-320 a transfection significantly decreased MMP2 and MMP7 proteins through downregulating SND1 or β-catenin in GBM cells, and the suppressive effects of miR-320 a could be reversed by SND1 or β-catenin overexpression.7. The transwell results showed that the migratory and invasive capabilities were significantly decreased in the SND1 knockdown sub-cell lines. The Smad2, Smad4 and MMP2 were largely decreased by SND1 knockdown gauged by q RT-PCR and Western blot. Notably, with TGFβ treatment, although P-Smad2 and MMP2 were slightly increased in SND1-knockdown cells, both basal and TGF-β-induced expression of MMP2 was largely reduced and abrogated by SND1 knockdown, indicating that SND1 knockdown was sufficient to counteract the induction of MMP2 triggered by TGF-β signaling activation. Conclusions:1. The aberrant decrease of miR-320 a is a common feature of human gliomas and glioblastoma cell lines. The expresson level of miR-320 a correlates perfectly with the pathological grades of gliomas, hence miR-320 a can serve as a potential biomarker for distinguishing malignant gliomas and as an important reference to glioma grading system.2. miR-320 a can potently inhibit the proliferation, migration and invasion of the GBM cells. The deregulation of miR-320 a contributes to the rapid growth and relentless invasion of GBM, hence plays a pivital role in the gliomagenesis and malignant progression.3. SND1 and β-catenin are direct targets of miR-320 a which downregulates their expression via inducing the degradation of their m RNAs in GBM cells. Therefore, miR-320 a inhibits the expression of SND1 and β-catenin at the post-transcriptional level.4. The expression levels of SND1 and β-catenin are inversely correlated with that of miR-320 a in the same batch of clinical tissue samples and thus provide a possible explanation for the abnormal overexpression of these factors in glioma cells. miR-320 a and its two targets well predict the survival of glioma patients. miR-320 a and SND1 are the independent predictors and β-catenin is an auxiliary predictor for patients’ survival.5. miR-320 a reduces the level of cyclin D1 and increases the level of p21WAF1 by silencing β-catenin and SND1, synergistically arrests glioma cells in G0/G1 phase and thus retards cell cycle progression, and eventually suppresses GBM cell proliferation.6. miR-320a-induced knockdown of SND1 and β-catenin reduces the endogenous expression and extracellular activities of MMP2 and MMP7, consequently suppressing migration and invasion of GBM cells.7. SND1 knockdown not only decreases the levels of critical factors in TGF-β signaling pathway, but also blunts GBM cells to TGF-β stimulus. Both basal and TGF-β-induced expression of MMP2 are largely reduced and abrogated by SND1 knockdown, unveiling a novel regulatory role of SND1 on MMP2 expression.8. miR-320 a can act as a versatile suppressor in glioma and exert its anti-glioma effects through multiple targets and pathways, which provides us a better understanding of the tumorigenesis and progression of gliomas. With the strong clinical relevance and delicate regulatory network, miR-320 a has potential value as a prognostic biomarker and therapeutic candidate of human gliomas. Our work sheds great light on the miR-based gene interference and targeted molecular therapy.