Study Of The Effects Of MiR-146b-5p Abnormal Decrease On Occurrence And Progress Of Gliomas As Well As The Mechanisms

Objectives:miR-146b-5p is a tumour suppressive miRNA (TS-miRNA) of gliomas. Gliomas are characterized by high rate of recurrence and poor prognosis, because they usually grow in an infiltrative manner and cannot be excised completely. MMP16is responsible for the invasive growth of several kinds of extracranial malignant tumours. Although bioinformatic prediction suggests that MMP16is a potential target of miR-146b-5p, little is known about whether miR-146b-5p and MMP16express differently in various grade of gliomas, what roles these expression changes play in glioma-genesis and progression, and whether and how miR-146b-5p modulate the invasion and migration of the glioma cells. The aim of this study is to explore the above questions in human glioma samples and human glioblastoma cell lines.Methods:(1) The expression levels of miR-146b-5p and MMP16in60gliomas of various malignant grades and10non-tumoural control brain tissues were measured by means of lock-nucleotide probe in situ hybridization and immunohistochemistry, respectively.(2) Luciferase assay, qRT-PCR and Western blot were adopted to confirm whether MMP16was the target of miR-146b-5p and further clarify the mechanism by which miR-146b-5p down-regulated the expression of MMP16.(3) The miR-146b-5p overexpressing sub-cell lines from human glioblastoma cell lines (U87MG, SNB19and TJ905) and their scrambled-sequence-overexpressing controls were established by plasmid transfection and G418screening, and their miR-146b-5p as well as MMP16mRNA and protein expression levels were detected by stem-loop qRT-PCR, regular qRT-PCR and Western blot, respectively.(4) Western blot and zymography of metalloproteinase were used to verify whether miR-146b-5p could suppress the expression of MM16and block the activation of pro-MMP2.(5) The migratory and invasive abilities of the miR-146b-5p overexpressing sub-cell lines and their corresponding controls were assessed by transwell in vitro migration and invasion assays and Matrigel3D culture, and targeting MMP16specific siRNA blocked the expression of MMP16and further validated whether miR-146b-5p could suppress the migration and invasion of the glioblastoma by down-regulating the expression of MMP16.(6) The apoptotic levels of the glioblastoma sub-cell lines were evaluated by cytometry (FCM) and single cell gel electrophoresis (SCGE).(7) The cell cycle distribution of the sub-cell lines was analyzed by FCM.Results:(1) The result of in situ hybridization showed that, miR-146b-5p labeling indexes (LI%) of the WHO Grade Ⅰ～Ⅱ, Grade Ⅲ and Grade Ⅳ group were13.34±0.14,8.60±0.17,2.90±1.26for respectively and all were significantly lower than that of the non-tumoural control brain tissues (34.23±1.58, P<0.0001). The expression level of miR-146b-5p decreased with the malignancy grades, and all the differences except for the one between the WHO Grade Ⅰ～Ⅱ and the Grade Ⅲ group were statistically significant (P<0.001). While the expression level of MMP16showed an opposite trend, which was lowest in the control brain tissues (0.40±0.55) and kept on increasing with the tumours’ malignancy grades (25.99±1.24,30.99±1.30,64.37±6.26, for the WHO Grade Ⅰ～Ⅱ, Grade Ⅲ and Grade Ⅳ group, respectively). All the differences except for the one between the WHO Grade Ⅰ～Ⅱ and Grade Ⅲ group were statistically significant (P<0.0001). Linear correlation analysis showed that the LI%s of the two molecules correlated negatively with each other (r=-0.634, P<0.0001).(2) Luciferase assay results showed that, miR-146b-5p could mediate the silencing effect of the expression of luciferase gene in glioblastoma cells by specially binding with the wild type3’UTR of MMP16mRNA. Because miR-146b-5p could not bind with the mutated type3’UTR of MMP16mRNA, miR-146b-5p could not mediate the silencing effect of the expression of luciferase gene when both co-tranfected glioma cells.(3) Compared with the corresponding controls, the expression levels of miR-146b-5p were dramatically higher in the miR-146b-5p overexpressing sub-cell lines, while the expression levels of MMP16mRNA and protein were significantly lower, suggesting that miR-146b-5p could block the expression of MMP16by directly degrading its mRNA.(4) miR-146b-5p did not affect pro-MMP2expression, but it could block the activation of extracellular pro-MMP2by suppressing MMP16expression.(5) miR-146b-5p overexpression effectively abated the migratory and invasive abilities of the glioblastoma cells by the mechanisms, while MMP16specific siRNA could partially mimic this anti-invasive effect, suggesting that miR-146b-5p/MMP16/MMP2pathway played an important role in miR-146b-5p-mediated inhibitory effect to the migration and invasion of glioma cells.(6) FCM and SCGE results demonstrated that the apoptotic levels of the miR-146b-5p overexpressing sub-cell lines were significantly higher than those of their corresponding controls, but the cell cycle distribution did not change, indicating that miR-146b-5p promoted the tumour cells to undergo apoptosis rather than halted their proliferation.Conclusions:(1) With the malignancy grade of gliomas arises, the expression level of miR-146b-5p decreases while that of MMP16increases, and the expression levels of these two molecules correlate inversely with each other. The expression level of miR-146b-5p and MMP16could be served as an important reference index of assessing glioma biological behavior and patients prognosis.(2) The miR-146b-5p overexpressing sub-cell lines established in this study can stably express high levels of miR-146b-5p, and therefore can be used as cell models to study the tumour suppressive effects of miR-146b-5p.(3) MMP16is the natural target gene of miR-146b-5p in glioma cell, and miR-146b-5p can down-regulate the expression of MMP16by directly degradating its mRNA,(4) miR-146b-5p does not affect the expression of pro-MMP2, but it can block the activation of the extracellular pro-MMP2by suppressing MMP16and thereby abate the invasive abilities of the glioma cells.(5) miR-146b-5p has no effect on tumour cell proliferation and cell cycle distribution, but it can suppress the migration and promote the apoptosis of the glioma cells. The molecular mechanism of the latter two regulatory role is not clear and should be further explored.(6) The abnormal decrease of miR-146b-5p expression is one of the important factors for glioma-genesis and malignant progression. Exogenous miR-146b-5p can reverse the abnormal and play the effective tumor suppression role, therefore it can exert important practical value as a multifunctional tumour suppressor in the gene therapy of malignant gliomas.