MicroRNA-132 Can Inhibit Glioma Cells Invasion And Migration By Target MMP-16 In Vitro

Background and Objective:Gliomas are the most common malignant primary brain tumors and are characterized by increased proliferation, robust angiogenesis, and invasion into the surrounding normal brain tissue. Despite aggressive surgery, radiation, and chemotherapy, the median survival of glioblastoma patients is only 12-15 months. Since tumor invasion is a major reason for treatment failure, the development of novel therapeutic strategies aimed at limiting or reducing the ability of the invasion of glioma cells could have a profound effect on patient survival. new clinical biomarkers and therapeutic targets are urgently required.Micro RNAs(mi RNAs) are a novel class of small non-coding RNAs(approximately 22 nucleotides in length) involved in the regulation of various biological processes. Here, by using real time PCR, micro RNA-132(mi R-132) was found to be significantly dysregulated in glioma tissues. Based on the prediction of the target genes of mi R-132, we hypothesized that there was a significant association between mi R-132 and MMP-16(MT-MP3), a protein of the matrix metalloproteinase(MMP) family. The over-expression of mi R-132 was shown to inhibit cell migration and invasion in the human glioma cell-lines A172, SHG44, and U87. Furthermore, the over-expression of mi R-132 reduced the expression of MMP-16 in A172, SHG44, and U87 cells. Collectively the results suggested that mi R-132 affected glioma cell migration and invasion by MMP-16 and implicated mi R-132 as a metastasis-inhibiting mi RNA in gliomas.Micro RNAs(mi RNAs) are short and single-stranded nucleotide RNA molecules that regulate gene expression by binding to the 3`-untranslated regions of their target m RNA molecules, which represses transcription or induces m RNA degradation. mi RNAs control cell growth, proliferation, metabolism, and apoptosis. Indeed, specific mi RNA dysregulation has been shown to correlate with particular types of cancer. For example, mi R-16 has a lower level of expression in glioma cells and suppresses Bcl-2. In addition, mi R-145 was over-expressed in metastatic glioma cells and suppressed expression of ADAM17.Matrix metalloprotease 16(MT3-MMP) is a membrane-type metalloprotease that functions by activating pro MMP-2(gelatinase A) into its active form as the zymogen is excreted out of the cell. Therefore, a zymogram depicting the gelatinase activity of activated MMP-2 would be an indirect mechanism for determining the activity of MMP-16. MMP-2 can cleave collagen IV of the basement membrane and is implicated in cancer metastasis. It is thus unsurprising that high MMP16 expression has been associated with increasing invasiveness in gastric cancer, hepatocellular carcinoma, prostate cancer, and melanoma cells.The aim of the present study was to explore mi R-132 expression in human brain gliomas and in three malignant glioma cell lines(A172, SHG44,and U87). as well as cell apoptosis and invasiveness. This study also aimed to lay the foundation for future in-depth study of the mechanisms of action of mi R-16 in human glioma.Materials and Methods1. Through utilising the micro RNA.org database and the Database on Predicted and Published Micro RNAs(mi RWalk), we previously identified mi RNA132(mi R-132) is most strongly correlated with malignancy in nearly all analyzed human tumors.2. Chemical synthesis mi R-132 oligonucleotide random sequence was transfected into the malignant glioma A172, SHG44, and U87 cell lines by the Lipofectamine2000 liposome.3. The transfection rates in human glioma cell lines A172, SHG44, and U87 were determined by flow cytometry and Green fluorescence.4. Real-time quantitative RT–PCR(q RT–PCR) was used to determine the expression of mi R-132 and MMP-16 in noncancerous brain tissue and Human glioma tissue samples respectively.5. Scratch migration assay and Transwell assay were used to detect invasive effect of mi R-132 in human glioma A172, SHG44, and U87.6. The expression of MMP-2 and MMP-16 proteins were detected by western blot.7. Luciferase reporter assay demonstrate that MMP-16 is a direct target of mi R-1328. Data are presented as the mean ± standard deviation. The data were analyzed using the SPSS version 12.0 Windows version software. Statistical analyses were performed using analysis of variance(ANOVA) or Student’s t-test. P values < 0.05 were considered statistically significant.Results1. The expression of mi R-132 was lower in glioma tissues compared to normal brain tissuesTo understand the role of mi R-132 in glioma tissues, real-time PCR analyses were performed to determine the expression of mi R-132 in 11 low-grade glioma tissues and 12 high-grade glioma tissues compared to 8 normal brain tissues. mi R-132 expression levels were significantly decreased in glioma tissues compared to normal brain tissues.The results also showed that the expression of mi R-132 was lower in glioma cell-lines compared to normal brain tissues. The down-regulation of mi R-132 in glioma tissues and glioma cell-lines suggested that mi R-132 may be a potential target in glioma therapy.2. mi R-132 inhibited the invasion of glioma cellsTo understand the effects of mi R-132 on glioma cell invasion and migration, mi R-132 mimic and inhibitors were used to infect human glioma A172, SHG44, and U87 cells. Seventy-two hours after transfection with mi R-132 or scrambled-mi RNA, A172, SHG44, and U87 cells were seeded into the upper chamber, and then cells that invaded through the extracellular matrix after 48 h were imaged and counted. In both cell-lines, mi R-132 decreased the number of cells that invaded compared to the controls. The collective data indicated that mi R-132 may be a regulatory molecule in cell migration and invasion in vitro.3. The expression of MMP-16 m RNA in glioma tissus.Real-time PCR analyses showed that the expression of MMP-16 m RNA was dramatically higher in 11 low-grade glioma tissues and 12 high-grade glioma tissues compared to 8 normal brain tissues. The results shown in figure demonstrated that MMP-16 m RNA had a higher expression in glioma tissues compared to mi R-132 in glioma tissues, suggesting that the expression of MMP-16 m RNA expression was inversely related to mi R-132.4. mi R-132 downregulate the expression of MMP-16 m RNA.To clarify the mechanism by which mi R-132 inhibited cell migration in glioma cells, mi R-132 targets were found using the algorithms Target Scan5 and mi RBase, which revealed that MMP-16 might play a role in cell migration and invasion. A RT-PCR assay and q PCR systems were used as well. Transfection of glioma cells with mi R-132 mimic significantly decreased the expression of MMP-16 m RNA in glioma cells compared to the levels in control mi R-expressing cells. The above results showed that MMP-16 may be a target gene of mi R-132, promoting glioma cell migration and invasion.5. mi R-132 can directly affect the expression of the target gene MMP-16To validate whether mi R-132 affected the expression levels of MMP-16 protein, the protein expression level of MMP-16 was measured in response to the effects of mi R-132 expression levels in A172, SHG44, and U87 following transfection. This showed that protein expression levels of MMP-16 were down-regulated, and by contrast it was up-regulated following transfection with the mi R-132 mimic and the mi R-132 inhibitor respectively. The luciferase assay revealed reduced relative luciferase activities in 293 T cells stably over-expressing mi R-132 following transfection with MMP-16 3’-UTR(p < 0.05).6. MMP-16 as an activator of MMP-2 activated the expression level of MMP-2 proteinmi R-132 was predicted to target MMP-16(MT3-MMP, membrane type 3 MMP)(www.micro RNA.org), which is a potential activator of MMP-2. Western blot was used to validate the hypothesis. The results showed that mi R-132 activated the expression of MMP-2 protein by regulating the expression of MMP-16. The over-expression of mi R-132 inhibited invasion in human glioma cells by directly down-regulating MMP-16 expression..Conclusions:The hsa-mi R-132 gene is located in chromosome 17(1953202-1953302) with 100 bp in the mi R base. In the last several years, many studies have found the deregulated expression of mi R-132, which has been implicated in the development and progression of various cancers. For instance, mi R-132 can act as a tumor suppressor and inhibit cell proliferation in breast cancer. Furthermore, the dysregulation of mi R-132 has been found in primary osteosarcoma, Alzheimer’s disease, prostate cancer, and pancreatic cancer. However, the function of mi R-132 in regulating glioma cell migration and invasion remained unexplored.The present study demonstrated lower levels of mi R-132 in glioma cancer tissues than in normal brain tissues and lower levels in glioma cells than in normal brain tissues. Furthermore, mi R-132 inhibited invasion and migration in glioma cells. Glioma cells transfected with mi R-132 mimic showed decreased tumor cell migration and invasion, while transfection with mi R-132 inhibitor produced the opposite result. Nevertheless, whether mi R-132 inhibits tumor the invasion and metastasis of glioma cells in vivo remains to be confirmed, because such a study is hampered at present by the lack of an experimental strategy for stably increasing or silencing mi RNAs over extended periods of time.The mi R-132 function of inhibiting glioma cell invasion and migration properties was further demonstrated by the reduced expression of a matrix metalloproteinase gene, MMP-16. MMP-16 is a membrane-anchored MMP that is able to activate MMP-2 for invasion. Since MMP-2 was activated by MMP-16, it was an indirect indication of the function of MMP-16. The present study’s results suggested the functional relevance of MMP-16 in the down-regulation of glioma cell migration. The previous report showed that decreasing MMP-16 levels efficiently inhibited glioma cell invasion. MMP-16 was also found to possess proteolytic activity against extracellular matrix(ECM) components such as type III collagen. It is possible that MT3-MMP was involved in the turnover of ECM in the normal brain and astrocytic tumor tissues.In conclusion, the data suggested that mi R-132 plays a key role in the malignancy of glioma cells possibly by directly regulating MMP-16 protein expression, which affects glioma cell migration and invasion. The study’s results suggested that mi R-132 may be a potential therapeutic target for preventing GBM invasion and metastasis. However, further study is needed to determine if MMP-16 activity is influenced by mi R-132 in vivo in glioma cells.