To validate microarray results, we performed real-time qRT-PCR expression analysis on one tumor samples analyzed by microarray and furthermore the other three new patients' samples. Including additional miR-917,21miR-128,9miR-204 and miR-214, the expression of 8 selected miRNAs genes were analyzed (Figure 2). The expression data obtained by qRT-PCR analysis are comparable to the microarray data. The qRT-PCR for miR-17, miR-100, miR-106b, and miR-218 confirmed results obtained by microarray analysis.
Given that biological significant of miRNA deregulation relies on their protein-coding gene targets, we analyzed the predicted targets of the most significantly down-regulated and up-regulated miRNAs: miR-17, miR-100, miR-106b, and miR-218. The analysis was done using the ensemble algorithm to predict human miRNA gene targets. Partial target genes (including oncogene and tumor suppressor gene) are showed in Table 3.
Although MB represent the most frequent highly malignant brain tumors in childhood, their molecular pathogenesis is only partially understood. Calin et al22 demonstrated that >50% of miRNA genes are located at chromosomal regions, such as fragile sites, and regions of deletion or amplification that are genetically altered in human cancer, suggesting that miRNAs may play an important role in the pathogenesis of human cancers as a novel class of oncogenes or tumor suppressor genes. So, we reasoned that alterations in miRNA expression might contribute to MB. As far as we know, there is only one group research reported about miRNAs expression in human MB and normal tissue.17 To investigate whether miRNAs are differentially expressed among different MB individuals, we performed a high throughput microRNA microarray in human primary MB specimens to investigate microRNA involvement in carcinogenesis.
Although northern blotting and gene clone technique are widely used method for miRNA analysis, they have some limitations, such as difficulty in detecting multiple miRNAs simultaneously. MiRNA microarray analysis has become a comprehensive technology to help us better understand the relationship between cancer tissues and normal tissues.23 In addition, miRNA microarray technology has been widely used to study the roles of miRNA in cancers.6,12 An oligonucleotide miRNAs array chip has been a most widely used high throughput screening method to detect the expression profiles of hundreds of miRNAs in human cancer simultaneously.21
In this study, to reduce the bias from individual difference, the final microarray chip results were derived from the intersection set of three different samples. And then we recognized differentially expressed miRNAs which are same expression tendency in different chips as the final results and validated their reliability by qRT-PCR among extended samples range. The criteria for aberrant expression miRNAs in MB were as follows: (1) up- or down-expression change tendency in all chips and significantly different (P <0.05); (2) the detected signals ratio of T-MB and N-MB more than 2 or less than 0.5 fold at least.
Rapidly detect miRNA expression, real-time PCR can also be employed to quantify miRNAs expression profiles and study the potential function of miRNAs in cancer pathogenesis.24 We analyzed six MB samples to identify miRNAs whose expression were significantly deregulated in cancer versus non-tumorous cerebellum tissues by qRT-PCR analysis comparable to the microarray data. We have indeed identified four miRNAs whose expression were significantly deregulated (at P <0.05). These results leave few doubts that aberrant expression of miRNA is indeed involved in human MBs. Three of them, miR-17, miR-100, and miR-106b, were up-regulated and miR-218, was down-regulated, suggesting that they may potentially act as oncogenes or tumor suppressor genes, respectively. In line with our study, Uziel et al25 demonstrated that 3 miR-17-92 cluster miRNAs family (miR-92, miR-19a and miR-20) were also overexpressed in mouse MB with a constitutively activated Sonic Hedgehog (SHH) signalling pathway which involves the carcionogenesis in MB. In addition, Ferretti et al17 also confirmed that miR-106b and miR-17-5p were upregulated in MB vs normal cerebellum.
For the up-regulated miRNAs, it may be expected that their tagets include tumor suppressor genes or genes encoding proteins with potential promote tumor development by negatively inhibiting oncogenes. Interestingly, the tumor suppressor genes, fas-activated serine, FASTK and the p53-binding protein 3, TOPORS were potential targets of both miR-17 and miR-106b. It has been reported that the function of miR-17-92 cluster and miR-106b might play a role as synergistic effect each other involved same intracellular biological procedure.30 And then we discussed the same targets of both of them. Consequently, bioinformatics studies indicate that numerous genes are the targets of miR-17, miR-106b, and miR-100. Reasonably, among putative targets, several genes act as potential tumor suppressor genes, such as BAMBI, TOPORS, BTG2, TOB1, CASP8, and WEE1 could be found. TOPORS is encoding gene for tumor suppressor p53-binding protein, which results in the suppression of cell growth by cell cycle arrest and/or by the induction of apoptosis and mediates p53-dependent cellular responses as a tumor suppressor.31 It has been already reported in gliomas.32 BAMBI is an encoded protein to function as tumor suppressor gene. It is confirmed that the member of transforming growth factor (TGF)-beta family play important roles as an oncogenes in signal transduction in MB pathological process.33,34 BAMBI is a pseudoreceptor of TGF type I receptors and acts as a negative regulator of TGF-beta signalling. The antiproliferative genes BTG2 encode nerve growth factor (NGF)-inducible anti-proliferative protein which involved in the regulation of the G1/S transition of the cell cycle and act as anti-proliferative tumor suppressor. MB originate from cerebellar granule cell precursors (GCPs), located in the external granular layer (EGL) of the cerebellum while BTG2 promotes cerebellar neurogenesis by inducing GCPs to shift from proliferation to differentiation. Farioli-Vecchioli et al35 demonstrated up-regulation of BTG2 in mice resulted in a decrease of MB incidence. In addition, the tumor suppressor genes SUFU, PTCH1, and RB1 which were associated with MB carcinogenesis were predicted to be targeted by miR-100.
On the other hand, it may be expected that targets of down-regulated miRNAs include oncogenic functions. The pro-oncogenes ROS1, EGFR, and BCL2L11 (Bcl-2), CTNND2 (β-catenin), EGFR, MAPK9, MYBL1, TNFRSF1A (TNF) were predicted to be targeted by miR-218. ERBB1, oncogenes EGFR encoded protein, play an important role as a pro-oncogene in gliomas and as one of the therapeutic targets in MB;36CTNND2 is the encoded gene for β-catenin which be involved in the activation of APC/Wnt signal transduction pathway in MB.37 So, down-regulated of miR-218 would result in over-expression of the predicted target EGFR, which can activate mitogen-activated protein kinases (MAPK) pathway and β-catenin to promote development and invasion of tumor cell.
In conclusion, results reported here increase our understanding of the molecular basis of MB and suggest that aberrant expression of miRNA genes may be important for the pathogenesis of MB. There is a long way to go before artificial and natural miRNAs therapy could be used as cancer therapeutic tools and strategies for the clinical service. Additional studies will be required to further characterize role of the miRNAs gene regulation in MB.
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