Integrated analysis of miRNA and mRNA expression in childhood medulloblastoma compared with neural stem cells

Medulloblastoma (MB) is the most common malignant brain tumor in children and a leading cause of cancer-related mortality and morbidity. Several molecular sub-types of MB have been identified, suggesting they may arise from distinct cells of origin. Data from animal models indicate that some MB sub-types arise from multipotent cerebellar neural stem cells (NSCs). Hence, microRNA (miRNA) expression profiles of primary MB samples were compared to CD133+ NSCs, aiming to identify deregulated miRNAs involved in MB pathogenesis. Expression profiling of 662 miRNAs in primary MB specimens, MB cell lines, and human CD133+ NSCs and CD133- neural progenitor cells was performed by qRT-PCR. Clustering analysis identified two distinct sub-types of MB primary specimens, reminiscent of sub-types obtained from their mRNA profiles. 21 significantly up-regulated and 12 significantly down-regulated miRNAs were identified in MB primary specimens relative to CD133+ NSCs (p<0.01). The majority of up-regulated miRNAs mapped to chromosomal regions 14q32 and 17q. Integration of the predicted targets of deregulated miRNAs with mRNA expression data from the same specimens revealed enrichment of pathways regulating neuronal migration, nervous system development and cell proliferation. Transient over-expression of a down-regulated miRNA, miR-935, resulted in significant down-regulation of three of the seven predicted miR-935 target genes at the mRNA level in a MB cell line, confirming the validity of this approach. This study represents the first integrated analysis of MB miRNA and mRNA expression profiles and is the first to compare MB miRNA expression profiles to those of CD133+ NSCs. We identified several differentially expressed miRNAs that potentially target networks of genes and signaling pathways that may be involved in the transformation of normal NSCs to brain tumor stem cells. Based on this integrative approach, our data provide an important platform for future investigations aimed at characterizing the role of specific miRNAs in MB pathogenesis.     

胃癌中显著下调的miR-433的作用靶点研究

目的为了筛选胃癌中miRNAs的表达标记,验证胃癌相关miRNAs的作用靶点,建立一种新的诊断和治疗胃癌的方法。方法运用基因芯片技术检测3个正常胃组织标本,24个胃癌组织标本,胃癌细胞SGC7901和正常胃黏膜细胞GES-1中328个miRNAs的表达情况。用以上方法检测出在胃癌组织和SGC7901中,miR-433的表达水平显著下调。为了确保结果的准确性,采用实时荧光定量PCR对其进行验证。并用基因克隆和Western印迹方法分析miR-433的作用靶点。结果共有26个miRNAs在胃癌标本（包括24个胃癌组织和SGC7901）中异常表达。其中19个miRNAs下调,7个miRNAs上调。实时荧光定量PCR检测出miR-433在胃癌标本中的表达水平显著下调,该结果和基因芯片检测结果一致。另外,在本实验中发现miR-433与Grb2（growth factor receptor—bound protein 2）的表达呈负相关。结论胃癌相关miRNAs已进行了初步筛选。其中,miR-433可能是胃癌中的标记性miRNAs之一,Grb2是其作用靶点。这为建立新的以miRNAs为基础的诊断和治疗胃癌的方法提供了相关信息。     

miRNA expression profile during osteogenic differentiation of human adipose-derived stem cells

Human adipose-derived stem cells (hADSC) are capable of differentiating into an osteogenic lineage. It is believed that microRNAs (miRNAs) play important roles in regulating this osteogenic differentiation of human adipose-derived cells, although its molecular mechanism remains unclear. We investigated the miRNA expression profile during osteogenic differentiation of hADSCs, and assessed the roles of involved miRNAs during the osteogenic differentiation. We obtained and cultured human adipose-derived stems cells from donors who underwent elective liposuction or other abdominal surgery at our institution. miRNA expression profiles pre- and post-osteogenic induction were obtained using microarray essay, and differently expressed miRNAs were verified using quantitative real-time polymerase chain reaction (qRT-PCR). The expression of osteogenic proteins was detected using an enzyme-linked immunosorbent assay. Putative targets of the miRNAs were predicted using online software MiRanda, TargetScan, and miRBase. Eight miRNAs were found differently expressed pre- and post-osteogenic induction, among which four miRNAs (miR-17, miR-20a, miR-20b, and miR-106a) were up-regulated and four miRNAs (miR-31, miR-125a-5p, miR-125b, and miR-193a) were down-regulated. qRT-PCR analysis further confirmed the results. Predicted target genes of the differentially expressed miRNAs based on the overlap from three public prediction algorithms: MiRanda, TargetScan, and miRBase Target have the known functions of regulating stem cell osteogenic differentiation, self-renewal, signal transduction, and cell cycle control. We identified a group of miRNAs that may play important roles in regulating hADSC cell differentiation toward an osteoblast lineage. Further study of these miRNAs may elucidate the mechanism of hADSC differentiation into adipose tissue, and thus provide basis for tissue engineering.     

The microRNA expression profiles of mouse mesenchymal stem cell during chondrogenic differentiation

MicroRNAs are potential key regulators in mesenchymal stem cells chondrogenic differentiation. However, there were few reports about the accurate effects of miRNAs on chondrogenic differentiation. To investigate the mechanisms of miRNAs-mediated regulation during the process, we performed miRNAs microarray in MSCs at four different stages of TGF-β3-induced chondrogenic differentiation. We observed that eight miRNAs were significantly up-regulated and five miRNAs were downregulated. Interestingly, we found two miRNAs clusters, miR-143/145 and miR-132/212, kept on down-regulation in the process. Using bioinformatics approaches, we analyzed the target genes of these differentially expressed miRNAs and found a series of them correlated with the process of chondrogenesis. Furthermore, the qPCR results showed that the up-regulated (or down-regulated) expression of miRNAs were inversely associated with the expression of predicted target genes. Our results first revealed the expression profiles of miRNAs in chondrogenic differentiation of MSCs and provided a new insight on complicated regulation mechanisms of chondrogenesis.     

Role of Deregulated microRNAs in Breast Cancer Progression Using FFPE Tissue

MicroRNAs (miRNAs) contribute to cancer initiation and progression by silencing the expression of their target genes, causing either mRNA molecule degradation or translational inhibition. Intraductal epithelial proliferations of the breast are histologically and clinically classified into normal, atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). To better understand the progression of ductal breast cancer development, we attempt to identify deregulated miRNAs in this process using Formalin-Fixed, Paraffin-Embedded (FFPE) tissues from breast cancer patients. Following tissue microdissection, we obtained 8 normal, 4 ADH, 6 DCIS and 7 IDC samples, which were subject to RNA isolation and miRNA expression profiling analysis. We found that miR-21, miR-200b/c, miR-141, and miR-183 were consistently up-regulated in ADH, DCIS and IDC compared to normal, while miR-557 was uniquely down-regulated in DCIS. Interestingly, the most significant miRNA deregulations occurred during the transition from normal to ADH. However, the data did not reveal a step-wise miRNA alteration among discrete steps along tumor progression, which is in accordance with previous reports of mRNA profiling of different stages of breast cancer. Furthermore, the expression of MSH2 and SMAD7, two important molecules involving TGF-β pathway, was restored following miR-21 knockdown in both MCF-7 and Hs578T breast cancer cells. In this study, we have not only identified a number of potential candidate miRNAs for breast cancer, but also found that deregulation of miRNA expression during breast tumorigenesis might be an early event since it occurred significantly during normal to ADH transition. Consequently, we have demonstrated the feasibility of miRNA expression profiling analysis using archived FFPE tissues, typically with rich clinical information, as a means of miRNA biomarker discovery.     

Expression of exosomal microRNAs during chondrogenic differentiation of human bone mesenchymal stem cells

The aim of the current study was to compare the expression of microRNAs (miRNAs) in exosomes derived from human bone mesenchymal stem cells (hBMSCs) with and without chondrogenic induction. Exosomes derived from hBMSCs were isolated and identified. Microarray analysis was performed to compare miRNA expression between exosomes derived from hBMSCs with and without chondrogenic induction, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the differentially expressed miRNAs. hBMSCs were transfected with miRNA mimic to extract miRNA-overexpressed exosomes. The results showed that most exosomes exhibited a cup-shaped or round-shaped morphology with a diameter of approximately 50-200 nm and expressed CD9 and CD63. We detected 141 miRNAs that were differentially expressed with and without chondrogenic induction by over a twofold change, including 35 upregulated miRNAs, such as miR-1246, miR-1290, miR-193a-5p, miR-320c, and miR-92a, and 106 downregulated miRNAs, such as miR-377-3p and miR-6891-5p. qRT-PCR analysis validated these results. Exosomes derived from hBMSCs overexpressing miR-320c were more efficient than normal exosomes derived from control hBMSCs at promoting osteoarthritis chondrocyte proliferation, down-regulated matrix metallopeptidase 13 and up-regulated (sex determining region Y)-box 9 expression during hBMSC chondrogenic differentiation. In conclusion, we identified a group of upregulated miRNAs in exosomes derived from hBMSCs with chondrogenic induction that may play an important role in mesenchymal stem cell-derived exosomes in cartilage regeneration and, ultimately, the treatment of arthritis. We demonstrated the potential of these modified exosomes in the development of novel therapeutic strategies.     

Loss of brain-enriched miR-124 microRNA enhances stem-like traits and invasiveness of glioma cells

miR-124 is a brain-enriched microRNA that plays a crucial role in neural development and has been shown to be down-regulated in glioma and medulloblastoma, suggesting its possible involvement in brain tumor progression. Here, we show that miR-124 is down-regulated in a panel of different grades of glioma tissues and in all of the human glioma cell lines we examined. By integrated bioinformatics analysis and experimental confirmation, we identified SNAI2, which is often up-regulated in glioma, as a direct functional target of miR-124. Because SNAI2 has been shown to regulate stem cell functions, we examined the roles of miR-124 and SNAI2 in glioma cell stem-like traits. The results showed that overexpression of miR-124 and knockdown of SNAI2 reduced neurosphere formation, CD133(+) cell subpopulation, and stem cell marker (BMI1, Nanog, and Nestin) expression, and these effects could be rescued by re-expression of SNAI2. Furthermore, enhanced miR-124 expression significantly inhibited glioma cell invasion in vitro. Finally, stable overexpression of miR-124 and knockdown of SNAI2 inhibited the tumorigenicity and invasion of glioma cells in vivo. These findings reveal, for the first time, that the tumor suppressor activity of miR-124 could be partly due to its inhibitory effects on glioma stem-like traits and invasiveness through SNAI2.     

MiR-483-5p suppresses the proliferation of glioma cells via directly targeting ERK1

MicroRNAs (miRNAs) exhibit tumor-specific expression signatures and play crucial roles in tumorigenesis by targeting oncogenes. Here, through analyzing the miRNA-array profiles of human glioblastoma tissues and the adjacent normal brain tissues, we found miR-483-5p was significantly down-regulated in gliomas, which was confirmed in both human glioma specimens and cell lines. The overexpression of miR-483-5p suppressed glioma cell proliferation and induced a G0/G1 arrest. In contrast, miR-483-5p inhibition promoted cell proliferation. Furthermore, by a dual-luciferase reporter assay and expression analysis, we identified extracellular signal-regulated kinase 1 (ERK1) as a direct target of miR-483-5p. ERK1 knockdown can block cell proliferation induced by miR-483-5p inhibition. Thus, our findings provide the first evidence that miR-483-5p can serve as a tumor suppressor in gliomas.     

MiR-628-5p decreases the tumorigenicity of epithelial ovarian cancer cells by targeting at FGFR2

Micro RNAs (miRNAs) are small non-coding RNAs which are 19–24 nucleotides in length. MiRNAs play a vital role in the whole process of tumour development, but how they influence the tumourigenecity of epithelial ovarian cancer (EOC)cells is rarely researched. In our study, it was verified that miR-628-5p decreased the stem like cell percentage of EOC cells by inducing their apoptosis. The animal experiments showed that miR-628-5p decreased the tumourigenecity of EOC cells. Besides, we found miR-628-5p targeted at and down-regulated the expression of fibroblast growth factor receptor 2 (FGFR2). FGFR2 expressed higher in ovarian cancer tissues and was correlated with worse prognosis. Our findings indicated that miR-628-5pplays an important role in ovarian cancer stem cell driven tumorigenesis.     

MicroRNA-4731-5p delivered by AD-mesenchymal stem cells induces cell cycle arrest and apoptosis in glioblastoma

Glioblastoma multiforme (GBM) exhibits the most malignant brain tumor with very poor prognosis. MicroRNAs (miRNAs) are regulatory factors that can downregulate the expression of multiple genes. Several miRNAs acting as tumor-suppressor genes have been identified so far. The delivery of miRNA by mesenchymal stem cell (MSC) due to their ability to specifically target tumors is a new, hopeful therapeutic approach for glioblastoma. The objective of our study is the investigation of the effect of lentivirus-mediated microRNA-4731 (miR-4731) genetic manipulated adipose-derived (AD)-MSC on GBM. The downregulation of miR-4731 in human GBM tumor was detected using the GEO dataset. To evaluate the function of miR-4731, we overexpressed miR-4731 using lentiviral vectors in U-87 and U-251 GBM cell lines. The effects of miR-4731 on cell proliferation and cell cycle of glioma cells were analyzed by wound test and flow-cytometry assay. miR-4731 inhibited the proliferation of GBM cancer cells. Coculturing was used to study the antiproliferative effect of miR-4731-AD-MSCs on GBM cell lines. Direct and indirect coculture of GBM cell lines with miR-4731-AD-MSCs induced cell cycle arrest and apoptosis. Our findings suggest that AD-MSCs expressing miR-4731 have favorable antitumor characteristics and should be further explored in future glioma therapy.     

The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44

Cancer stem cells (CSCs), or tumor-initiating cells, are involved in tumor progression and metastasis. MicroRNAs (miRNAs) regulate both normal stem cells and CSCs, and dysregulation of miRNAs has been implicated in tumorigenesis. CSCs in many tumors--including cancers of the breast, pancreas, head and neck, colon, small intestine, liver, stomach, bladder and ovary--have been identified using the adhesion molecule CD44, either individually or in combination with other marker(s). Prostate CSCs with enhanced clonogenic and tumor-initiating and metastatic capacities are enriched in the CD44(+) cell population, but whether miRNAs regulate CD44(+) prostate cancer cells and prostate cancer metastasis remains unclear. Here we show, through expression analysis, that miR-34a, a p53 target, was underexpressed in CD44(+) prostate cancer cells purified from xenograft and primary tumors. Enforced expression of miR-34a in bulk or purified CD44(+) prostate cancer cells inhibited clonogenic expansion, tumor regeneration, and metastasis. In contrast, expression of miR-34a antagomirs in CD44(-) prostate cancer cells promoted tumor development and metastasis. Systemically delivered miR-34a inhibited prostate cancer metastasis and extended survival of tumor-bearing mice. We identified and validated CD44 as a direct and functional target of miR-34a and found that CD44 knockdown phenocopied miR-34a overexpression in inhibiting prostate cancer regeneration and metastasis. Our study shows that miR-34a is a key negative regulator of CD44(+) prostate cancer cells and establishes a strong rationale for developing miR-34a as a novel therapeutic agent against prostate CSCs.     

Expression of Dicer and Its Related MiRNAs in the Progression of Prostate Cancer

Dicer is aberrantly expressed in several types of malignancies. Cleaved by Dicer, the small noncoding microRNAs (miRNAs) are considered potential tools for the diagnosis and prognosis of cancer. This study investigated the expression of miRNAs thought to target Dicer. Expression of 1,205 human miRNAs and miRNA*s were examined in four patients with prostate cancer (PCa) by miRNA array in which the threshold was set as two-fold. Seventy-three miRNAs and miRNA*s were significantly down-regulated while 10 were up-regulated in PCa tissues compared with matched histologically normal glands. Of these, miR-29b-1, miR-200a, miR-370, and miR-31, which were the most down/up-regulated and closely potentially target to the Dicer 3′ UTR, were investigated further. Tissues of primary tumors and matched normal prostate glands from 185 patients with PCa were collected for further investigation. Dicer mRNA levels were negatively correlated with miR-29b-1 (ρs = −0.177, p = 0.017), miR-200a (ρs = -0.489, p < 0.0001) and miR-31 (ρs = −0.314, p < 0.0001) expression. Compared with adjacent normal glands, PCa tissues showed significantly lower miR-200a and miR-31 expression levels. Furthermore, in metastatic PCa, the expression levels of miR-200a, miR-370, and miR-31 were dramatically higher than in localized PCa. Additionally, elevated expression levels of miR-200a and miR-31 appeared to be associated with castration-resistant PCa. These findings suggest possibilities that miR-200a and miR-31 target Dicer and are involved in the carcinogenesis, migration, and behavior of castration-resistant PCa, indicating that they could be potential biomarkers for monitoring PCa progression.     

miR-29b regulates migration of human breast cancer cells

microRNAs (miRNAs) are short non-coding RNAs that regulate gene expression by targeting mRNAs, inhibiting the expression of the associated proteins. Although a role for aberrant miRNA expression in cancer has been postulated, the pathophysiologic role and relevance of aberrantly expressed miRNAs in tumor biology has not been established. We evaluated the expression pattern of miRNAs in human breast cancer cells by qPCR, finding out an up-regulated miRNA miR-29b and studying its biological effect by migration assay. We defined a target gene PTEN by bioinformatics approach and western blot. In breast cancer cell line MDA-MB-231 cell, which migrate faster than MCF-7, we observed that miR-29b was highly over-expressed. Inhibition of miR-29b in cultured cells increased the expression of the phosphatase and tensin homolog (PTEN) tumor suppressor, promoting apoptosis, decreasing migration, and decreasing invasion. In contrast, enhanced miR-29b expression by transfection with pre-miR-29b decreased the expression of PTEN and impaired apoptosis, increasing tumor cell migration and invasion. Moreover, PTEN was shown to be a direct target of miR-29b and was also shown to contribute to the miR-29b-mediated effects on cell invasion. Modulation of miR-29b altered the role of PTEN involved in cell migration and invasion. Aberrant expression of miR-29b, which modulates PTEN expression, can contribute to migration, invasion, and anti-apoptosis.     

MicroRNA-135a Inhibits Cell Proliferation by Targeting Bmi1 in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal solid tumor due to the lack of reliable early detection markers and effective therapies. MicroRNAs (miRNAs), noncoding RNAs that regulate gene expression, are involved in tumorigenesis and have a remarkable potential for the diagnosis and treatment of malignancy. In this study, we investigated aberrantly expressed miRNAs involved in PDAC by comparing miRNA expression profiles in PDAC cell lines with a normal pancreas cell line and found that miR-135a was significantly down-regulated in the PDAC cell lines. The microarray results were validated by qRT-PCR in PDAC tissues, paired adjacent normal pancreatic tissues, PDAC cell lines, and a normal pancreas cell line. We then defined the tumor-suppressing significance and function of miR-135a by constructing a lentiviral vector to express miR-135a. The overexpression of miR-135a in PDAC cells decreased cell proliferation and clonogenicity and also induced G1 arrest and apoptosis. We predicted Bmi1 may be a target of miR-135a using bioinformatics tools and found that Bmi1 expression was markedly up-regulated in PDAC. Its expression was inversely correlated with miR-135a expression in PDAC. Furthermore, a luciferase activity assay revealed that miR-135a could directly target the 3''-untranslated region (3''-UTR) of Bmi1. Taken together, these results demonstrate that miR-135a targets Bmi1 in PDAC and functions as a tumor suppressor. miR-135a may offer a new perspective for the development of effective miRNA-based therapy for PDAC.     

Novel Anti-Apoptotic MicroRNAs 582-5p and 363 Promote Human Glioblastoma Stem Cell Survival via Direct Inhibition of Caspase 3, Caspase 9, and Bim

Glioblastoma is the most common and lethal primary brain tumor. Tumor initiation and recurrence are likely caused by a sub-population of glioblastoma stem cells, which may derive from mutated neural stem and precursor cells. Since CD133 is a stem cell marker for both normal brain and glioblastoma, and to better understand glioblastoma formation and recurrence, we looked for dys-regulated microRNAs in human CD133+ glioblastoma stem cells as opposed to CD133+ neural stem cells isolated from normal human brain. Using FACS sorting of low-passage cell samples followed by microRNA microarray analysis, we found 43 microRNAs that were dys-regulated in common in three separate CD133+ human glioblastomas compared to CD133+ normal neural stem cells. Among these were several microRNAs not previously associated with cancer. We then verified the microRNAs dys-regulated in glioblastoma using quantitative real time PCR and Taqman analysis of the original samples, as well as human GBM stem cell and established cell lines and many human specimens. We show that two candidate oncogenic microRNAs, miR-363 and miR-582-5p, can positively influence glioblastoma survival, as shown by forced expression of the microRNAs and their inhibitors followed by cell number assay, Caspase 3/7 assay, Annexin V apoptosis/fluorescence activated cell sorting, siRNA rescue of microRNA inhibitor treatment, as well as 3′UTR mutagenesis to show luciferase reporter rescue of the most successful targets. miR-582-5p and miR-363 are shown to directly target Caspase 3, Caspase 9, and Bim.