MiR-107 and MiR-185 Can Induce Cell Cycle Arrest in Human Non Small Cell Lung Cancer Cell Lines

Background

MicroRNAs (miRNAs) are short single stranded noncoding RNAs that suppress gene expression through either translational repression or degradation of target mRNAs. The annealing between messenger RNAs and 5′ seed region of miRNAs is believed to be essential for the specific suppression of target gene expression. One miRNA can have several hundred different targets in a cell. Rapidly accumulating evidence suggests that many miRNAs are involved in cell cycle regulation and consequentially play critical roles in carcinogenesis.

Methodology/Principal Findings

Introduction of synthetic miR-107 or miR-185 suppressed growth of the human non-small cell lung cancer cell lines. Flow cytometry analysis revealed these miRNAs induce a G1 cell cycle arrest in H1299 cells and the suppression of cell cycle progression is stronger than that by Let-7 miRNA. By the gene expression analyses with oligonucleotide microarrays, we find hundreds of genes are affected by transfection of these miRNAs. Using miRNA-target prediction analyses and the array data, we listed up a set of likely targets of miR-107 and miR-185 for G1 cell cycle arrest and validate a subset of them using real-time RT-PCR and immunoblotting for CDK6.

Conclusions/Significance

We identified new cell cycle regulating miRNAs, miR-107 and miR-185, localized in frequently altered chromosomal regions in human lung cancers. Especially for miR-107, a large number of down-regulated genes are annotated with the gene ontology term ‘cell cycle’. Our results suggest that these miRNAs may contribute to regulate cell cycle in human malignant tumors.     

Genistein Inhibits Prostate Cancer Cell Growth by Targeting miR-34a and Oncogenic HOTAIR

Objective

Genistein is a soy isoflavone that has antitumor activity both in vitro and in vivo. It has been shown that genistein inhibits many type of cancers including prostate cancer (PCa) by regulating several cell signaling pathways and microRNAs (miRNAs). Recent studies suggest that the long non-coding RNAs (lncRNAs) are also involved in many cellular processes. At present there are no reports about the relationship between gensitein, miRNAs and lncRNAs. In this study, we focused on miRNAs, lncRNA that are regulated by genistein and investigated their functional role in PCa.

Method

Microarray (SurePrint G3 Human GE 8×60K) was used for expression profiling of genistein treated and control PCa cells (PC3 and DU145). Functional assay (cell proliferation, migration, invasion, apoptosis and cell cycle assays) were performed with the PCa cell lines, PC3 and DU145. Both in vitro and in vivo (nude mouse) models were used for growth assays. Luciferase reporter assays were used for binding of miR-34a to HOTAIR.

Results

LncRNA profiling showed that HOTAIR was highly regulated by genistein and its expression was higher in castration-resistant PCa cell lines than in normal prostate cells. Knockdown (siRNA) of HOTAIR decreased PCa cell proliferation, migration and invasion and induced apoptosis and cell cycle arrest. miR-34a was also up-regulated by genistein and may directly target HOTAIR in both PC3 and DU145 PCa cells.

Conclusions

Our results indicated that genistein inhibited PCa cell growth through down-regulation of oncogenic HOTAIR that is also targeted by tumor suppressor miR-34a. These findings enhance understanding of how genistein regulates lncRNA HOTAIR and miR-34a in PCa.     

Down-Regulation of miR-126 Is Associated with Colorectal Cancer Cells Proliferation,Migration and Invasion by Targeting IRS-1 via the AKT and ERK1/2 Signaling Pathways

Background

Colorectal carcinoma (CRC) is one of the leading causes of cancer-related mortality worldwide. MicroRNAs (miRNAs, miRs) play important roles in carcinogenesis. MiR-126 has been shown to be down-regulated in CRC. In this study, we identified the potential effects of miR-126 on some important biological properties of CRC cells and clarified the regulation of insulin receptor substrate 1 (IRS-1) and its possible signaling pathway by miR-126.

Methods

The effect of miR-126 on IRS-1, AKT, and ERK1/2 expression was assessed in the CRC cell lines HT-29 and HCT-116 with a miR-126 mimic or inhibitor to increase or decrease miR-126 expression. Furthermore, the roles of miR-126 in regulation of the biological properties of CRC cells were analyzed with miR-126 mimic or inhibitor-transfected cells. The 3′-untranslated region (3′-UTR) of IRS-1 regulated by miR-126 was analyzed by using a dual-luciferase reporter assay.

Results

We found that IRS-1 is the functional downstream target of miR-126 by directly targeting the 3′-UTR of IRS-1. Endogenous miR-126 and exogenous miR-126 mimic inhibited IRS-1 expression. Furthermore, gain-of-function or loss-of-function studies showed that over-expression of miR-126 down-regulated IRS-1, suppressed AKT and ERK1/2 activation, CRC cells proliferation, migration, invasion, and caused cell cycle arrest, but had no effect on cell apoptosis. Knockdown of miR-126 promoted these processes in HCT-116 cells and promoted AKT and ERK1/2 activation by up-regulating the expression of the IRS-1 protein.

Conclusions

MiR-126 may play roles in regulation of the biological behavior of CRC cells, at least in part, by targeting IRS-1 via AKT and ERK1/2 signaling pathways.     

Up-regulation of microRNA-21 correlates with lower kidney cancer survival

Background

MicroRNA-21 is up-regulated in a variety of cancers like, breast, colorectal, lung, head and neck etc. However, the regulation of miR-21 in renal cell carcinoma (RCC) has not yet been studied systematically.

Methods and Results

We measured miR-21 levels in 54 pairs of kidney cancers and their normal matched tissues by real-time PCR. The expression level of miR-21 was correlated with 5 year survival and the pathological stage. Functional studies were done after inhibiting miR-21 in RCC cell lines. We studied in vitro and in vivo effects of the chemo preventive agent genistein on miR-21 expression. In 48 cases (90%), miR-21 was increased. All patients with low miR-21 expression survived 5 years, while with high miR-21 expression, only 50% survived. Higher expression of miR-21 is associated with an increase in the stage of renal cancer. Functional studies after inhibiting miRNA-21 in RCC cell lines show cell cycle arrest, induction of apoptosis and reduced invasive and migratory capabilities. Western blot analysis showed an increase in the expression of p21 and p38 MAP kinase genes and a reduction in cyclin E2. Genistein inhibited the expression of miR-21 in A-498 cells and in the tumors formed after injecting genistein treated A-498 cells in nude mice besides inhibiting tumor formation.

Conclusions

The current study shows a clear correlation between miR-21 expression and clinical characteristics of renal cancer. Thus we believe that miR-21 can be used as a tumor marker and its inhibition may prove to be useful in controlling cancers with up-regulated miR-21.     

MicroRNAs associated with metastatic prostate cancer

Objective

Metastasis is the most common cause of death of prostate cancer patients. Identification of specific metastasis biomarkers and novel therapeutic targets is considered essential for improved prognosis and management of the disease. MicroRNAs (miRNAs) form a class of non-coding small RNA molecules considered to be key regulators of gene expression. Their dysregulation has been shown to play a role in cancer onset, progression and metastasis, and miRNAs represent a promising new class of cancer biomarkers. The objective of this study was to identify down- and up-regulated miRNAs in prostate cancer that could provide potential biomarkers and/or therapeutic targets for prostate cancer metastasis.

Methods

Next generation sequencing technology was applied to identify differentially expressed miRNAs in a transplantable metastatic versus a non-metastatic prostate cancer xenograft line, both derived from one patient''s primary cancer. The xenografts were developed via subrenal capsule grafting of cancer tissue into NOD/SCID mice, a methodology that tends to preserve properties of the original cancers (e.g., tumor heterogeneity, genetic profiles).

Results

Differentially expressed known miRNAs, isomiRs and 36 novel miRNAs were identified. A number of these miRNAs (21/104) have previously been reported to show similar down- or up-regulation in prostate cancers relative to normal prostate tissue, and some of them (e.g., miR-16, miR-34a, miR-126*, miR-145, miR-205) have been linked to prostate cancer metastasis, supporting the validity of the analytical approach.

Conclusions

The use of metastatic and non-metastatic prostate cancer subrenal capsule xenografts derived from one patient''s cancer makes it likely that the differentially expressed miRNAs identified in this study include potential biomarkers and/or therapeutic targets for human prostate cancer metastasis.     

Identification of XMRV infection-associated microRNAs in four cell types in culture

Introduction

XMRV is a gammaretrovirus that was thought to be associated with prostate cancer (PC) and chronic fatigue syndrome (CFS) in humans until recently. The virus is culturable in various cells of human origin like the lymphocytes, NK cells, neuronal cells, and prostate cell lines. MicroRNAs (miRNA), which regulate gene expression, were so far not identified in cells infected with XMRV in culture.

Methods

Two prostate cell lines (LNCaP and DU145) and two primary cells, Peripheral Blood Lymphocytes [PBL] and Monocyte-derived Macrophages [MDM] were infected with XMRV. Total mRNA was extracted from mock- and virus-infected cells at 6, 24 and 48 hours post infection and evaluated for microRNA profile in a microarray.

Results

MicroRNA expression profiles of XMRV-infected continuous prostate cancer cell lines differ from that of virus-infected primary cells (PBL and MDMs). miR-193a-3p and miRPlus-E1245 observed to be specific to XMRV infection in all 4 cell types. While miR-193a-3p levels were down regulated miRPlus-E1245 on the other hand exhibited varied expression profile between the 4 cell types.

Discussion

The present study clearly demonstrates that cellular microRNAs are expressed during XMRV infection of human cells and this is the first report demonstrating the regulation of miR193a-3p and miRPlus-E1245 during XMRV infection in four different human cell types.     

MicroRNA-196a Is a Putative Diagnostic Biomarker and Therapeutic Target for Laryngeal Cancer

Background

MicroRNA (miRNA) is an emerging subclass of small non-coding RNAs that regulates gene expression and has a pivotal role for many physiological processes including cancer development. Recent reports revealed the role of miRNAs as ideal biomarkers and therapeutic targets due to their tissue- or disease-specific nature. Head and neck cancer (HNC) is a major cause of cancer-related mortality and morbidity, and laryngeal cancer has the highest incidence in it. However, the molecular mechanisms involved in laryngeal cancer development remain to be known and highly sensitive biomarkers and novel promising therapy is necessary.

Methodology/Principal Findings

To explore laryngeal cancer-specific miRNAs, RNA from 5 laryngeal surgical specimens including cancer and non-cancer tissues were hybridized to microarray carrying 723 human miRNAs. The resultant differentially expressed miRNAs were further tested by using quantitative real time PCR (qRT-PCR) on 43 laryngeal tissue samples including cancers, noncancerous counterparts, benign diseases and precancerous dysplasias. Significant expressional differences between matched pairs were reproduced in miR-133b, miR-455-5p, and miR-196a, among which miR-196a being the most promising cancer biomarker as validated by qRT-PCR analyses on additional 84 tissue samples. Deep sequencing analysis revealed both quantitative and qualitative deviation of miR-196a isomiR expression in laryngeal cancer. In situ hybridization confirmed laryngeal cancer-specific expression of miR-196a in both cancer and cancer stroma cells. Finally, inhibition of miR-196a counteracted cancer cell proliferation in both laryngeal cancer-derived cells and mouse xenograft model.

Conclusions/Significance

Our study provided the possibilities that miR-196a might be very useful in diagnosing and treating laryngeal cancer.     

MiR-371-5p Facilitates Pancreatic Cancer Cell Proliferation and Decreases Patient Survival

Background

microRNAs (miRNAs) play a critical role in tumorigenesis, either as a tumor suppressor or as an oncogenic miRNA, depending on different tumor types. To date, scientists have obtained a substantial amount of knowledge with regard to miRNAs in pancreatic cancer. However, the expression and function of miR-371-5p in pancreatic cancer has not been clearly elucidated. The aim of this study was to investigate the roles of miR-371-5p in pancreatic cancer and its association with the survival of patients with pancreatic cancer.

Methods

The expression of miR-371-5p was examined in pancreatic duct adenocarcinoma (PDAC) and their adjacent normal pancreatic tissues (ANPT) or in pancreatic cancer cell lines by qRT-PCR. The association of miR-371-5p expression with overall survival was determined. The proliferation and apoptosis of SW-1990 and Panc-1 cells, transfected with miR-371-5p mimics or inhibitor, were assessed using MTT assay and flow cytometry, respectively. The tumorigenicity was evaluated via mice xenograft experiments. miR-371-5p promoter interactions were analyzed by chromatin immunoprecipitation assays (ChIP). Protein expression was analyzed by Western blot.

Results

The expression level of miR-371-5p was dramatically upregulated in clinical PDAC tissues compared with ANPT. Patients with high miR-371-5p expression had a significantly shorter survival than those with low miR-371-5p expression. The in vitro and in vivo assays showed that overexpression of miR-371-5p resulted in cell proliferation and increased tumor growth, which was associated with inhibitor of growth 1 (ING1) downregulation. Interestingly, we also found that ING1, in turn, inhibited expression of miR-371-5p in the promoter region.

Conclusions

our study demonstrates a novel ING1-miR-371-5p regulatory feedback loop, which may have a critical role in PDAC. Thus miR-371-5p can prove to be a novel prognostic factor and therapeutic target for pancreatic cancer treatment.     

Prognostic significance of miR-205 in endometrial cancer

Purpose

microRNAs have emerged as key regulators of gene expression, and their altered expression has been associated with tumorigenesis and tumor progression. Thus, microRNAs have potential as both cancer biomarkers and/or potential novel therapeutic targets. Although accumulating evidence suggests the role of aberrant microRNA expression in endometrial carcinogenesis, there are still limited data available about the prognostic significance of microRNAs in endometrial cancer. The goal of this study is to investigate the prognostic value of selected key microRNAs in endometrial cancer by the analysis of archival formalin-fixed paraffin-embedded tissues.

Experimental Design

Total RNAs were extracted from 48 paired normal and endometrial tumor specimens using Trizol based approach. The expression of miR-26a, let-7g, miR-21, miR-181b, miR-200c, miR-192, miR-215, miR-200c, and miR-205 were quantified by real time qRT-PCR expression analysis. Targets of the differentially expressed miRNAs were quantified using immunohistochemistry. Statistical analysis was performed by GraphPad Prism 5.0.

Results

The expression levels of miR-200c (P<0.0001) and miR-205 (P<0.0001) were significantly increased in endometrial tumors compared to normal tissues. Kaplan-Meier survival analysis revealed that high levels of miR-205 expression were associated with poor patient overall survival (hazard ratio, 0.377; Logrank test, P = 0.028). Furthermore, decreased expression of a miR-205 target PTEN was detected in endometrial cancer tissues compared to normal tissues.

Conclusion

miR-205 holds a unique potential as a prognostic biomarker in endometrial cancer.     

MicroRNA Let-7a Inhibits Proliferation of Human Prostate Cancer Cells In Vitro and In Vivo by Targeting E2F2 and CCND2

Background

Previous work has shown reduced expression levels of let-7 in lung tumors. But little is known about the expression or mechanisms of let-7a in prostate cancer. In this study, we used in vitro and in vivo approaches to investigate whether E2F2 and CCND2 are direct targets of let-7a, and if let-7a acts as a tumor suppressor in prostate cancer by down-regulating E2F2 and CCND2.

Methodology/Principal

Findings Real-time RT-PCR demonstrated that decreased levels of let-7a are present in resected prostate cancer samples and prostate cancer cell lines. Cellular proliferation was inhibited in PC3 cells and LNCaP cells after transfection with let-7a. Cell cycle analysis showed that let-7a induced cell cycle arrest at the G1/S phase. A dual-luciferase reporter assay demonstrated that the 3′UTR of E2F2 and CCND2 were directly bound to let-7a and western blotting analysis further indicated that let-7a down-regulated the expression of E2F2 and CCND2. Our xenograft models of prostate cancer confirmed the capability of let-7a to inhibit prostate tumor development in vivo.

Conclusions/Significance

These findings help to unravel the anti-proliferative mechanisms of let-7a in prostate cancer. Let-7a may also be novel therapeutic candidate for prostate cancer given its ability to induce cell-cycle arrest and inhibit cell growth, especially in hormone-refractory prostate cancer.     

MicroRNA-147 Induces a Mesenchymal-To-Epithelial Transition (MET) and Reverses EGFR Inhibitor Resistance

Background

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer progression and may promote resistance to therapy. An analysis of patients (n = 71) profiled with both gene expression and a global microRNA assessment (∼415 miRs) identified miR-147 as highly anti-correlated with an EMT gene expression signature score and postulated to reverse EMT (MET).

Methods and Findings

miR-147 was transfected into colon cancer cells (HCT116, SW480) as well as lung cancer cells (A-549). The cells were assessed for morphological changes, and evaluated for effects on invasion, motility, and the expression of key EMT markers. Resistance to chemotherapy was evaluated by treating cells with gefitinib, an EGFR inhibitor. The downstream genes regulated by miR-147 were assayed using the Affymetrix GeneChip U133 Plus2.0 platform. miR-147 was identified to: 1. cause MET primarily by increasing the expression of CDH1 and decreasing that of ZEB1; 2. inhibit the invasion and motility of cells; 3. cause G1 arrest by up-regulating p27 and down-regulating cyclin D1. miR-147 also dramatically reversed the native drug resistance of the colon cancer cell line HCT116 to gefitinib. miR-147 significantly repressed Akt phosphorylation, and knockdown of Akt with siRNA induced MET. The morphologic effects of miR-147 on cells appear to be attenuated by TGF-B1, promoting a plastic and reversible transition between MET and EMT.

Conclusion

miR-147 induced cancer cells to undergo MET and induced cell cycle arrest, suggesting a potential tumor suppressor role. miR-147 strikingly increased the sensitivity to EGFR inhibitor, gefitinib in cell with native resistance. We conclude that miR-147 might have therapeutic potential given its ability to inhibit proliferation, induce MET, as well as reverse drug sensitivity.     

MicroRNA-145 regulates human corneal epithelial differentiation

Background

Epigenetic factors, such as microRNAs, are important regulators in the self-renewal and differentiation of stem cells and progenies. Here we investigated the microRNAs expressed in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, and their role in corneal epithelium.

Methodology/Principal Findings

Human LPC epithelia was extracted for small RNAs or dissociated for CEPC culture. By Agilent Human microRNA Microarray V2 platform and GeneSpring GX11.0 analysis, we found differential expression of 18 microRNAs against central corneal (CC) epithelia, which were devoid of CEPCs. Among them, miR-184 was up-regulated in CC epithelia, similar to reported finding. Cluster miR-143/145 was expressed strongly in LPC but weakly in CC epithelia (P = 0.0004, Mann-Whitney U-test). This was validated by quantitative polymerase chain reaction (qPCR). Locked nucleic acid-based in situ hybridization on corneal rim cryosections showed miR-143/145 presence localized to the parabasal cells of limbal epithelium but negligible in basal and superficial epithelia. With holoclone forming ability, CEPCs transfected with lentiviral plasmid containing mature miR-145 sequence gave rise to defective epithelium in organotypic culture and had increased cytokeratin-3/12 and connexin-43 expressions and decreased ABCG2 and p63 compared with cells transfected with scrambled sequences. Global gene expression was analyzed using Agilent Whole Human Genome Oligo Microarray and GeneSpring GX11.0. With a 5-fold difference compared to cells with scrambled sequences, miR-145 up-regulated 324 genes (containing genes for immune response) and down-regulated 277 genes (containing genes for epithelial development and stem cell maintenance). As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin β8 (ITGB8) expression in both human corneal epithelial cells and primary CEPCs.

Conclusion/Significance

We found expression of miR-143/145 cluster in human corneal epithelium. Our results also showed that miR-145 regulated the corneal epithelium formation and maintenance of epithelial integrity, via ITGB8 targeting.     

The Loss of miR-26a-Mediated Post-Transcriptional Regulation of Cyclin E2 in Pancreatic Cancer Cell Proliferation and Decreased Patient Survival

Background

miR-26a plays a critical role in tumorigenesis, either as a tumor suppressor or as an oncogenic miRNA, depending on different tumor types. However, the function of miR-26a in pancreatic cancer has not been clearly elucidated. The present study was designed to determine the roles of miR-26a in pancreatic cancer and its association with the survival of patients with pancreatic cancer.

Methods

The expression of miR-26a was examined in 15 pairs of pancreatic duct adenocarcinoma (PDAC) and their adjacent benign pancreatic tissues (ABPT), by qRT-PCR. The results were confirmed by in situ hybridization using two panels of 106 PDACs and their ABPT microarray. The association of miR-26a expression with overall survival was determined. The proliferation and cell cycle distribution of Capan-2, SW-1990, and Panc-1 cells, transfected with miR-26a mimics or a miR-26a inhibitor, were assessed using the Cell Counting Kit-8 assay and flow cytometry, respectively. The cell tumorigenicity was evaluated via murine xenograft experiments. Cyclin D2, E2, EZH2, and PCNA levels were analyzed by Western blot and immunohistochemistry.

Results

miR-26a was expressed in the cytoplasm of pancreatic ductal epithelial cells, whereas its expression was significantly downregulated in PDAC tissues compared with that of ABPT. Patients with low miR-26a expression had a significantly shorter survival than those with high miR-26a expression. The in vitro and in vivo assays showed that overexpression of miR-26a resulted in cell cycle arrest, inhibited cell proliferation, and decreased tumor growth, which was associated with cyclin E2 downregulation.

Conclusions

miR-26a is an important suppressor of pancreatic ductal carcinoma, and can prove to be a novel prognostic factor and therapeutic target for pancreatic cancer treatment.     

Epithelial to Mesenchymal Transition Is Mechanistically Linked with Stem Cell Signatures in Prostate Cancer Cells

Background

Current management of patients diagnosed with prostate cancer (PCa) is very effective; however, tumor recurrence with Castrate Resistant Prostate Cancer (CRPC) and subsequent metastasis lead to poor survival outcome, suggesting that there is a dire need for novel mechanistic understanding of tumor recurrence, which would be critical for designing novel therapies. The recurrence and the metastasis of PCa are tightly linked with the biology of prostate cancer stem cells or cancer-initiating cells that is reminiscent of the acquisition of Epithelial to Mesenchymal Transition (EMT) phenotype. Increasing evidence suggests that EMT-type cells share many biological characteristics with cancer stem-like cells.

Methodology/Principal Findings

In this study, we found that PCa cells with EMT phenotype displayed stem-like cell features characterized by increased expression of Sox2, Nanog, Oct4, Lin28B and/or Notch1, consistent with enhanced clonogenic and sphere (prostasphere)-forming ability and tumorigenecity in mice, which was associated with decreased expression of miR-200 and/or let-7 family. Reversal of EMT by re-expression of miR-200 inhibited prostasphere-forming ability of EMT-type cells and reduced the expression of Notch1 and Lin28B. Down-regulation of Lin28B increased let-7 expression, which was consistent with repressed self-renewal capability.

Conclusions/Significance

These results suggest that miR-200 played a pivotal role in linking the characteristics of cancer stem-like cells with EMT-like cell signatures in PCa. Selective elimination of cancer stem-like cells by reversing the EMT phenotype to Mesenchymal-Epithelial Transition (MET) phenotype using novel agents would be useful for the prevention of tumor recurrence especially by eliminating those cells that are the “Root Cause” of tumor development and recurrence.     

Identification of microRNAs as potential prognostic markers in ependymoma

Introduction

We have examined expression of microRNAs (miRNAs) in ependymomas to identify molecular markers of value for clinical management. miRNAs are non-coding RNAs that can block mRNA translation and affect mRNA stability. Changes in the expression of miRNAs have been correlated with many human cancers.

Materials and Methods

We have utilized TaqMan Low Density Arrays to evaluate the expression of 365 miRNAs in ependymomas and normal brain tissue. We first demonstrated the similarity of expression profiles of paired frozen tissue (FT) and paraffin-embedded specimens (FFPE). We compared the miRNA expression profiles of 34 FFPE ependymoma samples with 8 microdissected normal brain tissue specimens enriched for ependymal cells. miRNA expression profiles were then correlated with tumor location, histology and other clinicopathological features.

Results

We have identified miRNAs that are over-expressed in ependymomas, such as miR-135a and miR-17-5p, and down-regulated, such as miR-383 and miR-485-5p. We have also uncovered associations between expression of specific miRNAs which portend a worse prognosis. For example, we have identified a cluster of miRNAs on human chromosome 14q32 that is associated with time to relapse. We also found that miR-203 is an independent marker for relapse compared to the parameters that are currently used. Additionally, we have identified three miRNAs (let-7d, miR-596 and miR-367) that strongly correlate to overall survival.

Conclusion

We have identified miRNAs that are differentially expressed in ependymomas compared with normal ependymal tissue. We have also uncovered significant associations of miRNAs with clinical behavior. This is the first report of clinically relevant miRNAs in ependymomas.     

MicroRNA-21 in Pancreatic Ductal Adenocarcinoma Tumor-Associated Fibroblasts Promotes Metastasis

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is projected to rise to the second leading cause of U.S. cancer-related deaths by 2020. Novel therapeutic targets are desperately needed. MicroRNAs (miRs) are small noncoding RNAs that function by suppressing gene expression and are dysregulated in cancer. miR-21 is overexpressed in PDAC tumor cells (TC) and is associated with decreased survival, chemoresistance and invasion. Dysregulation of miR regulatory networks in PDAC tumor-associated fibroblasts (TAFs) have not been previously described. In this study, we show that miR-21 expression in TAFs promotes TC invasion.

Methods

In-situ hybridization for miR-21 was performed on the 153 PDAC patient UCLA tissue microarray and 23 patient-matched lymph node metastases. Stromal and TC histoscores were correlated with clinicopathologic parameters by univariate and multivariate Cox regression. miR-21 positive cells were further characterized by immunofluorescence for mesenchymal/epithelial markers. For in vitro studies, TAFs were isolated from freshly resected human PDAC tumors by the outgrowth method. miR-21 was overexpressed/inhibited in fibroblasts and then co-cultured with GFP-MiaPaCa TCs to assess TC invasion in modified Boyden chambers.

Results

miR-21 was upregulated in TAFs of 78% of tumors, and high miR-21 significantly correlated with decreased overall survival (P = 0.04). Stromal miR-21 expression was also significantly associated with lymph node invasion (P = 0.004), suggesting that it is driving TC spread. Co-immunofluorescence revealed that miR-21 colocalized with peritumoral fibroblasts expressing α-smooth muscle actin. Moreover, expression of miR-21 in primary TAFs correlated with miR-21 in TAFs from patient-matched LN metastases; evidence that PDAC tumor cells induce TAFs to express miR-21. miR-21 expression in TAFs and TCs promotes invasion of TCs and is inhibited with anti-miR-21.

Conclusions

miR-21 expression in PDAC TAFs is associated with decreased overall survival and promotes TC invasion. Anti-miR-21 may represent a novel therapeutic strategy for dual targeting of both tumor and stroma in PDAC.