TIMP4 expression is regulated by miR‐200b‐3p in prostate cancer cells

In prostate cancer TIMP4 expression level fluctuates with tumor progression. The mechanism and factors influencing its expression remain unclear. The aim of the study was to test the hypothesis on regulation of TIMP4 by microRNA‐200b‐3p. The levels of TIMP4 and miR‐200b‐3p expression were determined by real time PCR in 27 prostate carcinomas and eight benign prostatic hyperplasia samples. We found that miR‐200b‐3p positively correlated with TIMP4 expression in cancer samples (r = 0.46; p < 0.02). Moreover, mean miR‐200b‐3p level and TIMP4 expression were both higher in cancer tissues compared to benign prostatic hyperplasia samples (p > 0.05). Next, to test probable mechanisms of the regulation androgen‐sensitive human prostate adenocarcinoma cells (LNCaP) were transfected with synthetic‐miR‐200b‐3p or its synthetic antagonist. Modulation of miR‐200b‐3p in LNCaP cells had an impact on TIMP4 expression confirming the observation made in analyzed clinical samples. Two targets of miR‐200b‐3p: ZEB1 and ETS1 were investigated subsequently as potential regulators of TIMP4, however, no effect of their modulation on TIMP4 expression in LNCaP cells was found. Concluding, miR‐200b‐3p mediates regulation of TIMP4 expression in prostate cancer but exact mechanism needs to be investigated.     

miR‐1, regulated by LMP1, suppresses tumour growth and metastasis by targeting K‐ras in nasopharyngeal carcinoma

There is evidence to show that downregulation of miR‐1 expression is closely related to cancer progression, including in nasopharyngeal carcinoma (NPC). However, the molecular mechanisms underlying miR‐1 downregulation in NPC remain largely unknown, especially its association with Epstein–Barr virus (EBV). In this study we found that restoration of miR‐1 dramatically inhibited cell invasion in vitro, together with tumour growth and metastasis in vivo. Importantly, we found that LMP1, an Epstein–Barr virus (EBV)‐associated protein, suppressed miR‐1 expression. Furthermore, we identified K‐ras as a novel direct target of miR‐1. Our results demonstrated for the first time that miR‐1 was suppressed by LMP1 and its tumour‐suppressive effects were mediated chiefly by repressing K‐ras expression. We propose that miR‐1 could serve as an independent biomarker to identify patients with different clinical characteristics.     

MicroRNA‐137‐mediated Src oncogenic signaling promotes cancer progression

The tyrosine kinase c‐Src is frequently overexpressed and activated in a wide variety of human cancers. However, the molecular mechanisms responsible for the upregulation of c‐Src remain elusive. To examine whether microRNA‐mediated c‐Src upregulation promotes cancer progression, we screened miRNAs with complementarity to the 3′‐UTR of c‐Src mRNA. Among these miRNAs, down‐regulation of miR‐137 was tightly associated with c‐Src‐mediated tumor progression of human colon cancer cells/tissues. Re‐expression of miR‐137 in human colon cancer cells suppressed tumor growth and caused the disruption of focal contacts, suppression of cell adhesion, and invasion, although restoration of c‐Src in miR‐137‐treated cells could not fully rescue the tumor‐suppressive effect of miR‐137. We found that miR‐137 targets AKT2 and paxillin also and miR‐137‐mediated regulation of c‐Src /AKT2 is crucial for controlling tumor growth, whereas that of c‐Src/paxillin contributes to malignancy. miR‐137 suppressed Src‐related oncogenic signaling and changed the expression of miRNAs that are regulated by Src activation. miR‐137 controls the expression of c‐Src/AKT2/paxillin and synergistically suppresses Src oncogenic signaling evoked from focal adhesions. In various human cancers that harbor c‐Src upregulation, the dysfunction of this novel mechanism would serve as a critical trigger for tumor progression.     

Overexpression of miR‐17 in gastric cancer is correlated with proliferation‐associated oncogene amplification

The molecular mechanism underlying microRNA (miR)‐17 overexpression has not been clearly evaluated in gastric cancer. We aimed to evaluate the functional roles of miR‐17 in gastric cancer and test its viability as a therapeutic target. We conducted comparative genomic hybridization and expression array analyses on human gastric cancer tissue samples, as well as evaluating the functional roles of miR‐17 in gastric cancer cell lines and transgenic mice. miR‐17 overexpression in gastric cancer patients was associated with copy number gain of proliferation‐associated oncogenes such as MYC, CCNE1, ERBB2, and FGFR2. Copy number gain of MIR17HG gene (13q31.3) was rare, with an overall frequency of 2% in gastric cancers (1 of 51). miR‐17 knockdown suppressed the monolayer and anchorage‐independent growth of FGFR2‐amplified KATO‐III gastric cancer cells. mir‐17–92 TG/TG mice overexpressing the mir‐17–92 cluster under the villin promoter developed spontaneous benign tumors in the intestinal tract (log‐rank P for tumor‐free survival = 0.069). Taken together, miR‐17 overexpression in gastric cancer was rarely associated with MIR17HG gene amplification, but correlated with proliferation‐associated oncogene amplification. Therefore, miR‐17‐targeting approach may benefit patients with gastric cancers harboring proliferation‐associated oncogene amplification.     

Loss of miR‐182 affects B‐cell extrafollicular antibody response

MicroRNAs have been shown to play a role in B‐cell differentiation and activation. Here, we found miR‐182 to be highly induced in activated B cells. However, mice lacking miR‐182 have normal B‐cell and T‐cell development. Interestingly, mutant mice exhibited a defective antibody response at early time‐points in the immunization regimen when challenged with a T‐cell‐dependent antigen. Germinal centres were formed but the generation of extrafollicular plasma cells was defective in the spleens of immunized miR‐182‐deficient mice. Mutant mice were also not able to respond to a T‐cell‐independent type 2 antigen, which typically elicited an extrafollicular B‐cell response. Taken together, the data indicated that miR‐182 plays a critical role in driving extrafollicular B‐cell antibody responses.     

MicroRNA miR‐7 contributes to the control of Drosophila wing growth

Background : The control of organ growth is critical for correct animal development. From flies to mammals, the mechanisms regulating growth are conserved and the role of microRNAs in this process is emerging. The conserved miR‐7 has been described to control several aspects of development. Results: Here, we have analyzed the function of miR‐7 during Drosophila wing development. We found that loss of miR‐7 function results in a reduction of wing size and produces wing cells that are smaller than wild type cells. We also found that loss of miR‐7 function interferes with the cell cycle by affecting the G1 to S phase transition. Further, we present evidence that miR‐7 is expressed in the wing imaginal discs and that the inactivation of miR‐7 increases the expression of Cut and Senseless proteins in wing discs. Finally, our results show that the simultaneous inactivation of miR‐7 and either cut, Notch, or dacapo rescues miR‐7 loss of function wing size reduction phenotype. Conclusions: The results from this work reveal, for the first time, that miR‐7 functions to regulate Drosophila wing growth by controlling cell cycle phasing and cell mass through its regulation of the expression of dacapo and the Notch signaling pathway. Developmental Dynamics 244:21–30, 2015. © 2014 Wiley Periodicals, Inc.     

MiR‐23b and miR‐199a impair epithelial‐to‐mesenchymal transition during atrioventricular endocardial cushion formation

Background: Valve development is a multistep process involving the activation of the cardiac endothelium, epithelial‐mesenchymal transition (EMT) and the progressive alignment and differentiation of distinct mesenchymal cell types. Several pathways such as Notch/delta, Tgf‐beta and/or Vegf signaling have been implicated in crucial steps of valvulogenesis. We have previously demonstrated discrete changes in microRNAs expression during cardiogenesis, which are predicted to target Bmp‐ and Tgf‐beta signaling. We now analyzed the expression profile of 20 candidate microRNAs in atrial, ventricular, and atrioventricular canal regions at four different developmental stages. Results: qRT‐PCR analyses of microRNAs demonstrated a highly dynamic and distinct expression profiles within the atrial, ventricular, and atrioventricular canal regions of the developing chick heart. miR‐23b, miR‐199a, and miR‐15a displayed increased expression during early AVC development whereas others such as miR‐130a and miR‐200a display decreased expression levels. Functional analyses of miR‐23b, miR‐199a, and miR‐15a overexpression led to in vitro EMT blockage. Molecular analyses demonstrate that distinct EMT signaling pathways are impaired after microRNA expression, including a large subset of EMT‐related genes that are predicted to be targeted by these microRNAs. Conclusions: Our data demonstrate that miR‐23b and miR‐199a over‐expression can impair atrioventricular EMT. Developmental Dynamics 244:1259–1275, 2015. © 2015 Wiley Periodicals, Inc.     

Down‐regulation of miR‐301a suppresses pro‐inflammatory cytokines in Toll‐like receptor‐triggered macrophages

In many types of tumours, especially pancreatic adenocarcinoma, miR‐301a is over‐expressed. This over‐expression results in negative regulation of the target gene of miR‐301a, the nuclear factor‐κB (NF‐κB) repressing factor (NKRF), increasing the activation of NF‐κB and production of NF‐κB‐responsive pro‐inflammatory cytokines such as interleukin‐8, interferon‐β, nitric oxide synthase 2A and cytochrome oxidase subunit 2 (COX‐2). However, in immune cells, mechanisms that regulate miR‐301a have not been reported. Similar to tumour cells, Toll‐like receptor (TLR) ‐activated macrophages produce NF‐κB‐responsive pro‐inflammatory cytokines. Therefore, it is of considerable interest to determine whether miR‐301a regulates the secretion of cytokines by immune cells. In the present study, we demonstrate that the expression of miR‐301a was decreased in TLR‐triggered macrophages. Through targeting NKRF, miR‐301a affected the activity of NF‐κB and the expression of pro‐inflammatory genes downstream of NF‐κB such as COX‐2, prostaglandin E₂ and interleukin‐6. In addition, when lipopolysaccharide‐treated macrophages were simultaneously stimulated with trichostatin A, an inhibitor of histone deacetylases, the expression of miR‐301a increased, whereas NKRF and pro‐inflammatory cytokine expression decreased. However, further investigation revealed that there was no correlation between the induction of miR‐301a and the inhibitory effect of trichostatin A on lipopolysaccharide‐induced gene expression in macrophages. In summary, our study indicates a new mechanism by which miR‐301a regulates inflammatory cytokine expression in macrophages, which may clarify the regulatory role of microRNAs in immune‐mediated inflammatory responses.     

Expression of miR‐155 and miR‐126 in situ in cutaneous T‐cell lymphoma

Recently, miR‐155 has been implicated in cutaneous T‐cell lymphoma (CTCL). Thus, elevated levels of miR‐155 were observed in skin lesions from CTCL patients as judged from qPCR and micro‐array analysis and aberrant, high miR‐155 expression was associated with severe disease. Moreover, miR‐155 promoted proliferation of malignant T cells in vitro. Little is, however, known about which cell types express miR‐155 in vivo in CTCL skin lesions. Here, we study miR‐155 expression using in situ hybridization (ISH) with a miR‐155 probe, a negative control (scrambled), and a miR‐126 probe as a positive control in nine patients with mycosis fungoides, the most frequent subtype of CTCL. We provide evidence that both malignant and non‐malignant T cells stain weakly to moderately positive with the miR‐155 probe, but generally negative with the miR‐126 and negative control probes. Reversely, endothelial cells stain positive for miR‐126 and negative for miR‐155 and the control probe. Solitary T cells with a malignant morphology display brighter staining with the miR‐155 probe. Taken together, our findings suggest that both malignant and non‐malignant T cells express miR‐155 in situ in CTCL. Moreover, they indicate heterogeneity in miR‐155 expression among malignant T cells.     

Deregulation of miR‐92a expression is implicated in hepatocellular carcinoma development

MicroRNAs (miRNAs) belong to a class of the endogenously expressed non‐coding small RNAs which primarily function as gene regulators. Growing evidence suggests that miRNAs have a significant role in tumor development and may constitute robust biomarkers for cancer diagnosis and prognosis. The miR‐17‐92 cluster especially is markedly overexpressed in several cancers, and is associated with the cancer development and progression. In this study, we have demonstrated that miR‐92a is highly expressed in hepatocellular carcinoma (HCC). In addition, the proliferation of HCC‐derived cell lines was enhanced by miR‐92a and inhibited by the anti‐miR‐92a antagomir. On the other hand, we have found that the relative amount of miR‐92a in the plasmas from HCC patients is decreased compared with that from the healthy donors. Interestingly, the amount of miR‐92a was elevated after surgical treatment. Thus, although the physiological significance of the decrease of miR‐92a in plasma is still unknown, deregulation of miR‐92 expression in cells and plasma should be implicated in the development of HCC.     

Loss of miR‐101 expression promotes Wnt/β‐catenin signalling pathway activation and malignancy in colon cancer cells

Colorectal cancer (CRC) is the second leading cause of cancer‐related mortality in Western countries. Although the aberrant expression of several microRNAs (oncomiRs) is associated with CRC progression, the molecular mechanisms of this phenomenon are still under investigation. Here we show that miR‐101 expression is differentially impaired in CRC specimens, depending on tumour grade. miR‐101 re‐expression suppresses cell growth in 3D, hypoxic survival and invasive potential in CRC cells showing low levels of miR‐101. Additionally, we provide molecular evidence of a bidirectional regulatory mechanism between miR‐101 expression and important CRC pro‐malignant features, such as inflammation, activation of the Wnt/β‐catenin signalling pathway and epithelial–mesenchymal transition (EMT). We then propose that up‐regulated miR‐101 may function as a tumour suppressor in CRC and that its pharmacological restoration might hamper the aggressive behaviour of CRC in vivo. MiR‐101 expression may also represent a cancer biomarker for CRC diagnosis and prognosis. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Catenin‐δ1, negatively regulated by miR‐145, promotes tumour aggressiveness in gastric cancer

Increasing evidence supports the association of catenin‐δ1 (CTNND1, p120ctn) with tumour development and progression. However, the mechanism and clinical significance of CTNND1 deregulation in gastric cancer remain unknown. The expression level and cellular localization of CTNND1 were determined by immunohistochemistry in 126 human gastric cancer and 50 non‐tumourous tissues. The cellular localization of CTNND1 and epithelial cadherin (E‐cadherin) were detected by immunofluorescence. Cell proliferation, apoptosis, migration and invasion assays were performed to assess the effect of CTNND1 cDNA or CTNND1 siRNA transfection on gastric cancer cells. Luciferase assay, western blot analysis and in vivo assays were used to determine whether CTNND1 could be regulated by miR‐145. The results demonstrate that the cytoplasmic localization of CTNND1 protein, rather than expression level, was indicative of higher clinical stage, positive lymph node metastasis and poorer prognosis in gastric cancers. CTNND1 could promote gastric cancer cell migration and invasion with little effect on cellular proliferation and apoptosis. CTNND1 was proved to be a direct target gene for miR‐145. Besides suppressing cytoplasmic CTNND1 expression, miR‐145 could recover the membranous localization of CTNND1 and E‐cadherin. We conclude that cytoplasmic CTNND1 can serve as an independent prognostic factor for patients with gastric cancers. MiR‐145 inhibits invasion of gastric cancer cells not only by down‐regulating cytoplasmic CTNND1 expression but also by inducing the translocation of CTNND1 and E‐cadherin from the cytoplasm to the cell membrane through down‐regulating N‐cadherin. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

miR‐155 overexpression is followed by downregulation of its target gene,NFE2L2, and altered pattern of VEGFA expression in the liver of melanoma B16‐bearing mice at the premetastatic stage

MicroRNAs are involved in the control of tumour progression and in metastatic cascade dynamics. However, the role of microRNAs in distant organ reorganization at the premetastatic stage is less clear, although the process of premetastatic niche formation is a crucial event according to modern concepts of tumour dissemination. The role of the present study was to investigate the expression levels of miR‐155, miR‐21, miR‐205 and miR‐let7b, as well as that of their target genes, in target organs of melanoma metastasis at the premetastatic stage. The expression levels of both the pro‐oncogenic miR‐155 and the tumour suppressive miR‐205 were found to be altered in the premetastatic liver of melanoma B16‐bearing mice. Bioinformatics analysis identified the target genes of miR‐155 to be nuclear factor, erythroid 2 like 2 (NFE2L2), secretogranin II, miR‐205, semaphorin 5A and vascular endothelial growth factor A (VEGFA). Among those, the redox status regulatory factor NFE2L2 was downregulated, which corresponded to increased levels of miR‐155. Due to the ability of pro‐oxidative events to initiate angiogenesis, VEGFA levels were evaluated in the premetastatic liver by immunohistochemistry, which revealed increased VEGFA expression in the central parts of the organ and diminished expression in the periphery. Taken together, these findings may support the concept of functional organ reorganization due to melanoma progression.     

Molecular bases of aberrant miR‐182 expression in ovarian cancer

The molecular bases of miR‐182 deregulation in epithelial ovarian cancers (EOCs) remain unknown and its diagnostic or prognostic role in EOCs is still unclear. We performed miR‐182 expression analysis using a microarray approach and real‐time PCR (qPCR). We also used array comparative genomic hybridization and methylated DNA immunoprecipitation to study copy number changes and methylation aberrations within coding locus/promoter sequences of miR‐182 in EOC tissues, respectively. We have found that miR‐182 expression is significantly increased in EOC (P < 0.00001) and that higher miR‐182 expression in EOC is linked with significantly shorter overall survival (P = 0.026). The methylation of miR‐182 promoter was significantly associated with lower miR‐182 expression in EOC tissues (P = 0.045). miR‐182 over‐expression is connected with copy number (CN) gains of this miRNA coding sequences in EOC (P = 0.002), and the number of PRDM5 copies is significantly and inversely correlated with miR‐182 expression evaluated by qPCR (R = ?0.615, P = 0.009). We conclude that the aberrant miR‐182 expression in EOC may be due to CN gains within its coding locus. The miR‐182 promoter is rarely methylated in EOC, and its methylation status is associated with lower miR‐182 expression. Deletion of the PRDM5 locus may play a supportive role in miR‐182 overexpression in EOC. miR‐182 is an unfavorable prognostic factor in EOC. © 2016 Wiley Periodicals, Inc.     

MiR-182 overexpression in tumourigenesis of high-grade serous ovarian carcinoma

Molecular pathogenesis of high‐grade serous ovarian carcinoma (HG‐SOC) is poorly understood. Recent recognition of HG‐SOC precursor lesions, defined as serous tubal intraepithelial carcinoma (STIC) in fimbria, provides a new venue for the study of early genetic changes in HG‐SOC. Using microRNA profiling analysis, we found that miR‐182 expression was significantly higher in STIC than in matched normal Fallopian tube. Further study revealed that miR‐182 was significantly overexpressed in most HG‐SOC cases. To test whether miR‐182 plays a major role in early tumourigenesis of HG‐SOC, we overexpressed miR‐182 in immortalized ovarian surface, Fallopian tube secretory cells and malignant ovarian cell lines, and found that miR‐182 overexpression resulted in increased tumour transformation in vitro, and enhanced tumour invasiveness in vitro and metastasis in vivo. Mechanistically, we demonstrated that the oncogenic properties of miR‐182 in ovarian cancer were mediated in part by its impaired repair of DNA double‐strand breaks and negative regulation of breast cancer 1 (BRCA1) and metastasis suppressor 1 (MTSS1) expression as well as its positive regulation of the oncogene high‐mobility group AT‐hook 2 (HMGA2). Our findings suggest that miR‐182 dysregulation confers powerful oncogenic potential in the tumourigenesis of HG‐SOC. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.