MicroRNA‐30b functions as a tumour suppressor in human colorectal cancer by targeting KRAS,PIK3CD and BCL2

Colorectal cancer (CRC) is the third most common cancer in the USA. MicroRNAs play important roles in the pathogenesis of CRC. In this study, we investigated the role of miR‐30b in CRC and found that its expression was significantly lower in CRC tissues than that in normal tissues. We showed that a low expression level of miR‐30b was closely related to poor differentiation, advanced TNM stage and poor prognosis of CRC. Further experiments showed that over‐expression of miR‐30b suppressed CRC cell proliferation in vitro and tumour growth in vivo. Specifically, miR‐30b promoted G₁ arrest and induced apoptosis. Moreover, KRAS, PIK3CD and BCL2 were identified as direct and functional targets of miR‐30b. MiR‐30b directly targeted the 3′‐untranslated regions of their mRNAs and repressed their expression. This study revealed functional and mechanistic links between miRNA‐30b and oncogene KRAS, PIK3CD and BCL2 in the pathogenesis of CRC. MiR‐30b not only plays important roles in the regulation of cell proliferation and tumour growth in CRC, but is also a potential prognostic marker or therapeutic target for CRC. Restoration of miR‐30b expression may represent a promising therapeutic approach for targeting malignant CRC. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

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.     

Suppression of SPIN1‐mediated PI3K–Akt pathway by miR‐489 increases chemosensitivity in breast cancer

Drug resistance is one of the major obstacles for improving the prognosis of breast cancer patients. Increasing evidence has linked the association of aberrantly expressed microRNAs (miRNAs) with tumour development and progression as well as chemoresistance. Despite recent advances, there is still little known about the potential role and mechanism of miRNAs in breast cancer chemoresistance. Here we describe that 16 miRNAs were found to be significantly down‐regulated and 11 up‐regulated in drug‐resistant breast cancer tissues compared with drug‐sensitive tissues, using a miRNA microarray. The results also showed miR‐489 to be one of the most down‐regulated miRNAs in drug‐resistant tissues and cell lines, as confirmed by miRNA microarray screening and real‐time quantitative PCR. A decrease in miR‐489 expression was associated with chemoresistance as well as lymph node metastasis, increased tumour size, advanced pTNM stage and poor prognosis in breast cancer. Functional analysis revealed that miR‐489 increased breast cancer chemosensitivity and inhibited cell proliferation, migration and invasion, both in vitro and in vivo. Furthermore, SPIN1, VAV3, BCL2 and AKT3 were found to be direct targets of miR‐489. SPIN1 was significantly elevated in drug‐resistant and metastatic breast cancer tissues and inversely correlated with miR‐489 expression. High expression of SPIN1 was associated with higher histological grade, lymph node metastasis, advanced pTNM stage and positive progesterone receptor (PR) status. Increased SPIN1 expression enhanced cell migration and invasion, inhibited apoptosis and partially antagonized the effects of miR‐489 in breast cancer. PIK3CA, AKT, CREB1 and BCL2 in the PI3K–Akt signalling pathway, demonstrated to be elevated in drug‐resistant breast cancer tissues, were identified as downstream effectors of SPIN1. It was further found that either inhibition of SPIN1 or overexpression of miR‐489 suppressed the PI3K–Akt signalling pathway. These data indicate that miR‐489 could reverse the chemoresistance of breast cancer via the PI3K–Akt pathway by targeting SPIN1. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Oncogenic role of miR‐155 in anaplastic large cell lymphoma lacking the t(2;5) translocation

Anaplastic large cell lymphoma (ALCL) is a rare, aggressive, non‐Hodgkin's lymphoma that is characterized by CD30 expression and disease onset in young patients. About half of ALCL patients bear the t(2;5)(p23;q35) translocation, which results in the formation of the nucleophosmin‐anaplastic lymphoma tyrosine kinase (NPM–ALK) fusion protein (ALCL ALK⁺). However, little is known about the molecular features and tumour drivers in ALK‐negative ALCL (ALCL ALK^?), which is characterized by a worse prognosis. We found that ALCL ALK^?, in contrast to ALCL ALK⁺, lymphomas display high miR‐155 expression. Consistent with this, we observed an inverse correlation between miR‐155 promoter methylation and miR‐155 expression in ALCL. However, no direct effect of the ALK kinase on miR‐155 levels was observed. Ago2 immunoprecipitation revealed miR‐155 as the most abundant miRNA, and enrichment of target mRNAs C/EBPβ and SOCS1. To investigate its function, we over‐expressed miR‐155 in ALCL ALK⁺ cell lines and demonstrated reduced levels of C/EBPβ and SOCS1. In murine engraftment models of ALCL ALK^?, we showed that anti‐miR‐155 mimics are able to reduce tumour growth. This goes hand‐in‐hand with increased levels of cleaved caspase‐3 and high SOCS1 in these tumours, which leads to suppression of STAT3 signalling. Moreover, miR‐155 induces IL‐22 expression and suppresses the C/EBPβ target IL‐8. These data suggest that miR‐155 can act as a tumour driver in ALCL ALK^? and blocking miR‐155 could be therapeutically relevant. Original miRNA array data are to be found in the supplementary material (Table S1). © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

MicroRNA‐542‐3p inhibits tumour angiogenesis by targeting Angiopoietin‐2

Angiopoietin‐2 (Angpt2) plays a critical role in angiogenesis and tumour progression. Therapeutic targeting of Angpt2 has been implicated as a promising strategy for cancer treatment. Whereas miRNAs are emerging as important modulators of angiogenesis, regulation of Angpt2 by miRNAs has not been established. Here we firstly report that Ang2 is targeted by a microRNA, miRNA‐542‐3p, which inhibits tumour progression by impairing Ang2's pro‐angiogenic activity. In cultured endothelial cells, miR‐542‐3p inhibited translation of Angpt2 mRNA by binding to its 3′ UTR, and addition of miR‐542‐3p to cultured endothelial cells attenuated angiogenesis. Administration of miR‐542‐3p to tumour‐bearing mice reduced tumour growth, angiogenesis and metastasis. Furthermore, the level of miR‐542‐3p in primary breast carcinomas correlated inversely with clinical progression in primary tumour samples from stage III and IV patients. Together, these findings uncover a novel regulatory pathway whereby an anti‐angiogenic miR‐542‐3p directly targets the key angiogenesis‐promoting protein Angpt2, suggesting that miR‐542‐3p may represent a promising target for anti‐angiogenic therapy and a potential marker for monitoring disease progression. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

Exosomal miR‐24‐3p impedes T‐cell function by targeting FGF11 and serves as a potential prognostic biomarker for nasopharyngeal carcinoma

Recent studies have shown that extracellular microRNAs are not only potential biomarkers but are also involved in cell interactions to regulate the intercommunication between cancer cells and their microenvironments in various types of malignancies. In this study, we isolated exosomes from nasopharyngeal carcinoma (NPC) cell lines and patient sera (T‐EXOs), or control NP69 cells and healthy donor sera (HD‐EXOs). We found that miR‐24‐3p was markedly enriched in T‐EXOs as compared with HD‐EXOs; the serum exosomal miR‐24‐3p level was correlated with worse disease‐free survival of patients (p < 0.05). Knockdown of exosomal miR‐24‐3p (miR‐24‐3p‐sponge‐T‐EXOs) by a sponge RNA targeting miR‐24‐3p restored the T‐EXO‐mediated (control‐sponge‐T‐EXO) inhibition of T‐cell proliferation and Th1 and Th17 differentiation, and the induction of regulatory T cells (Tregs). Mechanistic analyses revealed that administration of exosomal miR‐24‐3p increased P‐ERK, P‐STAT1 and P‐STAT3 expression while decreasing P‐STAT5 expression during T‐cell proliferation and differentiation. Moreover, by in vivo and in vitro assessments, we found FGF11 to be a direct target of miR‐24‐3p. However, both miR‐24‐3p‐sponge‐T‐EXOs and T‐EXOs (control‐sponge‐T‐EXOs) impeded proliferation and Th1 and Th17 differentiation, but induced Treg differentiation, of lenti‐shFGF11‐transfected T cells. The levels of phosphorylated ERK and STAT proteins were different in lenti‐ScshRNA‐transfected T cells and lenti‐shFGF11‐transfected T cells following administration of miR‐24‐3p‐sponge‐T‐EXO. Interestingly, tumour FGF11 expression was positively correlated with the number of CD4⁺ and CD8⁺ T cells in vivo, and predicted favourable patient DFS (p < 0.05). Additionally, hypoxia increased cellular and exosomal miR‐24‐3p levels and enhanced the inhibitory effect of T‐EXO on T‐cell proliferation and differentiation. Collectively, our findings suggest that exosomal miR‐24‐3p is involved in tumour pathogenesis by mediating T‐cell suppression via repression of FGF11, and may serve as a potential prognostic biomarker in NPC. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Methylation‐induced loss of miR‐484 in microsatellite‐unstable colorectal cancer promotes both viability and IL‐8 production via CD137L

Colorectal cancer (CRC) exhibiting MSI (microsatellite instability) represents a well‐defined subtype characterized by a deficient mismatch repair pathway and typical clinico‐pathological features. Our objective was to identify the entire miRNome and its molecular pathological roles in MSI CRCs. We profiled miRNA expression in MSI CRCs and compared it with MSS counterparts. Microarray and qRT‐PCR analysis identified eight miRNAs that could distinguish the MSI status of CRCs. MiR‐484 was the most significantly decreased miRNA in MSI CRCs, primarily mediated by the CpG island methylator phenotype. MiR‐484 functions as a tumour suppressor to inhibit MSI CRC cell viability in vitro and in vivo. Moreover, miR‐484 repressed CD137L expression and thereby attenuated IL‐8 production by MSI CRC cells. Our results contribute to a better understanding of the roles of dysregulated miRNAs in the distinct phenotypic features of MSI CRCs and indicate an option for early diagnosis and gene therapy for these patients. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

miRNA‐199a‐5p regulates VEGFA in endometrial mesenchymal stem cells and contributes to the pathogenesis of endometriosis

It is believed that endometrial miRNAs contribute to the aetiology of endometriosis in stem cells; however, the mechanisms remain unclear. Here we collected serum samples from patients with or without endometriosis and characterized the miRNA expression profiles of these two groups. MicroRNA‐199a‐5p (miR‐199a‐5p) was dramatically down‐regulated in patients with endometriosis compared with control patients. In addition, we found that the tumour suppressor gene, SMAD4, could elevate miR‐199a‐5p expression in ectopic endometrial mesenchymal stem cells. Up‐regulation of miR‐199a‐5p suppressed cell proliferation, motility and angiogenesis of these ectopic stem cells by targeting the 3′ untranslated region of VEGFA. Furthermore, we established an animal model of endometriosis and found that miR‐199a‐5p could decrease the size of endometriotic lesions in vivo. Taken together, this newly identified miR‐199a‐5p module provides a new avenue to the understanding of the processes of endometriosis development, especially proliferation, motility and angiogenesis, and may facilitate the development of potential therapeutics against endometriosis. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

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.     

MiR‐26b is down‐regulated in carcinoma‐associated fibroblasts from ER‐positive breast cancers leading to enhanced cell migration and invasion

Carcinoma‐associated fibroblasts (CAFs) influence the behaviour of cancer cells but the roles of microRNAs in this interaction are unknown. We report microRNAs that are differentially expressed between breast normal fibroblasts and CAFs of oestrogen receptor‐positive cancers, and explore the influences of one of these, miR‐26b, on breast cancer biology. We identified differentially expressed microRNAs by expression profiling of clinical samples and a tissue culture model: miR‐26b was the most highly deregulated microRNA. Using qPCR, miR‐26b was confirmed as down‐regulated in fibroblasts from 15 of 18 further breast cancers. Next, we examined whether manipulation of miR‐26b expression changed breast fibroblast behaviour. Reduced miR‐26b expression caused fibroblast migration and invasion to increase by up to three‐fold in scratch‐closure and trans‐well assays. Furthermore, in co‐culture with MCF7 breast cancer epithelial cells, fibroblasts with reduced miR‐26b expression enhanced both MCF7 migration in trans‐well assays and MCF7 invasion from three‐dimensional spheroids by up to five‐fold. Mass spectrometry was used to identify expression changes associated with the reduction of miR‐26b expression in fibroblasts. Pathway analyses of differentially expressed proteins revealed that glycolysis/TCA cycle and cytoskeletal regulation by Rho GTPases are downstream of miR‐26b. In addition, three novel miR‐26b targets were identified (TNKS1BP1, CPSF7, COL12A1) and the expression of each in cancer stroma was shown to be significantly associated with breast cancer recurrence. MiR‐26b in breast CAFs is a potent regulator of cancer behaviour in oestrogen receptor‐positive cancers, and we have identified key genes and molecular pathways that act downstream of miR‐26b in CAFs. © 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Low expression of MicroRNA‐126 is associated with poor prognosis in colorectal cancer

MicroRNA‐126 (miR‐126) has been reported to be a tumor suppressor that targets CXCR4 in colorectal cancer (CRC) cells. This study investigated whether miR‐126 has any prognostic impact in patients with CRC. MiR‐126 and CXCR4 mRNA expression in 92 pairs of CRC and adjacent nontumorous tissues was examined using quantitative real‐time PCR, and CXCR4 protein expression was assessed by immunohistochemistry (IHC) and Western blotting. The correlation between miR‐126 and CXCR4 protein expression and clinicopathological features and overall survival rate was determined. MiR‐126 was downregulated in CRC tissues that expressed high levels of CXCR4 mRNA. IHC and Western blotting detected high expression of CXCR4 protein in CRC tissues. An inverse correlation was observed between miR‐126 and CXCR4 protein expression in CRC tissues. Moreover, low miR‐126 and high CXCR4 protein expression was associated with distant metastasis, clinical TNM stage, and poor survival. Multivariate analysis indicated that miR‐126 was an independent prognostic factor for overall survival, suggesting its clinical significance as a prognostic predictor in CRC patients. © 2014 Wiley Periodicals, Inc.     

Up‐regulation of miR‐455‐5p by the TGF‐β–SMAD signalling axis promotes the proliferation of oral squamous cancer cells by targeting UBE2B

MicroRNAs (miRNAs) are involved in the tumourigenesis of various cancers by regulating their downstream targets. To identify the changes of miRNAs in oral squamous cell carcinoma (OSCC), we investigated the expression profiles of miRNAs in 40 pairs of OSCC specimens and their matched non‐tumour epithelial tissues. Our data revealed higher miR‐455‐5p expression in the tumour tissues than in the normal tissues; the expression was also higher in oral cancer cell lines than in normal keratinocyte cell lines. MiR‐455‐5p knockdown reduced both the anchorage‐independent growth and the proliferative ability of oral cancer cells, and these factors increased in miR‐455‐5p‐overexpressing cells. Furthermore, by analysing the array data of patients with cancer and cell lines, we identified ubiquitin‐conjugating enzyme E2B (UBE2B) as a target of miR‐455‐5p, and further validated this using 3′‐untranslated region luciferase reporter assays and western blot analysis. We also demonstrated that UBE2B suppression rescued the impaired growth ability of miR‐455‐5p‐knockdown cells. Furthermore, we observed that miR‐455‐5p expression was regulated, at least in part, by the transforming growth factor‐β (TGF‐β) pathway through the binding of SMAD3 to specific promoter regions. Notably, miR‐455‐5p expression was associated with the nodal status, stage, and overall survival in our patients, suggesting that miR‐455‐5p is a potential marker for predicting the prognosis of patients with oral cancer. In conclusion, we reveal that miR‐455‐5p expression is regulated by the TGF‐β‐dependent pathway, which subsequently leads to UBE2B down‐regulation and contributes to oral cancer tumourigenesis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.