MicroRNAs (miRNAs) can function as either oncogenes or tumor suppressor genes via regulation of cell proliferation and/or apoptosis. MiR-221 and miR-222 were discovered to induce cell growth and cell cycle progression via direct targeting of p27 and p57 in various human malignancies. However, the roles of miR-221 and miR-222 have not been reported in human gastric cancer. In this study, we examined the impact of miR-221 and miR-222 on human gastric cancer cells, and identified target genes for miR-221 and miR-222 that might mediate their biology.
The human gastric cancer cell line SGC7901 was transfected with AS-miR-221/222 or transduced with pMSCV-miR-221/222 to knockdown or restore expression of miR-221 and miR-222, respectively. The effects of miR-221 and miR-222 were then assessed by cell viability, cell cycle analysis, apoptosis, transwell, and clonogenic assay. Potential target genes were identified by Western blot and luciferase reporter assay.
Upregulation of miR-221 and miR-222 induced the malignant phenotype of SGC7901 cells, whereas knockdown of miR-221 and miR-222 reversed this phenotype via induction of PTEN expression. In addition, knockdonwn of miR-221 and miR-222 inhibited cell growth and invasion and increased the radiosensitivity of SGC7901 cells. Notably, the seed sequence of miR-221 and miR-222 matched the 3'UTR of PTEN, and introducing a PTEN cDNA without the 3'UTR into SGC7901 cells abrogated the miR-221 and miR-222-induced malignant phenotype. PTEN-3'UTR luciferase reporter assay confirmed PTEN as a direct target of miR-221 and miR-222.
These results demonstrate that miR-221 and miR-222 regulate radiosensitivity, and cell growth and invasion of SGC7901 cells, possibly via direct modulation of PTEN expression. Our study suggests that inhibition of miR-221 and miR-222 might form a novel therapeutic strategy for human gastric cancer.
Core-binding factor leukemia (CBFL) is a subgroup of acutemyeloid leukemia (AML) characterized by genetic mutations involving the subunits of the core-binding factor (CBF). The leukemogenesis model for CBFL posits that one, or more, gene mutations inducing increased cell proliferation and/or inhibition of apoptosis cooperate with CBF mutations for leukemia development. One of the most commonmutations associated with CBF mutations involves the KIT receptor. A high expression of KIT is a hallmark of a high proportion of CBFL. Previous studies indicate that microRNA (MIR) 222/221 targets the 3′ untranslated region of the KIT messenger RNA and our observation that AML1 can bind the MIR-222/221 promoter, we hypothesized that MIR-222/221 represents the link between CBF and KIT. Here, we show that MIR-222/221 expression is upregulated after myeloid differentiation of normal bone marrow AC133+ stem progenitor cells. CBFL blasts with either t(8;21) or inv(16) CBF rearrangements with high expression levels of KIT (CD117) display a significantly lower level of MIR-222/221 expression than non-CBFL blasts. Consistently, we found that the t(8;21) AML1-MTG8 fusion protein binds the MIR-222/221 promoter and induces transcriptional repression of a MIR-222/221-LUC reporter. Because of the highly conserved sequence homology, we demonstrated concomitant MIR-222/221 down-regulation and KIT up-regulation in the 32D/WT1 mouse cell model carrying the AML1-MTG16 fusion protein. This study provides the first hint that CBFL-associated fusion proteins may lead to up-regulation of the KIT receptor by down-regulating MIR-222/221, thus explaining the concomitant occurrence of CBF genetic rearrangements and overexpression of wild type or mutant KIT in AML.
A rising body of evidence suggests that silencing microRNAs (miRNAs) with oncogenic potential may represent a successful therapeutic strategy for human cancer. We investigated the therapeutic activity of miR-221/222 inhibitors against human multiple myeloma (MM) cells. Enforced expression of miR-221/222 inhibitors triggered in vitro anti-proliferative effects and up-regulation of canonic miR-221/222 targets, including p27Kip1, PUMA, PTEN and p57Kip2, in MM cells highly expressing miR-221/222. Conversely, transfection of miR-221/222 mimics increased S-phase and down-regulated p27Kip1 protein expression in MM with low basal miR-221/222 levels. The effects of miR-221/222 inhibitors was also evaluated in MM xenografts in SCID/NOD mice. Significant anti-tumor activity was achieved in xenografted mice by the treatment with miR-221/222 inhibitors, together with up-regulation of canonic protein targets in tumors retrieved from animals. These findings provide proof of principle that silencing the miR-221/222 cluster exerts significant therapeutic activity in MM cells with high miR-221/222 level of expression, which mostly occurs in TC2 and TC4 MM groups. These findings suggest that MM genotyping may predict the therapeutic response. All together our results support a framework for clinical development of miR-221/222 inhibitors-based therapeutic strategy in this still incurable disease.
miR-221; miR-222; microRNA; miRNA; multiple myeloma; plasma cell leukemia
Presently, neurodegenerative diseases and cancer are the most clinically problematic age-related diseases worldwide. Although being distinct disorders, their developments share common cellular mechanisms. Oncogenesis and neurodegeneration arise from the deregulation of signaling pathways, as a consequence of the resulting imbalance in cellular homeostasis. The epidermal growth factor receptor (EGFR) belongs to an important cellular signaling pathway, which regulates proliferation, differentiation, cell cycle and migration. As transcriptional targets of EGFR, the microRNAs-221/222 (miR-221/222) are important expression regulators. Dysfunctions in their networks are associated with cellular disruptions. The transcriptional activation of these microRNAs (miRNAs) seems to be involved in cell cycle, apoptosis, metastization, and in the acquisition of resistance to therapies. The up-regulation of miR-221/222 is associated with increased expression levels of matrix metalloproteinases (MMPs) and repression of cell cycle inhibitors, which are key molecules in oncogenesis and neurodegeneration processes. The interaction loop between proliferative signaling pathways and miRNA expression could reveal new targets for controlling the molecular behavior of age-related diseases.
EGFR; miRNAs; miR-221/222; age-related diseases; cancer; neurodegenerative diseases
MiR-221 and miR-222 are two highly homologous microRNAs whose upregulation has been recently described in several types of human tumors, for some of which their oncogenic role was explained by the discovery of their target p27, a key cell cycle regulator. We previously showed this regulatory relationship in prostate carcinoma cell lines in vitro, underlying the role of miR-221/222 as inducers of proliferation and tumorigenicity.
Here we describe a number of in vivo approaches confirming our previous data. The ectopic overexpression of miR-221 is able, per se, to confer a high growth advantage to LNCaP-derived tumors in SCID mice. Consistently, the anti-miR-221/222 antagomir treatment of established subcutaneous tumors derived from the highly aggressive PC3 cell line, naturally expressing high levels of miR-221/222, reduces tumor growth by increasing intratumoral p27 amount; this effect is long lasting, as it is detectable as long as 25 days after the treatment. Furthermore, we provide evidence in favour of a clinical relevance of the role of miR-221/222 in prostate carcinoma, by showing their general upregulation in patient-derived primary cell lines, where we find a significant inverse correlation with p27 expression.
These findings suggest that modulating miR-221/222 levels may have a therapeutic potential in prostate carcinoma.
MicroRNAs (miRNAs) are noncoding RNAs that impact almost every aspect of biology and disease. Until now, the cell-specific effects of miRNAs in cardiovascular system have not been established. In the current study, the cellular functions of miR-221 and miR-222 (miR-221/222) in vascular smooth muscle cells (VSMCs) and vascular endothelial cells (ECs) were compared. In cultured cells, we identified that the effects of miR-221/222 on proliferation, migration, and apoptosis are opposite between VSMCs and ECs. In VSMCs, miR-221/222 had effects of pro-proliferation, pro-migration, and anti-apoptosis. In contrast, miR-221/222 had effects of anti-proliferation, anti-migration, and pro-apoptosis in ECs. The different expression profiles of their target genes, p27(Kip1), p57(kip2), and c-kit between the two cell types might be related to the opposite effects. Finally, the opposite cellular effects of miR-221/222 were verified in vivo in balloon-injured rat carotid artery as demonstrated by different consequences in neointimal growth and re-endothelialization. The results suggest that the biological functions of miR-221/222 in vascular walls are cell-specific. The opposite cellular effects of miR-221/222 on VSMCs and ECs may have important therapeutic applications in many vascular diseases such as atherosclerosis and restenosis after angioplasty.
microRNAs; smooth muscle cells; endothelial cells; cell-specific effects; vascular disease
MicroRNAs (miRNAs) are potent negative regulators of gene expression involved in all aspects of cell biology. They finely modulate virtually all physiological pathways in metazoans, and are deeply implicated in all main pathologies, among which cancer. Mir-221 and miR-222, two closely related miRNAs encoded in cluster from a genomic region on chromosome X, are strongly upregulated in several forms of human tumours. In this work, we report that the ectopic modulation of NF-kB modifies miR-221/222 expression in prostate carcinoma and glioblastoma cell lines, where we had previously shown their oncogenic activity. We identify two separate distal regions upstream of miR-221/222 promoter which are bound by the NF-kB subunit p65 and drive efficient transcription in luciferase reporter assays; consistently, the site-directed mutagenesis disrupting p65 binding sites or the ectopical inhibition of NF-kB activity significantly reduce luciferase activity. In the most distal enhancer region, we also define a binding site for c-Jun, and we show that the binding of this factor cooperates with that of p65, fully accounting for the observed upregulation of miR-221/222. Thus our work uncovers an additional mechanism through which NF-kB and c-Jun, two transcription factors deeply involved in cancer onset and progression, contribute to oncogenesis, by inducing miR-221/222 transcription.
MiR-221 and miR-222 (miR-221/222), upregulated in gliomas, can regulate glioma cell cycle progression and apoptosis, respectively. However, the association of miR-221/222 with glioma cell invasion and survival remains unknown.
Invasion capability of miR-221/222 was detected by mutiple analyses, including diffusion tensor imaging (DTI), transwell, wound healing and nude mouse tumor xenograft model assay. Further, the target of miR-221/222 was determined by luciferase reporter, western blot and gene rescue assay. The association of miR-221/222 with outcome was examined in fifty glioma patients.
MiR-221/222 expression was significantly increased in high-grade gliomas compared with low-grade gliomas, and positively correlated with the degree of glioma infiltration. Over-expression of miR-221/222 increased cell invasion, whereas knockdown of miR-221/222 decreased cell invasion via modulating the levels of the target, TIMP3. Introduction of a TIMP3 cDNA lacking 3’ UTR abrogated miR-221/222-induced cell invasion. In addition, knockdown of miR-221/222 increased TIMP3 expression and considerably inhibited tumor growth in a xenograft model. Finally, the increased level of miR-221/222 expression in high-grade gliomas confers poorer overall survival.
The present data indicate that miR-221 and miR-222 directly regulate cell invasion by targeting TIMP3 and act as prognostic factors for glioma patients.
MiRNA; TIMP3; Glioblastoma; Cell invasion; Prognosis
Lung and liver cancers are among the most deadly types of cancer. Despite improvements in treatment over the past few decades, patient survival remains poor, underlining the need for development of targeted therapies. MicroRNAs represent a class of small RNAs, frequently deregulated in human malignancies. We now report that miR221&222 are over-expressed in aggressive non small cell lung cancer and hepatocarcinoma cells, as compared with less invasive and/or normal lung and liver cells. We show that miR-221&222, by targeting PTEN and TIMP3 tumor suppressors, induce TRAIL resistance and enhance cellular migration through the activation of the AKT pathway and metallopeptidases. Finally, we demonstrate that the MET oncogene is involved in miR-221&222 activation, through the c-Jun transcription factor.
MicroRNAs are often aberrantly expressed in human neoplasms and are postulated to play a role in neoplastic initiation and progression. miR-221 and miR-222 negatively regulate expression of CDKN1B (p27)and CDKN1C (p57), two cell cycle regulators expressed in ovarian surface epithelium and down-regulated in ovarian carcinomas. We characterized miR-221 and miR-222 expression in 49 sporadic high grade ovarian carcinomas and determined whether somatic mutation or epigenetic alterations explained the differences in expression of these miRNAs. We correlated these findings with protein expression of CDKN1B and CDKN1C as assessed by immunohistochemistry. Expression of miR-221 and miR-222 were closely correlated with each other (P=0.0001). Interestingly, a lower ratio of miR-221 to miR-222 expression was significantly correlated with worse overall survival (P=0.01) and remained a significant predictor of overall survival in multivariate analysis using the co-variate adequacy of surgical cytoreduction (P=0.03). Higher miR-222 and miR-221 expression were significantly associated with decreased CDKN1C expression (P=0.009 and 0.01). In contrast, CDKN1B expression was not associated with miR-221 or miR-222 expression. Neither somatic mutations nor methylation of the studied region explained the alterations in miR-221 and miR-222 expression in most carcinomas.
MicroRNAs are small noncoding RNA molecules that negatively regulate gene expression via degradation or translational repression of their targeted mRNAs. It is known that aberrant microRNA expression can play important roles in cancer, but the role of microRNAs in autoimmune diseases is only beginning to emerge. In this study, the expression of selected microRNAs is examined in rheumatoid arthritis.
Total RNA was isolated from peripheral blood mononuclear cells obtained from patients with rheumatoid arthritis, and healthy and disease control individuals, and the expression of miR-146a, miR-155, miR-132, miR-16, and microRNA let-7a was analyzed using quantitative real-time PCR.
Rheumatoid arthritis peripheral blood mononuclear cells exhibited between 1.8-fold and 2.6-fold increases in miR-146a, miR-155, miR-132, and miR-16 expression, whereas let-7a expression was not significantly different compared with healthy control individuals. In addition, two targets of miR-146a, namely tumor necrosis factor receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK-1), were similarly expressed between rheumatoid arthritis patients and control individuals, despite increased expression of miR-146a in patients with rheumatoid arthritis. Repression of TRAF6 and/or IRAK-1 in THP-1 cells resulted in up to an 86% reduction in tumor necrosis factor-α production, implicating that normal miR-146a function is critical for the regulation of tumor necrosis factor-α production.
Recent studies have shown that synovial tissue and synovial fibroblasts from patients with rheumatoid arthritis exhibit increased expression of certain microRNAs. Our data thus demonstrate that microRNA expression in rheumatoid arthritis peripheral blood mononuclear cells mimics that of synovial tissue/fibroblasts. The increased microRNA expression in rheumatoid arthritis patients is potentially useful as a marker for disease diagnosis, progression, or treatment efficacy, but this will require confirmation using a large and well defined cohort. Our data also suggest a possible mechanism contributing to rheumatoid arthritis pathogenesis, whereby miR-146a expression is increased but unable to properly function, leading to prolonged tumor necrosis factor-α production in patients with rheumatoid arthritis.
The epithelial-to-mesenchymal transition (EMT) is a highly conserved physiological program involved in development and tissue repair; however, its aberrant activation has been implicated in accelerating the progression of a variety of cancers. In breast cancer, the microRNAs (miRNAs) miR-221 and miR-222 (miR-221/222) are differentially expressed in the clinically more aggressive basal-like subtype compared to luminal subtype of breast cancer and upregulation of miR-221/222 induces the EMT by targeting the 3′ untranslated region (3′UTR) of the GATA family transcriptional repressor TRPS1 (tricho-rhino-phalangeal syndrome type 1). The complete mechanism through which miR-221/222 promotes the EMT, however, is not fully understood. We identified adiponectin receptor 1 (ADIPOR1), a receptor for the adipocytokine adiponectin, as a direct target of miR-221/222. ADIPOR1 is expressed at higher levels in the luminal compared to the basal-like subtype of breast cancer cell lines, which can be reduced by miR-221/222 targeting of its 3’UTR. In addition, miR-221/222 were negatively correlated with ADIPOR1 expression across breast cancer cell lines and tumors. ADIPOR1 depletion by siRNA in MCF10A cells induced the EMT and increased cell invasion. Depletion of ADIPOR1 by siRNA induced activation of the canonical nuclear factor-kappaB (NF-κB) and subsequent phosphorylation of signal transducer and activator of transcription 3 (STAT3) in an interleukin 6 (IL6)-dependent manner. Finally, overexpression of ADIPOR1 in the basal-like cell line, MDA-MB-231, attenuated cell invasion and promoted the mesenchymal-to-epithelial transition (MET). We conclude that ADIPOR1 negatively regulates EMT in breast cancer and provides an additional node by which miR-221/222 induces the EMT. These results suggest that ADIPOR1 may play an important role in breast cancer progression and metastasis, and could potentially offer an alternative therapeutic strategy for basal-like breast cancer patients.
MiRNAs are small non-coding RNAs of ~24 nt that can block mRNA translation and/or negatively regulate its stability. There is a large body of evidence that dysregulation of miRNAs is a hallmark of cancer. miRNAs are often aberrantly expressed and their function is linked to the regulation of oncogenes and/or tumor suppressor genes involved in cell signaling pathway. MiR-221 and miR-222 are two highly homologous microRNAs, whose upregulation has been recently described in several types of human tumors. MiR-221/222 have been considered to act as oncogenes or tumor suppressors, depending on tumor system. Silencing oncomiRs or gene therapy approaches, based on re-expression of miRNAs that are down-regulated in cancer cells, could represent a novel anti-tumor approach for integrated cancer therapy. Here we will review the role of miR-221/222 in cancer progression and their use as prognostic and therapeutic tools in cancer.
microRNA; cancer; cancer therpay
MicroRNAs constitute a large family of small non-coding RNAs that have emerged as key post-transcriptional regulators in a wide variety of organisms. Because any one miRNA can potentially regulate expression of a distinct set of genes, differential miRNA expression can shape the repertoire of proteins that are actually expressed during development, differentiation or disease. Here, we have used mast cells as a model to investigate the role of miRNAs in differentiated innate immune cells, and found that miR-221-222 are significantly up-regulated upon mast cell activation. Using both bioinformatics and experimental approaches, we identified some signaling pathways, transcription factors and potential cis-regulatory regions that control miR-221-222 transcription. Overexpression of miR-221-222 in a model mast cell-line perturbed cell morphology and cell cycle regulation without altering viability. While in stimulated cells miR-221-222 partially counteracted expression of the cell-cycle inhibitor p27kip1, we found that in the mouse alternative splicing results in two p27kip1 mRNA isoforms that differ in their 3′ UTR, only one of which is subject to miR-221-222 regulation. In addition, transgenic expression of miR-221-222 from BAC clones in embryonic stem cells dramatically reduced cell-proliferation and severely impaired their accumulation. Our study provides further insights on miR-221-222 transcriptional regulation as well as evidences that miR-221-222 regulates cell-cycle checkpoints in mast cells in response to acute activation stimuli.
cell cycle; mast cells; microRNAs; transcription; proliferation
MicroRNAs (miRNA) have tumor suppressive and oncogenic potential in human cancer, but whether and how miRNAs control cell cycle progression is not understood. To address this question, we carried out a comprehensive analysis of miRNA expression during serum stimulation of quiescent human cells. Time course analyses revealed that four miRNAs are up-regulated and >100 miRNAs are down-regulated, as cells progress beyond the G1-S phase transition. We analyzed the function of two up-regulated miRNAs (miR-221 and miR-222) that are both predicted to target the cell growth suppressive cyclin-dependent kinase inhibitors p27 and p57. Our results show that miR-221 and miR-222 both directly target the 3′ untranslated regions of p27 and p57 mRNAs to reduce reporter gene expression, as well as diminish p27 and p57 protein levels. Functional studies show that miR-221 and miR-222 prevent quiescence when elevated during growth factor deprivation and induce precocious S-phase entry, thereby triggering cell death. Thus, the physiologic upregulation of miR-221 and miR-222 is tightly linked to a cell cycle checkpoint that ensures cell survival by coordinating competency for initiation of S phase with growth factor signaling pathways that stimulate cell proliferation.
The use of new, deep sequencing technologies has greatly accelerated microRNA discovery. We have applied this approach to the identification of chicken microRNAs and to the comparison of microRNAs in chicken embryo fibroblasts (CEF) infected with Marek's disease virus (MDV) to those present in uninfected CEF.
We obtained 125,463 high quality reads that showed an exact match to the chicken genome. The majority of the reads corresponded to previously annotated chicken microRNAs; however, the sequences of many potential novel microsRNAs were obtained. A comparison of the reads obtained in MDV-infected and uninfected CEF indicates that infection does not significantly perturb the expression profile of microRNAs. Frequently sequenced microRNAs include miR-221/222, which are thought to play a role in growth and proliferation. A number of microRNAs (e.g., let-7, miR-199a-1, 26a) are expressed at lower levels in MDV-induced tumors, highlighting the potential importance of this class of molecules in tumorigenesis.
Deep sequencing technology is highly suited for small RNA discovery. This approach is independent of comparative sequence analysis, which has been the primary method used to identify chicken microRNAs. Our results have confirmed the expression of many microRNAs identified by sequence similarity and identified a pool of candidate novel microRNAs.
Recent evidence shows that certain microRNAs (miRNAs) play a role in both obesity and prostate cancer recurrence, but the association between the expression of these miRNAs and obesity in prostate cancer recurrence is unknown. In this study, we examined the effect of the interaction between obesity and miR-21, miR-221 or miR-222 expression on prostate cancer recurrence among 28 recurrent and 37 non-recurrent prostate cancer cases. miRNA expression was determined using quantitative real-time polymerase chain reaction. Cox proportional hazard models adjusting for age at diagnosis, clinical stage and Gleason score were used to estimate hazard ratios (HR) and 95% confidence intervals (95% CI) for recurrence free survival. A significantly (P=0.014) higher proportion of recurrent cases (78.6%) than non-recurrent cases (48.6%) had a low expression of miR-21 and the difference was more prominent in obese than non-obese patients. Multivariate analysis showed that the expression of miR-21 was an independent risk factor for recurrence in obese (HR=6.15, 95% CI=1.04–36.48, P=0.045), but not in non-obese (HR=1.28, 95% CI=0.30–5.49, P=0.74) cases. A significant association with recurrence was not observed for the expression of miR-221 and miR-222. In summary, our findings show that miR-21 is associated with prostate cancer recurrence after radical prostatectomy and suggest that the differential expression of miR-21 is more prominent in obese than in non-obese cases. Future larger studies are warranted to confirm these initial findings and to elucidate the mechanisms involved.
miR-21; miR-221; miR-222; miRNA; obesity; prostate carcinoma; recurrence
MicroRNAs comprise a novel class of endogenous, small, noncoding RNAs that negatively regulate gene expression. Functionally, an individual miRNA is as important as a transcription factor because it is able to regulate the expression of its multiple target genes. Recently, miR-221 and miR-222 have been found to play a critical role in cancer cell proliferation. However, their roles in vascular smooth muscle cell (VSMC) biology are currently unknown. In the current study, the time course changes and cellular distribution of miR-221 and miR-222 expression were identified in rat carotid arteries after angioplasty, in which their expression was upregulated and localized in VSMCs in the injured vascular walls. In cultured VSMCs, miR-221 and miR-222 expression was increased by growth stimulators. Knockdown of miR-221 and miR-222 resulted in decreased VSMC proliferation in vitro. Using both gain-of-function and loss-of-function approaches, we found that p27(Kip1) and p57(Kip2) were two target genes that were involved in miR-221 and miR-222-mediated effect on VSMC growth. Finally, knockdown of miR-221 and miR-222 in rat carotid arteries suppressed VSMC proliferation in vivo and neointimal lesion formation after angioplasty. The results indicate that miR-221 and miR-222 are novel regulators for VSMC proliferation and neointimal hyperplasia. These findings may also represent promising therapeutic targets in proliferative vascular diseases.
MicroRNAs; vascular smooth muscle cells; gene regulation; proliferation; vascular disease
MicroRNAs are required for maintenance of pluripotency as well as differentiation, but since more microRNAs have been computationally predicted in genome than have been found, there are likely to be undiscovered microRNAs expressed early in stem cell differentiation.
SOLiD ultra-deep sequencing identified >107 unique small RNAs from human embryonic stem cells (hESC) and neural-restricted precursors that were fit to a model of microRNA biogenesis to computationally predict 818 new microRNA genes. These predicted genomic loci are associated with chromatin patterns of modified histones that are predictive of regulated gene expression. 146 of the predicted microRNAs were enriched in Ago2-containing complexes along with 609 known microRNAs, demonstrating association with a functional RISC complex. This Ago2 IP-selected subset was consistently expressed in four independent hESC lines and exhibited complex patterns of regulation over development similar to previously-known microRNAs, including pluripotency-specific expression in both hESC and iPS cells. More than 30% of the Ago2 IP-enriched predicted microRNAs are new members of existing families since they share seed sequences with known microRNAs.
Extending the classic definition of microRNAs, this large number of new microRNA genes, the majority of which are less conserved than their canonical counterparts, likely represent evolutionarily recent regulators of early differentiation. The enrichment in Ago2 containing complexes, the presence of chromatin marks indicative of regulated gene expression, and differential expression over development all support the identification of 146 new microRNAs active during early hESC differentiation.
Fulvestrant is a selective estrogen receptor downregulator (SERD) and highly effective antagonist to hormone-sensitive breast cancers following failure of previous tamoxifen or aromatase inhibitor therapies. However, after prolonged fulvestrant therapy, acquired resistance eventually occurs in the majority of breast cancer patients, due to poorly understood mechanisms. To examine a possible role(s) of aberrantly expressed microRNAs (miRNAs) in acquired fulvestrant resistance, we compared antiestrogen-resistant and -sensitive breast cancer cells, revealing the over-expression of miR-221/222 in the SERD-resistant cell lines. Fulvestrant treatment of estradiol (E2)- and fulvestrant-sensitive MCF7 cells resulted in increased expression of endogenous miR-221/222. Ectopic upregulation of miR-221/222 in estrogen receptor-α (ERα)-positive cell lines counteracted the effects of E2 depletion or fulvestrant-induced cell death, thus also conferring hormone-independent growth and fulvestrant resistance. In cells with acquired resistance to fulvestrant, miR-221/222 expression was essential for cell growth and cell cycle progression. To identify possible miR-221/222 targets, miR-221- or miR-222- induced alterations in global gene expression profiles and target gene expression at distinct time points were determined, revealing that miR-221/222 overexpression resulted in deregulation of multiple oncogenic signaling pathways previously associated with drug resistance. Activation of β-catenin by miR-221/222 contributed to estrogen-independent growth and fulvestrant resistance, whereas TGF-β-mediated growth inhibition was repressed by the two miRNAs. This first in-depth investigation into the role of miR-221/222 in acquired fulvestrant resistance, a clinically important problem, demonstrates that these two ‘oncomirs’ may represent promising therapeutic targets for treating hormone-independent, SERD-resistant breast cancer.
microRNAs; breast cancer; antiestrogen; drug resistance; fulvestrant
To identify and study targets of microRNA biomarkers of glioblastoma survival across events (death and recurrence) and phases (life expectancy or post-diagnostic) using functional and network analyses.
Materials and Methods
microRNAs associated with glioblastoma survival within and across race, gender, recurrence, and therapy cohorts were identified using 253 individuals, 534 microRNAs, Cox survival model, cross-validation, discriminant analyses, and cross-study comparison.
All 45 microRNAs revealed were confirmed in independent cancer studies and 25 in glioblastoma studies. Thirty-nine and six microRNAs (including hsa-miR-222) were associated with one and multiple glioblastoma survival indicators, respectively. Nineteen and 26 microRNAs exhibited cohort-dependent (including hsa-miR-10b with therapy and hsa-miR-486 with race) and independent associations with glioblastoma, respectively.
Sensory perception and G protein-coupled receptor processes were enriched among microRNA gene targets also associated with survival and network visualization highlighted their relations. These findings can help to improve prognostic tools and personalized treatments.
Glioblastoma; microRNA; biomarkers; hazard; clinical cohort; gender; race
The importance of TNF-alpha in arthritis is well documented. It may be that TNF-alpha is also markedly involved in muscle inflammation (myositis). An animal model where this can be investigated is needed. A newly developed rabbit myositis model involving pronounced muscle overuse and local injections of substances having proinflammatory effects was therefore used in the present study. The aim was to investigate the patterns of TNF-alpha expression in the developing myositis and to evaluate the usefulness of this myositis model for further TNF-alpha research. Human rheumatoid arthritis (RA) synovial tissue was examined as a reference. TNF-alpha immunoexpression and TNF-alpha mRNA, visualized via in situ hybridization, were detected in cells in the inflammatory infiltrates of the affected muscle (soleus muscle). Coexistence of TNF-alpha and CD68 immunoreactions was noted, suggesting that the TNF-alpha reactive cells are macrophages. Expression of TNF-alpha mRNA was also noted in muscle fibers and blood vessel walls in areas with inflammation. These findings demonstrate that TNF-alpha is highly involved in the myositis process. The model can be used in further studies evaluating the importance of TNF-alpha in developing myositis.
MicroRNAs play an important functional role in post-transcriptional gene regulation. One of the largest known microRNA clusters is located within the imprinted Dlk1/Gtl2 region on human chromosome 14 and mouse chromosome 12. This cluster contains more than 40 microRNA genes that are expressed only from the maternal chromosome in mouse.
To shed light on the function of these microRNAs and possible crosstalk between microRNA-based gene regulation and genomic imprinting, we performed extensive in silico analyses of the microRNAs in this imprinted region and their predicted target genes.
Bioinformatic analysis reveals that these microRNAs are highly conserved in both human and mouse. Whereas the microRNA precursors at this locus mostly belong to large sequence families, the mature microRNAs sequences are highly divergent.
We developed a target gene prediction approach that combines three widely used prediction methods and achieved a sufficiently high prediction accuracy. Target gene sets predicted for individual microRNAs derived from the imprinted region show little overlap and do not differ significantly in their properties from target genes predicted for a group of randomly selected microRNAs. The target genes are enriched with long and GC-rich 3' UTR sequences and are preferentially annotated to development, regulation processes and cell communication. Furthermore, among all analyzed human and mouse genes, the predicted target genes are characterized by consistently higher expression levels in all tissues considered.
Our results suggest a complex evolutionary history for microRNA genes in this imprinted region, including an amplification of microRNA precursors in a mammalian ancestor, and a rapid subsequent divergence of the mature sequences. This produced a broad spectrum of target genes. Further, our analyses did not uncover a functional relation between imprinted gene regulation of this microRNA-encoding region, expression patterns or functions of predicted target genes. Specifically, our results indicate that these microRNAs do not regulate a particular set of genes. We conclude that these imprinted microRNAs do not regulate a particular set of genes. Rather, they seem to stabilize expression of a variety of genes, thereby being an integral part of the genome-wide microRNA gene regulatory network.
Human T-cell leukemia virus type I (HTLV-1) can cause an aggressive malignancy known as adult T-cell leukemia/lymphoma (ATL) as well as inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A transgenic mouse that expresses HTLV-1 Tax also develops T-cell leukemia/lymphoma and an inflammatory arthropathy that resembles rheumatoid arthritis. The aim of this study was to identify the primary T-cell subsets involved in the development of arthropathy in Tax transgenic mice.
By 24 months of age, Tax transgenic mice developed severe arthropathy with a cumulative incidence of 22.8%. The pathological findings of arthropathy in Tax transgenic mice were similar to those seen in human rheumatoid arthritis or mouse models of rheumatoid arthritis, with synovial proliferation and a positive rheumatoid factor. Before the onset of spontaneous arthropathy, young and old Tax transgenic mice were not sensitive to collagen and did not develop arthritis after immunization with type II collagen. The arthropathic Tax transgenic mice showed a significantly decreased proportion of splenic CD4+ T cells, whereas the proportion of splenic CD8+ T cells was increased. Regulatory T cells (CD4+CD25+Foxp3+) were significantly decreased and CD8+ T cells that expressed the chemokine receptor CCR4 (CD8+CCR4+) were significantly increased in arthropathic Tax transgenic mice. The expression of tax mRNA was strong in the spleen and joints of arthropathic mice, with a 40-fold increase compared with healthy transgenic mice.
Our findings reveal that Tax transgenic mice develop rheumatoid-like arthritis with proliferating synovial cells in the joints; however, the proportion of different splenic T-cell subsets in these mice was completely different from other commonly used animal models of rheumatoid arthritis. The crucial T-cell subsets in arthropathic Tax transgenic mice appear to resemble those in HAM/TSP patients rather than those in rheumatoid arthritis patients.
To investigate the levels for some specified microRNAs in human’s peripheral blood so as to determine whether they can serve as biomarkers for metastatic non-small-cell lung cancer.
Use a quantitative stem-loop RT-PCR method to examine the serum levels for certain microRNAs including has-miR-125a-5p, has-miR-126, has-miR-183, has-miR-200, has-miR-221, and has-miR-222 from the patients with Stage IV, Stage I/II non-small-cell lung cancer and the controls.
There was statistical difference in the serum levels for hsa-miR-126, hsa-miR-183, and hsa-miR-222 between the controls and the Stage IV patients, but not for has-miR-125a-5p, has-miR-200 and has-miR-221. It also showed statistical difference for hsa-miR-126 and hsa-miR-183 between the Stage I/II patients and Stage IV patients, but not between the controls and Stage I/II patients.
Hsa-miR-126 and hsa-miR-183 may serve as potential serum biomarkers for metastatic non-small-cell lung cancer.
microRNAs; Non-small-cell lung cancer; Stem-loop RT-PCR; Biomarker