Differences In miR Expression Research Articles

MicroRNA expression profiling of endocrine sensitive and resistant breast cancer cell lines

BackgroundMicroRNAs (miRs) regulate gene expression through translation inhibition of target mRNAs. One of the most promising approaches for cancer therapy is through mimicking or antagonizing the action of miRs. In this report, we analyzed the miRnome profile of several human breast cancer cell lines to determine the influence of estrogen receptor (ER) silencing previously shown to result in epithelial to mesenchymal transition (EMT) and enhanced tumor invasion. MethodsMicroRNA extracted from MDA-MB-231 (de novo ER-) and ER-silenced (acquired ER-) pII and IM-26 or ER-expressing (YS1.2) siRNA transfected derivatives of MCF7 cells was deep sequenced on Illumina NextSeq500. Respective miRnomes were compared with edgeR package in R and Venny2.1 and target prediction performed with miRTarBase. Mimics and inhibitors of selected differentially expressed miRs associated with EMT mediators (miR-200c-3p targeting ZEB1, miR-449a targeting δ-catenin and miR-29a-3p) were transfected into pII cells and mRNA targets, as well as E-cadherin and keratin 19 (epithelial and mesenchymal markers respectively) were measured using taqman PCR. ResultsEach cell line expressed about 20% of the total known human miRnome; There was a high degree of similarity between the 3 tested ER-lines. Out of these expressed miRs, 50–60% were significantly differentially expressed between ER- and ER + lines. Transfection of miR-200c-3p mimic into pII cells down regulated ZEB1 and vimentin, and increased E-cadherin and keratin 19 with accompanying morphological changes, and reduced cell motility, reflecting a reversal back into an epithelial phenotype. On the other hand, transfecting pII with miR-449a inhibitor reduced cell invasion but did not induce EMT. Transfecting pII cell line with the mimic or inhibitor of miR-29a-3p showed no change in EMT markers or cell invasion suggesting that the EMT induced by loss of ER function can be reversed by blocking some but not just any random EMT-associated genes. ConclusionsThese data suggest that differences in miR expression can be exploited not only as mediators (using mimics) and targets (using miR antagonists) for general cancer therapies aimed at regulating either individual or multiple mRNAs, but also to re-sensitize endocrine resistant breast cancers by turning them back into a type that will be susceptible to endocrine agents.

Abstract 12403: Type 1 Diabetes Alters Estrogen-Stimulated microRNA Expression and Impairs Nitric Oxide Production in Human Coronary Artery Endothelial Cells

Introduction: Women developed CAD about 1 decade after men. This advantage is erased in T1DM women, and the mechanisms remain unclear. Estrogen (E2) stimulates endothelial NO production via estrogen receptors, and microRNA (miR) have emerged as critical regulators of endothelial function. Whether E2 stimulation of NO production is impaired in T1DM and whether E2 stimulates different miR expression patterns are unknown. Hypothesis: E2-stimulated NO production will be impaired in T1DM endothelial cells which will also express differences in miR expression with E2 exposure. Methods: All experiments were done with and without incubation with 1 nM 17β-estradiol for 24 hours. MicroRNA was isolated from culture human coronary artery endothelial cells (HCAECs) from a woman with and a woman without T1DM. MicroRNA was isolated from HCAECs with four different exposure conditions (T1DM ± 1 nM 17β-estradiol for 24 hr and non-T1DM± 1 nM 17β-estradiol for 24 hr, N=4 biological replicates for all exposures). Fold changes in expression comparing T1DM and non-T1DM cells with and without estradiol stimulation were generated at significance testing performed using DESeq2 with FDR set at 5%. NO production was measured in HCAECs under the same 4 conditions using DAF2-DA and HPLC. Results: 249 miRs were differentially expressed inT1DM HCAECs compared to non-T1DM HCAECs in non-E2 stimulated samples. In E2-stimulated samples, 201 miRs were differentially expressed. Cross-referencing these comparisons showed 16 miRs differentially expressed in T1DM HCAECs relative to non-T1DM HCAECs both with and without E2 stimulation. 4 of these (miRs 548-ar-3p, 625-3p, 369-3p, and 376a-5p) were significantly changed in the opposite direction with estrogen stimulation compared to unstimulated HCAECs, suggesting changes in the expression were due to changes in E2 signaling in T1DM HCAECs. Further, estradiol stimulation of T1DM HCAECS demonstrates a significantly blunted NO production compared to non-T1DM HCAECs (N=6, P<0.05). Conclusions: E2-stimulated NO production is impaired and E2-stimulated changes in miR expression differ in the T1DM endothelium. Further work is necessary to determine whether these differences in miR expression impact E2-stimulated NO production in T1DM.

MicroRNA-155 contributes to plexiform neurofibroma growth downstream of MEK.

MicroRNAs (miRs) are small non-coding RNAs that can have large impacts on oncogenic pathways. Possible functions of dysregulated miRs have not been studied in neurofibromatosis type 1 (NF1) plexiform neurofibromas (PNFs). In PNFs, Schwann cells (SCs) have biallelic NF1 mutations necessary for tumorigenesis. We analyzed a miR-microarray comparing to normal and PNF SCs and identified differences in miR expression, and we validated in mouse PNFs versus normal mouse SCs by qRT-PCR. Among these, miR-155 was a top overexpressed miR, and its expression was regulated by RAS/MAPK signaling. Overexpression of miR-155 increased mature Nf1−/− mouse SC proliferation. In SC precursors, which model tumor initiating cells, pharmacological and genetic inhibition of miR-155 decreased PNF-derived sphere numbers in vitro and we identified Maf as a miR-155 target. In vivo, global deletion of miR-155 significantly decreased tumor number and volume, increasing mouse survival. Fluorescent nanoparticles entered PNFs, suggesting that an anti-miR might have therapeutic potential. However, treatment of established PNFs using anti-miR-155 peptide nucleic acid-loaded nanoparticles marginally decreased tumor numbers and did not reduce tumor growth. These results suggest that miR-155 plays a functional role in PNF growth and/or SC proliferation, and that targeting neurofibroma miRs is feasible, and might provide novel therapeutic opportunities.

Tissue microRNA expression profiling in hepatic and pulmonary metastatic melanoma.

Malignant melanoma has a propensity for the development of hepatic and pulmonary metastases. MicroRNAs (miRs) are small, noncoding RNA molecules containing about 22 nucleotides that mediate protein expression and can contribute to cancer progression. We aim to identify clinically useful differences in miR expression in metastatic melanoma tissue. RNA was extracted from formalin-fixed, paraffin-embedded samples of hepatic and pulmonary metastatic melanoma, benign, nevi, and primary cutaneous melanoma. Assessment of miR expression was performed on purified RNA using the NanoString nCounter miRNA assay. miRs with greater than twofold change in expression when compared to other tumor sites (P value ≤ 0.05, modified t-test) were identified as dysregulated. Common gene targets were then identified among dysregulated miRs unique to each metastatic site. Melanoma metastatic to the liver had differential expression of 26 miRs compared to benign nevi and 16 miRs compared to primary melanoma (P < 0.048). Melanoma metastatic to the lung had differential expression of 19 miRs compared to benign nevi and 10 miRs compared to primary melanoma (P < 0.024). Compared to lung metastases, liver metastases had greater than twofold upregulation of four miRs, and 4.2-fold downregulation of miR-200c-3p (P < 0.0081). These findings indicate that sites of metastatic melanoma have unique miR profiles that may contribute to their development and localization. Further investigation of the utility of these miRs as diagnostic and prognostic biomarkers and their impact on the development of metastatic melanoma is warranted.

Nicotine Changes the microRNA Profile to Regulate the FOXO Memory Program of CD8+ T Cells in Rheumatoid Arthritis.

Objective: Smoking suppresses PD-1 expression in patients with rheumatoid arthritis (RA). In this study, we assess if smoking changed the epigenetic control over CD8+ T cell memory formation through a microRNA (miR) dependent mechanism.Methods: Phenotypes of CD8+ T cells from smokers and non-smokers, RA and healthy, were analyzed by flow cytometry. A microarray analysis was used to screen for differences in miR expression. Sorted CD8+ cells were in vitro stimulated with nicotine and analyzed for transcription of miRs and genes related to memory programming by qPCR.Results: CD27+CD107a−CD8+ T cells, defining a naïve-memory population, had low expression of PD-1. Additionally, the CD27+ population was more frequent in smokers (p = 0.0089). Smokers were recognized by differential expression of eight miRs. Let-7c-5p, let-7d-5p and let-7e-5p, miR-92a-3p, miR-150-5p, and miR-181-5p were up regulated, while miR-3196 and miR-4723-5p were down regulated. These miRs were predicted to target proteins within the FOXO-signaling pathway involved in CD8+ memory programming. Furthermore, miR-92a-3p was differentially expressed in CD8+ cells with naïve-memory predominance. Nicotine exposure of CD8+ cells induced the expression of miR-150-5p and miR-181a-5p in the naïve-memory cells in vitro. Additionally, nicotine exposure inverted the ratio between mRNAs of proteins in the FOXO pathway and their targeting miRs.Conclusions: Smokers have a high prevalence of CD8+ T cells with a naïve-memory phenotype. These cells express a miR profile that interacts with the memory programming conducted through the FOXO pathway.

MiR-744-5p contributes to ocular inflammation in patients with primary Sjogrens Syndrome

In primary Sjögren’s syndrome (pSS) the exocrine glands become infiltrated with lymphocytes instigating severe damage to the salivary and lacrimal glands causing dry eyes and dry mouth. Previous investigations have suggested that dysregulated localized and systemic inflammation contributes to the development and pathogenesis of pSS. A miR microarray performed in primary human conjunctival epithelial cells (PECs) demonstrated significant differences in miR expression at the ocular surface between pSS patients and healthy controls. MicroRNA-744-5p (miR-744-5p) was identified as being of particular interest, as its top predicted target is Pellino3 (PELI3), a known negative regulator of inflammation. Validation studies confirmed that miR-744-5p expression is significantly increased in PECs from pSS patients, whilst PELI3 was significantly reduced. We validated the miR-744 binding site in the 3’ untranslated region (UTR) of PELI3 and demonstrated that increasing PELI3 levels with a miR-744-5p antagomir in an inflammatory environment resulted in reduced levels of IFN dependent chemokines Rantes (CCL5) and CXCL10. These results reveal a novel role for miR-744-5p in mediating ocular inflammation via Pellino3 expression in pSS patients and suggest that miR-744-5p may be a potential therapeutic target for the management of severe dry eye disease and ocular inflammation in pSS patients.

X-chromosome-linked miR548am-5p is a key regulator of sex disparity in the susceptibility to mitochondria-mediated apoptosis

Sex dimorphism in cell response to stress has previously been investigated by different research groups. This dimorphism could be at least in part accounted for by sex-biased expression of regulatory elements such as microRNAs (miRs). In order to spot previously unknown miR expression differences we took advantage of prior knowledge on specialized databases to identify X chromosome-encoded miRs potentially escaping X chromosome inactivation (XCI). MiR-548am-5p emerged as potentially XCI escaper and was experimentally verified to be significantly up-regulated in human XX primary dermal fibroblasts (DFs) compared to XY ones. Accordingly, miR-548am-5p target mRNAs, e.g. the transcript for Bax, was differently modulated in XX and XY DFs. Functional analyses indicated that XY DFs were more prone to mitochondria-mediated apoptosis than XX ones. Experimentally induced overexpression of miR548am-5p in XY cells by lentivirus vector transduction decreased apoptosis susceptibility, whereas its down-regulation in XX cells enhanced apoptosis susceptibility. These data indicate that this approach could be used to identify previously unreported sex-biased differences in miR expression and that a miR identified with this approach, miR548am-5p, can account for sex-dependent differences observed in the susceptibility to mitochondrial apoptosis of human DFs.

236: Micro RNA expression profile in pregnant women complicated fetuses with muscular ventricular septal defects

Ventricular septal defects (VSD) account for 20% of congenital heart defects (CHDs) and the pathogenesis and molecular mechanism have not been elucidated. MicroRNAs (miRs) are small single-stranded, non-coding RNA molecules that regulate gene expression at the post-transcriptional level. Recent research demonstrated that miRs help regulate embryonic cardiac development, cardiac mophogenesis, and cardiomyocyte growth and differentiation and are closely related to the occurrence of CHDs. This study aimed to measure differences in miR expression in maternal serum in patients complicated with fetal muscular VSD. This was a prospective study including fetuses at risk for CHD (pregestational diabetes, anticonvulsant use, or history of cardiac defects) during 2nd trimester echocardiographic examination. Fetuses without cardiac abnormalities were marked as control. Fetuses with isolated mVSD were marked as abnormal. Maternal venous serum was collected, RNA extracted, and an expression profile of 797 human miRs was measured using NanoString nCounter technology as counts (molecules/100ng RNA). Expression levels of >2fold increase or <50% decrease compared to control were considered up- or downregulated, respectively. Placental-derived miRs within the entire expression profile were also evaluated separately. miRs profiling was obtained in 6 controls and 7 mVSDs cases. Expression levels ranged from 3-198 counts, indicating relatively low expression. The median count did not vary between normal (20.7) and mVSD (21.4). Of the 797, 12 miRs were significantly upregulated (Fig. 1) and 36 miRs downregulated (P<0.01 for both). There were no differences in expression among the 114 known placentally-derived miRs between groups. This study identifies a select serum miR expression profile in women with mVSD fetuses in the 2nd trimester that differs from women with normal fetuses. This approach may provide a quantitative screening tool for confirming fetal CHDs diagnosed by echocardiography. This will be valuable as a biomarker approach for early detection of fetal CHDs in the first trimester to aid diagnosis by fetal echocardiography.

Alterations in patient plasma microRNA expression profiles following resection of metastatic melanoma.

MicroRNAs (miRs) are noncoding RNAs that regulate protein translation and melanoma progression. Changes in plasma miR expression following surgical resection of metastatic melanoma are under-investigated. We hypothesize differences in miR expression exist following complete surgical resection of metastatic melanoma. Blood collection pre- and post-surgical resection was performed in six individuals with solitary melanoma metastases. miR expression in extracted RNA was quantified using the NanoString nCounter Digital Analyzer. Pre- and post-surgical plasma samples contained 216 miRs with expression above baseline. Comparison of postsurgical to preresection samples revealed differential expression of 25 miRs: miR-let-7a, miR-let7g, miR-15a, miR-16, miR-22, miR-30b, miR-126, miR-140, miR-145, miR-148a, miR-150-5p, miR-191, miR-378i, miR-449c, miR-494, miR-513b, miR-548aa, miR-571, miR-587, miR-891b, miR-1260a, miR 1268a, miR-1976, miR-4268, miR-4454 (P < 0.05). Utilizing P < 0.0046 as a cutoff to control for one false positive among the 216 miRs revealed that postsurgical melanoma plasma samples had upregulation of miR-1260a (P = 0.0007) and downregulation of miR-150-5p (P = 0.0026) relative to pre-surgical samples. Differential expression of miR-150-5p and miR-1260a is present in plasma following surgical resection of metastatic melanoma in this small sample (n = 6) of melanoma patients. Therefore, further investigation of these plasma miRs as noninvasive biomarkers for melanoma is warranted.

Comparison of left versus right atrial myocardium in patients with sinus rhythm or atrial fibrillation - an assessment of mitochondrial function and microRNA expression.

Several of the cellular alterations involved in atrial fibrillation (AF) may be linked to mitochondrial function and altered microRNA (miR) expression. A majority of studies on human myocardium involve right atrial (RA) tissue only. There are indications that AF may affect the two atria differentially. This study aimed to compare interatrial differences in mitochondrial respiration and miR expression in the RA versus left atrium (LA) within patients with sinus rhythm (SR) and AF. Thirty‐seven patients with AF (n = 21) or SR (n = 16), undergoing coronary artery bypass surgery and/or heart valve surgery, were included. Myocardial biopsies were obtained from RA and LA appendages. Mitochondrial respiration was assessed in situ in permeabilized myocardium. MiR array and real‐time quantitative polymerase chain reaction were performed to evaluate miR expression. Mitochondrial respiratory rates were similar in RA versus LA. Expression of miR‐100, ‐10b, ‐133a, ‐133b, ‐146a, ‐155, ‐199a‐5p, ‐208b, and ‐30b were different between the atria in both SR and AF patients. In contrast, differential expression was observed between RA versus LA for miR‐93 in patients with SR only, and for miR‐1, ‐125b, ‐142‐5p, ‐208a, and ‐92b within AF patients only. These results indicate that mitochondrial respiratory capacity is similar in the RA and LA of patients with SR and AF. Differences in miR expressional profiles are observed between the RA versus LA in both SR and AF, and several interatrial differences in miR expression diverge between SR and AF. These findings may contribute to the understanding of how AF pathophysiology may affect the two atria differently.

MicroRNA profiling in ocular adnexal extranodal marginal zone lymphoma and diffuse large B‐cell lymphoma

AbstractPurpose To investigate changes in microRNA (miR) expression in ocular adnexal extranodal marginal zone lymphoma (EMZL) and diffuse large B‐cell lymphoma (DLBCL), and to perform a pathway analysis of differentially expressed miRs.Methods Global miR expression profiling was performed using miRCURYTM LNA miR‐arrays, v. 11.0, (Exiqon) on formalin‐fixed paraffin‐embedded tissue from 18 ocular adnexal EMZLs and 25 DLBCLs. The result of microarray was confirmed using quantitative real time (RT)‐PCR. Predicted and validated targets of the significantly differentially expressed miRs were obtained from MiR‐walk. Transcription factor (TF)‐miR interactions were obtained from the TransmiR database.Results The microarray results revealed two up‐regulated and 41 down‐regulated miRs in the DLBCLs compared to the EMZLs. Further, the differentially expressed miRs separated EMZLs and DLBCLs into two distinct entities using a supervised clustering analysis. These findings were confirmed by quantitative RT‐PCR. Five of the down‐regulated miRs (let‐7g, miR‐26a, miR‐29a, miR‐29c and miR‐221) were identified to be regulated negatively by MYC. A small set of miRs (let‐7g, miR‐199a‐5, miR‐26a, miR‐27a* and miR‐342‐3p) that regulate NFKB1 were all down‐regulated in the DLBCLs compared to the EMZL samples. Further, the NFKB1 regulated miRs (miR‐29a/b/c and miR‐125b) were down‐regulated significantly in the DLBCLs compared with the EMZLs (log2 fold‐changes: miR‐29a/b/c &lt; ‐1.9 and ‐1.1 for miR‐125b).Conclusion We demonstrated that fundamental differences in miR expression exist between ocular adnexal EMZLs and DLBCLs, mainly due to differences in MYC and NF‐kB regulatory pathways.

Circulating micro‐RNA expression profiles in early stage nonsmall cell lung cancer

Circulating micro-RNA (miR) profiles have been proposed as promising diagnostic and prognostic biomarkers for cancer, including lung cancer. We have developed methods to accurately and reproducibly measure micro-RNA levels in serum and plasma. Here, we study paired serum and plasma samples from 220 patients with early stage nonsmall cell lung cancer (NSCLC) and 220 matched controls. We use qRT-PCR to measure the circulating levels of 30 different miRs that have previously been reported to be differently expressed in lung cancer tissue. Duplicate RNA extractions were performed for 10% of all samples, and micro-RNA measurements were highly correlated among those duplicates. This demonstrates high reproducibility of our assay. The expressions of miR-146b, miR-221, let-7a, miR-155, miR-17-5p, miR-27a and miR-106a were significantly reduced in the serum of NSCLC cases, while miR-29c was significantly increased. No significant differences were observed in plasma of patients compared with controls. Overall, expression levels in serum did not correlate well with levels in plasma. In secondary analyses, reduced plasma expression of let-7b was modestly associated with worse cancer-specific mortality in all patients, and reduced serum expression of miR-223 was modestly associated with cancer-specific mortality in stage IA/B patients. MiR profiles also showed considerable differences comparing African American and European Americans. In summary, we found significant differences in miR expression when comparing cases and controls and find evidence that expression of let-7b is associated with prognosis in NSCLC.

Abstract 3036: Comparison of miRNA expression between human osteoblasts and osteosarcoma cells

Abstract Osteosarcoma is the most common primary malignant bone tumor that most frequently affects young adults. There is increasing evidence that miRNAs (miRs) are important modulators of gene expression and their deregulation has been reported in various human tumors. This study investigated differences in miR expression in normal human osteoblast cells versus malignant human osteosarcoma cells differ in their miR expression. Analysis of miRNA expression in normal osteoblast cell lines (hOBc, NHOST, hFOB) and osteosarcoma cell lines (KHOS, Saos, U-2OS) were completed using miR microarray technology with unsupervised hierarchical clustering analysis. The relative expression levels of these miRs were confirmed by real-time quantitative RT-PCR. A number of miRs were found to have different expression in the osteosarcoma cells when compared to normal human osteoblasts. Three miRs, miR-199a-3p, miR-127-3p and miR-376c were significantly decreased in osteosarcoma cell lines while miR-151-3p and miR-191 were increased in osteosarcoma cell lines in comparison to osteoblasts. Because miR-199a-3p has been suggested to play a vital role in lung and liver cancer, we further characterized its biological functional role in osteosarcomas. The precursor (miR-199a-1) of miR-199a-3p was stably transfected into osteosarcoma cell lines KHOS and U-2OS, resulting in a decrease of cellular proliferation. The expression of miR-199a-3p targeted genes in transfected cells was evaluated by Western blot. These targets included MET and mTOR, two genes that were observed to be overexpressed in osteosarcoma tissues and various cell lines. Following miR-199a-3p transfection, a decrease in MET and mTOR expression was observed in KHOS and U-2OS cells. This study demonstrates that various miRs are differentially expressed in osteosarcomas, and miR-199a-3p, in particular may play a functional role in osteosarcoma cell growth and proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3036.

Development of a micro-array to detect human and mouse microRNAs and characterization of expression in human organs

MicroRNAs (miRNAs) are believed to play important roles in developmental and other cellular processes by hybridizing to complementary target mRNA transcripts. This results in either cleavage of the hybridized transcript or negative regulation of translation. Little is known about the regulation or pattern of miRNA expression. The predicted presence of numerous miRNA sequences in higher eukaryotes makes it highly likely that the expression levels of individual miRNA molecules themselves should play an important role in regulating multiple cellular processes. Therefore, determining the pattern of global miRNA expression levels in mammals and other higher eukaryotes is essential to help understand both the mechanism of miRNA transcriptional regulation as well as to help identify miRNA regulated gene expression. Here, we describe a novel method to detect global processed miRNA expression levels in higher eukaryotes, including human, mouse and rats, by using a high-density oligonucleotide array. Array results have been validated by subsequent confirmation of mir expression using northern-blot analysis. Major differences in mir expression have been detected in samples from diverse sources, suggesting highly regulated mir expression, and specific gene regulatory functions for individual miRNA transcripts. For example, five different miRNAs were found to be preferentially expressed in human kidney compared with other human tissues. Comparative analysis of surrounding genomic sequences of the kidney-specific miRNA clusters revealed the occurrence of specific transcription factor binding sites located in conserved phylogenetic foot prints, suggesting that these may be involved in regulating mir expression in kidney.