Post-transcriptional Down-regulation Research Articles

Role of Small RNAs in Trypanosomatid Infections.

Trypanosomatid parasites survive and replicate in the host by using mechanisms that aim to establish a successful infection and ensure parasite survival. Evidence points to microRNAs as new players in the host-parasite interplay. MicroRNAs are small non-coding RNAs that control proteins levels via post-transcriptional gene down-regulation, either within the cells where they were produced or in other cells via intercellular transfer. These microRNAs can be modulated in host cells during infection and are among the growing group of small regulatory RNAs, for which many classes have been described, including the transfer RNA-derived small RNAs. Parasites can either manipulate microRNAs to evade host-driven damage and/or transfer small RNAs to host cells. In this mini-review, we present evidence for the involvement of small RNAs, such as microRNAs, in trypanosomatid infections which lack RNA interference. We highlight both microRNA profile alterations in host cells during those infections and the horizontal transfer of small RNAs and proteins from parasites to the host by membrane-derived extracellular vesicles in a cell communication mechanism.

MiR-1180 promotes apoptotic resistance to human hepatocellular carcinoma via activation of NF-κB signaling pathway.

Apoptosis resistance in human hepatocellular carcinoma (HCC) is a significant factor in carcinogenesis. Therefore, understanding the molecular mechanisms involved in apoptosis resistance is crucial for developing anticancer therapies. Importantly, small non-coding microRNAs (miRNAs) have been reported as key biomarkers for detecting tumour onset and progression. In the present study, we demonstrate that miR-1180 is upregulated in HCC. Ectopic expression of miR-1180 has an anti-apoptotic effect in HCC, while miR-1180 inhibition increases cell apoptosis, both in vitro and in vivo. Moreover, our results show that miR-1180 directly targets key inhibitors of the nuclear factor (NF)-κB signaling pathway (i.e., OTUD7B and TNIP2) and the pro-apoptotic Bcl-2 associated death promoter (BAD) protein by post-transcriptional downregulation. Therefore, the anti-apoptotic function of miR-1180 in HCC may occur through NF-κB pathway activation via downregulation of its negative regulators. In conclusion, our study reveals the critical role of miR-1180 during apoptosis resistance in HCC.

Multiple microRNA regulation of lipoprotein lipase gene abolished by 3′UTR polymorphisms in a triglyceride-lowering haplotype harboring p.Ser474Ter

BackgroundLipoprotein lipase (LPL) is a key enzyme in triglyceride (TG) metabolism. LPL gene single nucleotide polymorphisms (SNPs) are associated with TG concentrations however the functionality of many of these SNPs remains poorly understood. MicroRNAs (miR) exert post-transcriptional down-regulation and their target sequence on the 3′UTR may be altered by SNPs. We therefore investigated whether LPL 3′UTR SNPs could modulate plasma TG concentration through the alteration of miR binding-sites. Methods and resultsWe performed genetic association studies of LPL 3′UTR SNPs with TG concentrations in 271 type 2 diabetic patients and in general population samples (2997 individuals). A specific LPL haplotype (Hap4) was associated with lower plasma TG concentration (TG-0.18, IC95% [−0.30, −0.07] mmol/L or logTG-0.13, IC95% [−0.18, −0.08], p = 4.77·10−8) in the meta-analysis. Hap4 comprises seven 3′UTR SNP minor alleles and p.Ser474Ter (rs328) a well-documented nonsense mutation associated with low TG concentration although by an unknown mechanism so far. Bio-informatic studies identified several putative miRNA binding-sites on the wild-type Hap1 haplotype, lost on Hap4. Functional validation performed in HEK-293T cells using luciferase expression constructs with various LPL 3′UTR allele combinations demonstrated a binding of miR-29, miR-1277 and miR-410 on Hap1, lost on Hap4. This loss of specific miR binding-site in presence of Hap4 was independent of the allelic variation of p.Ser474Ter (rs328). ConclusionsWe report the regulation of LPL by the miR-29, miR-1277 and miR-410 that is lost in presence of Hap4, a specific LPL TG-lowering haplotype. Consequently p.Ser474Ter association with TG concentration could be at least partially explained by its strong linkage disequilibrium with these functional 3′UTR SNPs.

Epigenetics in oral cancer-neoteric biomarker

A cancer develops when a cell acquires specific growth advantages through the stepwise accumulation of heritable changes in gene function. Cancer genes may be changed by several mechanisms, which potentially alter the protein encoding nucleotide template, change the copy number of genes or leads to increased gene transcription. Epigenetic alterations are increasingly being recognized for their roles in carcinogenesis. Deregulation of gene expression is a hallmark of cancer. Although genetic lesions have been the focus of cancer research for many years, it has become increasingly recognized that aberrant epigenetic modifications also play major roles in the tumorigenic process. These modifications imposed on chromatin, do not change the nucleotide sequence of DNA, and are manifested by specific patterns of gene expression. The field of cancer epigenetics is evolving rapidly on several fronts. Advances in our understanding of chromatin structure, histone modification, and transcriptional activity and DNA methylation have resulted in an increasingly integrated view of epigenetics. Whilst genetic alterations in oral cancer have long been documented, the appreciation of epigenetic changes is more recent. Epigenetic changes alter expression of tumour suppressor genes without changes in DNA sequence. Epigenetic mechanisms such as DNA methylation, histone methylation and deacetylation have been shown to silence key genes involved in cell proliferation, differentiation and genome integrity and clearly have a central role in oral cancer. Epigenetics is another major player in multistep carcinogenesis of oral cancers. In this article we discuss current literature in the field of the epigenetics of oral cancer, placing a great deal of emphasis on DNA methylation, histone modification and post-transcriptional gene down-regulation by microRNAs.

Altered Expression/Activity of Cytochrome P450 (CYP) 3A4 Enzyme: Implications in Drug Safety and Efficacy

Cytochrome P450 (CYP) 3A4 enzyme alterations during diseases. Transcriptional or post-transcriptional down-regulation of hepatic CYP enzymes is a characteristic feature of many diseases including infections; cancer, cardiovascular diseases and liver disorders [1-5]. This disrupts CYP-mediated drug metabolism and clearance in these patients. The major drug metabolizing enzyme (DME) in human liver is CYP3A4, which metabolizes ~60% of drugs [6,7]. Recent studies in pediatric patients show that critical illness is the primary determinant of the clearance of the CYP3A4 substrate, midazolam [8]. Furthermore, diabetic kidney transplant patients had low metabolism of the immunosuppressant, cyclosporine A, likely due to reduced CYP3A4 expression [9]. These clinical observations warrant further studies to understand the molecular mechanism by which human CYP enzymes are down-regulated during diseases.

Abstract 181: miR-134 targets programmed cell death 7 (PDCD7) gene to modulate the pathogenesis of head and neck carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is one of the prevalent malignancies in the world. microRNAs (miRNAs) are involved in the neoplastic process of various malignancies including HNSCC to mediate post-transcriptional down-regulation of target genes. This study shows that miR-134 enhances the proliferation, migration and invasion of HNSCC cells. In silico modules predict that programmed cell death 7 (PDCD7) gene could be one of the targets of miR-134. In stable HNSCC cell subclones, ectopic miR-134 expression decreased PDCD7 expression; while the ectopic miR-134Zip expression increased PDCD7 expression. Reporter assays confirmed the binding of miR-134 to the wild type 3′UTR of PDCD7, which decreased reporter activity. The targeting of miR-134 to the 3′UTR of PDCD7 was abolished by the mutations in targeted sequences. Moreover, miR-134Zip expression increased the activity of wild type reporter. Down-regulation of PDCD7 was identified in around 70% of HNSCC tumors, which was significantly opposite to miR-134 expression. A trend of reverse expression between miR-134 and PDCD7 was also present in HNSCC cell lines. PDCD7 was up-regulated following the apoptosis induced by cis-platin. Ectopic PDCD7 expression decreased the proliferation and migration of HNSCC cells, but it was unable to affect apoptosis or drug resistance status. Moreover, miR-134 associated oncogenicity was attenuated by PDCD7 expression. Taking together, this study concludes that the oncogenic potential of miR-134 in head and neck carcinoma is able to be mediated by targeting PDCD7. Citation Format: Shih-Yuan Peng, Kuo-Wei Chang, Shu-Chun Lin, Hsi-Feng Tu. miR-134 targets programmed cell death 7 (PDCD7) gene to modulate the pathogenesis of head and neck carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 181. doi:10.1158/1538-7445.AM2015-181

MiR-204 mediates post-transcriptional down-regulation of PHOX2B gene expression in neuroblastoma cells

Neuroblastoma (NB) is a rare childhood cancer of the peripheral sympathetic nervous system and accounts for approximately 10% of all pediatric tumors. Heterozygous PHOX2B mutations have been found in association with NB development in familial, sporadic and syndromic cases. In addition, the PHOX2B gene is widely over-expressed both in tumor samples and NB cell lines. Post-transcriptional gene regulation is known to be involved in mRNA stability and, in NB, microRNAs (miRNAs) seem to be responsible for altered expression of genes driving differentiation, apoptosis, and migration. To assess the possible impact of post-transcriptional regulation in NB cell lines, we have focused on the PHOX2B mRNA stability by both in silico analysis and functional studies on its 3′untranslated region (3′UTR). PHOX2B gene expression has resulted under post-transcriptional control, as suggested by: i) instability of PHOX2B mRNA, demonstrated by short mRNA half-life levels in both IMR32 and LAN-1 cell lines, ii) role of the PHOX2B-3′UTR, confirmed by the activity of proper reporter constructs, and iii) miRNA-204, shown to enhance the PHOX2B 3′UTR mediated down-regulation of the reporter construct activity. Finally, miRNA-204 has resulted to decrease the stability of the PHOX2B mRNA at different extents in the presence of different SNP rs1063611 alleles. Therefore, post-transcriptional down-regulation of the PHOX2B gene takes place in NB cell lines and miRNA-204 participates in such a 3′UTR mediated control.

MicroRNA hsa-miR-4717-5p regulates RGS2 and may be a risk factor for anxiety-related traits.

Regulator of G-protein Signaling 2 (RGS2) is a key regulator of G-protein-coupled signaling pathways involved in fear and anxiety. Data from rodent models and genetic analysis of anxiety-related traits and disorders in humans suggest down-regulation of RGS2 expression to be a risk factor for anxiety. Here we investigated, whether genetic variation in microRNAs mediating posttranscriptional down-regulation of RGS2 may be a risk factor for anxiety as well. 75 microRNAs predicted to regulate RGS2 were identified by four bioinformatic algorithms and validated experimentally by luciferase reporter gene assays. Specificity was confirmed for six microRNAs (hsa-miR-1271-5p, hsa-miR-22-3p, hsa-miR-3591-3p, hsa-miR-377-3p, hsa-miR-4717-5p, hsa-miR-96-5p) by disrupting their seed sequence at the 3' untranslated region of RGS2. Hsa-miR-4717-5p showed the most robust effect on RGS2 and regulated two other candidate genes of anxiety disorders (CNR1 and IKBKE) as well. Two SNPs (rs150925, rs161427) within and 1,000 bp upstream of the hostgene of hsa-miR-4717-5p (MIR4717) show a minor allele frequency greater than 0.05. Both were in high linkage disequilibrium (r(2) = 1, D' = 1) and both major (G) alleles showed a trend for association with panic disorder with comorbid agoraphobia in one of two patient/control samples (combined n(patients) = 497). Dimensional anxiety traits, as described by Anxiety Sensitivity Index (ASI) and Agoraphobic Cognitions Questionnaire (ACQ) were significantly higher among carriers of both major (G) alleles in a combined patient/control sample (n(combined) = 831). Taken together, data indicate that MIR4717 regulates human RGS2 and contributes to the genetic risk towards anxiety-related traits.

Micro RNA-214 contributes to proteasome independent downregulation of beta catenin in Huntington's disease knock-in striatal cell model STHdhQ111/Q111

Role of beta catenin in Huntington's disease (HD) is not clear. Previous studies on HD reported varied levels of beta catenin. In the present study we showed that beta catenin is post transcriptionally down-regulated in mutant huntingtin knock-in cell model STHdhQ111/Q111. This in turn leads to decreased level of wnt/beta catenin responsive genes. We observed that Gsk3beta or Gsk3beta (phospho Ser 9) is unaltered in HD and this down-regulation of beta catenin is independent of proteasomal degradation. Finally, we showed that the overexpression of miR-214 leads to the down-regulation of beta catenin at protein level only and reduces its transcriptional activity. We concluded that, miR-214 contributes to the processes that result in proteasome independent post transcriptional down-regulation of beta catenin in STHdhQ111/Q111, probably through inhibition of protein synthesis from beta catenin mRNA.

Abstract B45: A search for ideal siRNA targets involved in pathway cross-talks for combinational silencing in human cancer cells

Abstract The heterogeneity in the pathways involved in enhanced cell proliferation and survival mechanisms, as well as the mechanisms playing a major role in development of drug resistance, is an important obstacle in cancer treatment. Signaling axes such as PI3K-AKT, Ras-Raf, MEK-ERK, and JAK-STAT pathways have not only been established as major processes involved in enhanced proliferation and activation of the transcription of multiple anti-apoptosis proteins, but are also shown to be interconnected in forming a vast intracellular signaling network. RNA interference, and more specifically, small interfering RNA (siRNA), is a post-transcriptional down-regulation of the expression of a specific protein, and has been studied extensively in the last decade as not only an investigational tool, but also as a therapeutic approach especially in cancer treatment. In the present study, we undertook a systematic approach to simultaneous silencing of two proteins involved in intracellular signaling network in order to inhibit more than one pathway involved in proliferation and survival of cancer cells. After carefully selecting the proteins with pivotal roles in cell survival through diverse pathways, we studied silencing each protein individually and in all possible dual combinations, and evaluated the cell response as the mRNA level of the selected proteins as well as the viable cell number. Our studies reveled that silencing JAK2, STAT3, and JUN have a significant effect on the expression level of anti-apoptotic proteins, e.g., Mcl-1 and survivin, and could negatively impact the survival of MDA435 cells. These results indicate a promising potential for combinational siRNA silencing as an effective anticancer strategy. Citation Format: Hamidreza Montazeri Aliabadi, Parvin Mahdipoor, Hasan Uludag. A search for ideal siRNA targets involved in pathway cross-talks for combinational silencing in human cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B45.

The Pim Kinase Inhibitor AZD1208 Enhances Apoptosis Induction By Clinically Active FLT3 Inhibitors in FLT3-ITD Acute Myeloid Leukemia Cells in Vitro and in Vivo through Synergistic Downregulation of Mcl-1 and of Bcl-xL

Internal tandem duplication (ITD) mutations of the receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3) are present in acute myeloid leukemia (AML) cells in 30% of cases and are associated with high relapse rate and short disease-free survival. FLT3 inhibitors have clinical activity, but their activity is limited and transient. New therapeutic approaches combining FLT3 inhibitors and inhibitors of downstream or parallel signaling pathways may increase depth and duration of responses.The Pim-1 serine/threonine kinase is transcriptionally upregulated by FLT3-ITD. We previously demonstrated that Pim-1 phosphorylates and stabilizes FLT3 and thereby promotes its signaling in a positive feedback loop. Pim kinase inhibitors are in clinical trials. Here we studied the effect of combinations of the Pim kinase inhibitor AZD1208 and clinically active FLT3 inhibitors on AML with FLT3-ITD in vitro and in vivo.Ba/F3-ITD cells, with FLT3-ITD, were grown in medium with the Pim kinase inhibitor AZD1208 at 1 μM and/or the FLT3 inhibitors quizartinib (Q), sorafenib (S) or crenolanib (C) at their IC50values of 1, 2.5 and 20 nM, respectively, and viable cells were measured at serial time points. While Q, S, C or AZD1208 treatments reduced cell numbers, compared to DMSO control, combined AZD1208 and Q, S or C treatments abrogated proliferation. Because FLT3-ITD cells remain responsive to FLT3 ligand (FLT3L) despite constitutive FLT3 activation and increased FLT3L levels following chemotherapy have been hypothesized to contribute to relapse, we repeated the proliferation experiments in the presence of 0, 1, 3 and 10 ng/ml FLT3L. FLT3L produced a concentration-dependent increase in proliferation and, while Q, S, C or AZD1208 treatments individually reduced cell numbers, combined AZD1208 and Q, S or C abrogated proliferation at all FLT3L concentrations tested, suggesting that these combinations overcome growth stimulation by FLT3L.To understand the anti-proliferative effect of combined Pim-1 and FLT3 inhibitors, we first studied cell cycle effects of AZD1208 and Q, S or C in Ba/F3-ITD cells and of AZD1208 and Q in the additional FLT3-ITD cell lines 32D-ITD, MV4-11 and MOLM14. We found a progressive increase in sub-G1 phase cells at 24, 48 and 72 hours, consistent with induction of apoptosis. Synergistic induction of apoptosis was confirmed by Annexin V/propidium iodide labeling of Ba/F3-ITD and 32D-ITD cells treated for 48 hours with AZD1208 combined with Q (p<0.0001), S (p<0.0001) or C (p<0.001), and of MV4-11 (p<0.0001) and MOLM14 (p<0.05) cells treated with AZD1208 combined with Q, in relation to each drug alone. Apoptosis was additionally confirmed by loss of mitochondrial membrane potential. Synergistic induction of apoptosis was not seen in Ba/F3-WT or 32D-WT cells, with wild-type FLT3, indicating a FLT3-ITD-specific effect. Synergistic (p<0.01) induction of apoptosis was seen in three FLT3-ITD AML patient samples treated in vitro with AZD1208 combined with Q. In an in vivo model, synergistic decrease in tumor volume was seen with combined AZD1208 and Q therapy in mice with subcutaneously implanted MV4-11 cells, with FLT3-ITD, but not with KG1a cells, with wild-type FLT3.Mechanistically, combined AZD1208 and Q treatment in vitro did not increase reactive oxygen species, compared to each drug alone, but increased both cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP) levels, and caspase 3 cleavage was reduced by co-incubation with the pan-caspase inhibitor Z-VAD. Moreover, combined AZD1208 and Q treatment caused a synergistic decrease in expression of the anti-apoptotic Mcl-1 and of Bcl-xL proteins, but did not significantly alter Bim-1, p-Bad, Bad, Bax, Bak or Bcl-2, pro- and anti-apoptotic protein levels. Bcl-xL mRNA expression decreased along with protein levels, but Mcl-1 mRNA levels remain unchanged, indicating post-transcriptional down-regulation of Mcl-1 by the combination treatment.In summary, synergistic cytotoxicity of AZD1208 and clinically active FLT3 inhibitors was demonstrated in FLT3-ITD cell lines and patient samples in vitro and in cell lines in vivo, via caspase-mediated apoptosis, associated with a synergistic decrease in Mcl-1 and Bcl-xL expression. Our data suggest clinical promise for combination therapy with Pim kinase and FLT3 inhibitors in patients with AML with FLT3-ITD. DisclosuresNo relevant conflicts of interest to declare.

Prediction of siRNA Efficacy Using BP Neural Network and Support Vector Machine

RNA interference (RNAi) is a mechanism for sequence-specific, post-transcriptional down-regulation of gene expression. The success of RNAi gene silencing depends on siRNA feature design. The shortcoming of previously reported methods which design siRNA sequences based on limited rules is that they are difficult to accurately predict the efficacy that a candidate siRNA sequence will silence the target gene. With validated siRNA databases have been developed in recent years, machine learning methods can be applied to predict siRNA accuracy and optimize design. This paper proposed a combined prediction method of BP neural network and support vector machine (SVM) for selecting effective siRNA sequences. With SVM, siRNA sequences were classified into effective or ineffective siRNAs. Subsequently, BP neural network model with great learning ability selected highly effective candidate sequences from effective siRNAs. We applied this method to published siRNAs datasets, and the experimental results confirmed good prediction capability.

The microRNA 424/503 cluster reduces CDC25A expression during cell cycle arrest imposed by transforming growth factor β in mammary epithelial cells.

Recently, we demonstrated that the microRNA 424(322)/503 [miR-424(322)/503] cluster is transcriptionally controlled by transforming growth factor β (TGF-β) in the mammary epithelium. Induction of this microRNA cluster impacts mammary epithelium fate by regulating apoptosis and insulin-like growth factor 1 (IGF1) signaling. Here, we expanded our finding to demonstrate that miR-424(322)/503 is an integral component of the cell cycle arrest mediated by TGF-β. Mechanistically, we showed that after TGF-β exposure, increased levels of miR-424(322)/503 reduce the expression of the cell cycle regulator CDC25A. miR-424(322)/503-dependent posttranscriptional downregulation of CDC25A cooperates with previously described transcriptional repression of the CDC25A promoter and proteasome-mediated degradation to reduce the levels of CDC25A expression and to induce cell cycle arrest. We also provide evidence that the TGF-β/miR-424(322)/503 axis is part of the mechanism that regulates the proliferation of hormone receptor-positive (HR(+)) mammary epithelial cells in vivo.

Identification of RISC-associated adenoviral microRNAs, a subset of their direct targets, and global changes in the targetome upon lytic adenovirus 5 infection.

Adenoviruses encode a set of highly abundant microRNAs (mivaRNAs), which are generated by Dicer-mediated cleavage of the larger noncoding virus-associated RNAs (VA RNAs) I and II. We performed deep RNA sequencing to thoroughly investigate the relative abundance of individual single strands of mivaRNA isoforms in human A549 cells lytically infected with human adenovirus 5 (Ad5) at physiologically relevant multiplicities of infection (MOIs). In addition, we investigated their relative abundance in the endogenous RNA-induced silencing complexes (RISCs). The occupation of endogenous RISCs by mivaRNAs turned out to be pronounced but not as dominant as previously inferred from experiments with AGO2-overexpressing cells infected at high MOIs. In parallel, levels of RISC-incorporated mRNAs were investigated as well. Analysis of mRNAs enriched in RISCs in Ad5-infected cells revealed that only mRNAs with complementarity to the seed sequences of mivaRNAs derived from VA RNAI but not VA RNAII were overrepresented among them, indicating that only mivaRNAs derived from VA RNAI are likely to contribute substantially to the posttranscriptional downregulation of host gene expression. Furthermore, to generate a comprehensive picture of the entire transcriptome/targetome in lytically infected cells, we determined changes in cellular miRNA levels in both total RNA and RISC RNA as well, and bioinformatical analysis of mRNAs of total RNA/RISC fractions revealed a general, genome-wide trend toward detargeting of cellular mRNAs upon infection. Lastly, we identified the direct targets of both single strands of a VA RNAI-derived mivaRNA that constituted one of the two most abundant isoforms in RISCs of lytically infected A549 cells. Viral and cellular miRNAs have been recognized as important players in virus-host interactions. This work provides the currently most comprehensive picture of the entire mRNA/miRNA transcriptome and of the complete RISC targetome during lytic adenovirus infection and thus represents the basis for a deeper understanding of the interplay between the virus and the cellular RNA interference machinery. Our data suggest that, at least in the model system that was employed, lytic infection by Ad5 is accompanied by a measurable global net detargeting effect on cellular mRNAs, and analysis of RISC-associated viral small RNAs revealed that the VA RNAs are the only source of virus-encoded miRNAs. Moreover, this work allows to assess the power of individual viral miRNAs to regulate cellular gene expression and provides a list of proven and putative direct targets of these miRNAs, which is of importance, given the fact that information about validated targets of adenovirus-encoded miRNAs is scarce.

Abstract 4362: miR-211 is contributive to the progression of oral carcinoma through targeting transcription factor 12 (Tcf12)

Abstract Oral squamous cell carcinoma (OSCC) is one of the prevalent malignancies worldwide. MicroRNAs are short RNA molecules that regulate pathogenesis, including OSCC carcinogenesis, by post-transcriptional down-regulation of target genes. Our previous study has identified a relationship between high miR-211 expression and the invasion of OSCC. This study further dissects the molecular mechanisms underlying the progression of OSCC as affected by miR-211. Tcf12 is a helix-loop-helix transcription factor with unclear implications in oral malignancies. MiR-211 was predicted to target the 3′UTR of transcription factor 12 (Tcf12). Reporter assays confirmed the direct targeting of miR-211 on Tcf12. MiR-211 expression decreased both endogenous and exogenous Tcf12 expression. Exogenous Tcf12 expression suppressed proliferation, migration and anchorage-independent colony formation of OECM-1 OSCC cells. On the contrary, knockdown of endogenous Tcf12 expression enhanced these oncogenic phenotypes. Furthermore, in tumor cells with miR-211 overexpression, Tcf12 can reduce its oncogenic potential; whereas miR-211 can also rescue the suppression of Tcf12 to tumor cells. Down-regulation of Tcf12 was identified in 75% (27/36) human OSCC samples. To elucidate the in vivo oncogenic roles of miR-211 in OSCC carcinogenesis, K14 promoter driven murine miR-211 transgenic mouse lines (K14-GFP- miR-211) were established. The Tcf12 expression in squamous epithelium was down-regulated in transgenic mice relative to wild type mice. After tumor induction by 4-nitroquinoline 1-oxide treatment, the transgenic mice exhibited more advanced tongue and esophageal tumorigenesis comparing to wild type mice. In summary, this study reveals that Tcf12 is suppressive to OSCC. MiR-211 regulates OSCC progression by targeting Tcf12. The K14-GFP-miR-211 transgenic mice will help future investigation of the pathogenic mechanisms underlying OSCC progression and the development of therapeutic strategy. Citation Format: Yi-Fen Chen, Kuo-Wei K. Chang, Shu-Chun Lin. miR-211 is contributive to the progression of oral carcinoma through targeting transcription factor 12 (Tcf12). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4362. doi:10.1158/1538-7445.AM2014-4362

Abstract 4360: Hypoxia-induced miR-372 targets p62 to affect the progression of oral carcinoma

Abstract Oral squamous cell carcinoma (OSCC) is one of the prevalent neoplasms worldwide. Recent studies have shown that miRNAs were involved in the pathogenesis of various human malignancies, which function by post-transcriptional down-regulation of target genes. Also, hypoxia is a feature of most tumors and plays an important role in regulating tumor progression and resistance to therapy. In our previous study, we found that miR-372 was highly expressed in OSCC tissues. Other studies have shown the oncogenic roles of miR-372 in regulating the cell cycle and apoptosis in tumor cells by targeting different targets. This study identified that miR-372 was up-regulated by hypoxia in OSCC cells. The plasma level of miR-372 in OSCC patients significantly increased with the advance of OSCC. p62 is a multidomain protein that interacts with key components to drive diverse impacts on signaling regulation including detoxification pathway. Using in silico prediction modules, qRT-PCR analysis, Western blot analysis, reporter assay and exogenous expression construct, we discovered that p62 was a novel target of miR-372. Ectopic miR-372 expression and knockdown of p62 both enhanced the migration of OSCC cells, but it did not affect the proliferation of OSCC cells. Furthermore, we demonstrated that p62 was able to induce the expression of phase II enzyme NQO1, which resulted in the attenuation of reactive oxygen species and migration in OSCC cells. Furthermore, correlation among the expression of miR-372, p62 and NQO1 was found in OSCC tissues. This study concludes that hypoxia-induced miR-372 expression modulates the progression of OSCC by targeting p62, which disrupts the ROS homeostasis in tumor cells. Citation Format: Li-Yin Yeh, Shu-Chun Lin, Chung-Ji Liu, Yong-Kie Wong, Kuo-Wei Chang. Hypoxia-induced miR-372 targets p62 to affect the progression of oral carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4360. doi:10.1158/1538-7445.AM2014-4360

Copy number variations burden on miRNA genes reveals layers of complexities involved in the regulation of pathways and phenotypic expression.

MicroRNAs are involved in post-transcriptional down-regulation of gene expression. Variations in miRNA genes can severely affect downstream-regulated genes and their pathways. However, population-specific burden of CNVs on miRNA genes and the complexities created towards the phenotype is not known. From a total of 44109 CNVs investigated from 1715 individuals across 12 populations using high-throughput arrays, 4007 miRNA-CNVs (∼9%) consisting 6542 (∼5%) miRNA genes with a total of 333 (∼5%) singleton miRNA genes were identified. We found miRNA-CNVs across the genomes of individuals showing multiple hits in many targets, co-regulated under the same pathway. This study proposes four mechanisms unraveling the many complexities in miRNA genes, targets and co-regulated miRNA genes towards establishment of phenotypic diversity.

Downregulation of Smurf2, a tumor-suppressive ubiquitin ligase, in triple-negative breast cancers: Involvement of the RB-microRNA axis

BackgroundThe HECT family ubiquitin ligase Smurf2 regulates cell polarity, migration, division, differentiation and death, by targeting diverse substrates that are critical for receptor signaling, cytoskeleton, chromatin remodeling and transcription. Recent studies suggest that Smurf2 functions as a tumor suppressor in mice. However, no inactivating mutation of SMURF2 has been reported in human, and information about Smurf2 expression in human cancer remains limited or complicated. Here we demonstrate that Smurf2 expression is downregulated in human breast cancer tissues, especially of the triple-negative subtype, and address the mechanism of Smurf2 downregulation in triple-negative breast cancer cells.MethodsHuman breast cancer tissues (47 samples expressing estrogen receptor (ER) and 43 samples with triple-negative status) were examined by immunohistochemistry for the expression of Smurf2. Ten widely-studied human breast cancer cell lines were examined for the expression of Smurf2. Furthermore, microRNA-mediated regulation of Smurf2 was investigated in triple-negative cancer cell lines.ResultsImmunohistochemical analysis showed that benign mammary epithelial cells expressed high levels of Smurf2, so did cells in ductal carcinomas in situ. In contrast, invasive ductal carcinomas showed focal or diffuse decrease in Smurf2 expression, which was observed more frequently in triple-negative tumors than in ER-positive tumors. Consistently, human triple-negative breast cancer cell lines such as BT549, MDA-MB-436, DU-4475 and MDA-MB-468 cells showed significantly lower expression of Smurf2 protein, compared to ER + or HER2+ cell lines. Studies using quantitative PCR and specific microRNA inhibitors indicated that increased expression of miR-15a, miR-15b, miR-16 and miR-128 was involved in Smurf2 downregulation in those triple-negative cancer cell lines, which have mutations in the retinoblastoma (RB) gene. Forced expression of RB increased levels of Smurf2 protein with concomitant decreases in the expression of the microRNAs.ConclusionsThis study provides evidence of posttranscriptional downregulation of Smurf2 in triple-negative breast cancers, and demonstrates that the loss of RB function is involved in microRNA-mediated interference with Smurf2 translation. The new link from RB inactivation to Smurf2 downregulation is likely to play a role in malignant phenotypes of triple-negative breast cancer cells.

Spatial correlation between patterns of wall permeability and patterns of multidirectional disturbed blood flow in rabbit aorta

Spatial correlation between patterns of wall permeability and patterns of multidirectional disturbed blood flow in rabbit aorta

An APOA5 3′ UTR Variant Associated with Plasma Triglycerides Triggers APOA5 Downregulation by Creating a Functional miR-485-5p Binding Site

APOA5 c.*158C>T (rs2266788), located in the 3' UTR, belongs to APOA5 haplotype 2 (APOA5*2), which is strongly associated with plasma triglyceride levels and modulates the occurrence of both moderate and severe hypertriglyceridemia. Individuals with APOA5*2 display reduced APOA5 expression at the posttranscriptional level. However, the functionality of this haplotype remains unclear. We hypothesized that the hypertriglyceridemic effects of APOA5*2 could involve miRNA regulation in the APOA5 3' UTR. Bioinformatic studies have identified the creation of a potential miRNA binding site for liver-expressed miR-485-5p (MIRN485-5p) in the mutant APOA5 3' UTR with the c.*158C allele. In human embryonic kidney 293T (HEK293T) cells cotransfected with an APOA5 3' UTR luciferase reporter vector and a miR485-5p precursor, c.*158C allele expression was significantly decreased. Moreover, in HuH-7 cells endogenously expressing miR-485-5p, we observed that luciferase activity was significantly lower in the presence of the c.*158C allele than in the presence of the c.*158T allele, which was completely reversed by a miR-485-5p inhibitor. We demonstrated that the rare c.*158C APOA5 allele creates a functional target site for liver-expressed miR-485-5p. Therefore, we propose that the well-documented hypertriglyceridemic effect of APOA5*2 involves an APOA5 posttranscriptional downregulation mediated by miR-485-5p.