Dysregulation of hsa-miR-34a and hsa-miR-449a leads to overexpression of PACS-1 and loss of DNA damage response (DDR) in cervical cancer.

We have observed overexpression of PACS-1, a cytosolic sorting protein in primary cervical tumors. Absence of exonic mutations and overexpression at the RNA level suggested a transcriptional and/or posttranscriptional regulation. University of California Santa Cruz genome browser analysis of PACS-1 micro RNAs (miR), revealed two 8-base target sequences at the 3' terminus for hsa-miR-34a and hsa-miR-449a. Quantitative RT-PCR and Northern blotting studies showed reduced or loss of expression of the two microRNAs in cervical cancer cell lines and primary tumors, indicating dysregulation of these two microRNAs in cervical cancer. Loss of PACS-1 with siRNA or exogenous expression of hsa-miR-34a or hsa-miR-449a in HeLa and SiHa cervical cancer cell lines resulted in DNA damage response, S-phase cell cycle arrest, and reduction in cell growth. Furthermore, the siRNA studies showed that loss of PACS-1 expression was accompanied by increased nuclear γH2AX expression, Lys382-p53 acetylation, and genomic instability. PACS-1 re-expression through LNA-hsa-anti-miR-34a or -449a or through PACS-1 cDNA transfection led to the reversal of DNA damage response and restoration of cell growth. Release of cells post 24-h serum starvation showed PACS-1 nuclear localization at G1-S phase of the cell cycle. Our results therefore indicate that the loss of hsa-miR-34a and hsa-miR-449a expression in cervical cancer leads to overexpression of PACS-1 and suppression of DNA damage response, resulting in the development of chemo-resistant tumors.

Comparative analyses of long non-coding RNA profiles in vivo in cystic fibrosis lung airway and parenchyma tissues.

BACKGROUND: Recent advances in the functional analyses of endogenous non-coding RNA (ncRNA) molecules, including long non-coding RNAs (LncRNAs), have provided a new perspective on the crucial roles of RNA in gene regulation. Consequently, LncRNA deregulation is a key factor in various diseases, including pulmonary disorders like Cystic Fibrosis (CF). CF is the most common life limiting recessive disease in the U.S., and is due to mutations in the CFTR gene. CF mutations, of which the most common is F508del-CFTR, prevents correct folding, trafficking and function of the mutant CFTR protein and is further manifested by the hyper-expression of pro-inflammatory cytokines and chemokines into the airway lumen leading to bronchiectasis and culminating in lung destruction. METHODS: Here we report a distinct LncRNA signature and corresponding mRNAs that distinguishes CF lung (airway and parenchyma) tissues from matched non-CF controls (n = 4 each group), generated by microarray specific for LncRNAs which includes corresponding mRNA expressions. In silico analyses of the cellular processes that are impacted by these LncRNAs was performed using Gene Ontology (GO). A selected subset of LncRNAs were validated by quantitative real-time PCR. RESULTS: We have identified 636 LncRNAs differentially expressed in CF airway epithelium and 1974 in CF lung parenchyma compared to matched non-CF controls (fold change ≥2, p < 0.05), majority of which (> 50%) are intergenic. Interestingly, 15 of these differentially expressed LncRNAs and 9 coding mRNAs are common to airway and parenchyma tissues. GO analyses indicates that signaling pathways and cell membrane functions are significantly affected by the alteration in LncRNA expressions in CF lung tissues. Seven of the differentially expressed LncRNAs, exhibit similar expression trends in CFBE41o- compared to control cells. CONCLUSION: Understanding the mechanisms by which these LncRNAs regulate CF disease phenotype will help develop novel therapeutic targets for CF and related pulmonary diseases, such as COPD and Asthma.

RPTOR, a novel target of miR-155, elicits a fibrotic phenotype of cystic fibrosis lung epithelium by upregulating CTGF.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the most frequent of which is F508del-CFTR. CF is characterized by excessive secretion of pro-inflammatory mediators into the airway lumen, inducing a highly inflammatory cellular phenotype. This process triggers fibrosis, causing airway destruction and leading to high morbidity and mortality. We previously reported that miR-155 is upregulated in CF lung epithelial cells, but the molecular mechanisms by which miR-155 affects the disease phenotype is not understood. Here we report that RPTOR (regulatory associated protein of mTOR, complex 1) is a novel target of miR-155 in CF lung epithelial cells. The suppression of RPTOR expression and subsequent activation of TGF-ß signaling resulted in the induction of fibrosis by elevating connective tissue growth factor (CTGF) abundance in CF lung epithelial cells. Thus, we propose that miR-155 might regulate fibrosis of CF lungs through the increased CTGF expression, highlighting its potential value in CF therapy.

Genetic heterogeneity revealed by sequence analysis of Mycobacterium tuberculosis isolates from extra-pulmonary tuberculosis patients.

BACKGROUND: Tuberculosis remains a major public health problem. Clinical tuberculosis manifests often as pulmonary and occasionally as extra-pulmonary tuberculosis. The emergence of drug resistant tubercle bacilli and its association with HIV is a formidable challenge to curb the spread of tuberculosis. There have been concerted efforts by whole genome sequencing and bioinformatics analysis to identify genomic patterns and to establish a relationship between the genotype of the organism and clinical manifestation of tuberculosis. Extra-pulmonary TB constitutes 15-20 percent of the total clinical cases of tuberculosis reported among immunocompetent patients, whereas among HIV patients the incidence is more than 50 percent. Genomic analysis of M. tuberculosis isolates from extra pulmonary patients has not been explored. RESULTS: The genomic DNA of 5 extra-pulmonary clinical isolates of M. tuberculosis derived from cerebrospinal fluid, lymph node fine needle aspirates (FNAC) / biopsies, were sequenced. Next generation sequencing approach (NGS) was employed to identify Single Nucleotide Variations (SNVs) and computational methods used to predict their consequence on functional genes. Analysis of distribution of SNVs led to the finding that there are mixed genotypes in patient isolates and that many SNVs are likely to influence either gene function or their expression. Phylogenetic relationship between the isolates correlated with the origin of the isolates. In addition, insertion sites of IS elements were identified and their distribution revealed a variation in number and position of the element in the 5 extra-pulmonary isolates compared to the reference M. tuberculosis H37Rv strain. CONCLUSIONS: The results suggest that NGS sequencing is able to identify small variations in genomes of M. tuberculosis isolates including changes in IS element insertion sites. Moreover, variations in isolates of M. tuberculosis from non-pulmonary sites were documented. The analysis of our results indicates genomic heterogeneity in the clinical isolates.

Regulation of miR-155 biogenesis in cystic fibrosis lung epithelial cells: antagonistic role of two mRNA-destabilizing proteins, KSRP and TTP.

Cystic fibrosis (CF) is characterized by a massive pro-inflammatory phenotype in the lung arising from profound expression of inflammatory genes, including interleukin-8 (IL-8). We have previously reported that IL-8 mRNA is stabilized in CF lung epithelial cells, resulting in concomitant hyper-expression of IL-8 protein through elevated expression of miR-155. We therefore investigated what factors promote the enhanced aberrant expression of miR-155 in CF. Here we report for the first time, the role of mRNA-destabilizing inflammatory RNA-binding proteins, KSRP and TTP, in the regulation of miR-155 biogenesis in CF lung epithelial cells. We find that KSRP and TTP have an antagonistic role in miR-155 biogenesis. While KSRP promotes enhanced processing of miR-155 precursors to mature miR-155, over-expression of TTP in the CF lung epithelial cells suppresses expression of miR-155. We find that TTP induces the expression of miR-1, which appears to be a regulator of miR-155 biogenesis in CF lung epithelial cells. These data provide novel insights into the mechanisms that induce hyper-inflammatory phenotype of CF, and are potential candidates for anti-inflammatory therapeutics for CF.

Prdm16 is a physiologic regulator of hematopoietic stem cells.

Fetal liver and adult bone marrow hematopoietic stem cells (HSCs) renew or differentiate into committed progenitors to generate all blood cells. PRDM16 is involved in human leukemic translocations and is expressed highly in some karyotypically normal acute myeloblastic leukemias. As many genes involved in leukemogenic fusions play a role in normal hematopoiesis, we analyzed the role of Prdm16 in the biology of HSCs using Prdm16-deficient mice. We show here that, within the hematopoietic system, Prdm16 is expressed very selectively in the earliest stem and progenitor compartments, and, consistent with this expression pattern, is critical for the establishment and maintenance of the HSC pool during development and after transplantation. Prdm16 deletion enhances apoptosis and cycling of HSCs. Expression analysis revealed that Prdm16 regulates a remarkable number of genes that, based on knockout models, both enhance and suppress HSC function, and affect quiescence, cell cycling, renewal, differentiation, and apoptosis to various extents. These data suggest that Prdm16 may be a critical node in a network that contains negative and positive feedback loops and integrates HSC renewal, quiescence, apoptosis, and differentiation.

SFPQ rescues F508del-CFTR expression and function in cystic fibrosis bronchial epithelial cells.

Cystic fibrosis (CF) occurs as a result of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which lead to misfolding, trafficking defects, and impaired function of the CFTR protein. Splicing factor proline/glutamine-rich (SFPQ) is a multifunctional nuclear RNA-binding protein (RBP) implicated in the regulation of gene expression pathways and intracellular trafficking. Here, we investigated the role of SFPQ in the regulation of the expression and function of F508del-CFTR in CF lung epithelial cells. We find that the expression of SFPQ is reduced in F508del-CFTR CF epithelial cells compared to WT-CFTR control cells. Interestingly, the overexpression of SFPQ in CF cells increases the expression as well as rescues the function of F508del-CFTR. Further, comprehensive transcriptome analyses indicate that SFPQ plays a key role in activating the mutant F508del-CFTR by modulating several cellular signaling pathways. This is the first report on the role of SFPQ in the regulation of expression and function of F508del-CFTR in CF lung disease. Our findings provide new insights into SFPQ-mediated molecular mechanisms and point to possible novel epigenetic therapeutic targets for CF and related pulmonary diseases.

Comparative RNA-seq analysis reveals dys-regulation of major canonical pathways in ERG-inducible LNCaP cell progression model of prostate cancer.

Prostate Cancer (CaP) is the second leading cause of cancer related death in USA. In human CaP, gene fusion between androgen responsive regulatory elements at the 5'-untranslated region of TMPRSS2 and ETS-related genes (ERG) is present in at least 50% of prostate tumors. Here we have investigated the unique cellular transcriptome associated with over-expression of ERG in ERG-inducible LNCaP cell model system of human CaP. Comprehensive transcriptome analyses reveal a distinct signature that distinguishes ERG dependent and independent CaP in LNCaP cells. Our data highlight a significant heterogeneity among the transcripts. Out of the 526 statistically significant differentially expressed genes, 232 genes are up-regulated and 294 genes are down-regulated in response to ERG. These ERG-associated genes are linked to several major cellular pathways, cell cycle regulation being the most significant. Consistently our data indicate that ERG plays a key role in modulating the expression of genes required for G1 to S phase transition, particularly those that affect cell cycle arrest at G1 phase. Moreover, cell cycle arrest in response to ERG appears to be promoted by induction of p21 in a p53 independent manner. These findings may provide new insights into mechanisms that promote growth and progression of CaP.

Digitoxin Inhibits Epithelial-to-Mesenchymal-Transition in Hereditary Castration Resistant Prostate Cancer.

Castration Resistant Prostate Cancer (CRPC) is thought to be driven by a collaborative mechanism between TNFα/NFκB and TGFß signaling, leading to inflammation, Epithelial-to-Mesenchymal-Transition (EMT), and metastasis. Initially, TGFß is a tumor suppressor, but in advanced metastatic disease it switches to being a tumor promoter. TGFBR2 may play a critical role in this collaboration, as its expression is driven by NFκB and it is the primary receptor for TGFß. We have previously reported that the cardenolide drug digitoxin blocks TNFα/NFκB-driven proinflammatory signaling. We therefore hypothesized that digitoxin might break the collaborative process between NFκB and TGFß by also inhibiting expression of TGFBR2. We therefore tested whether TGFß-driven EMT and resulting metastases would be suppressed. Here we show, in vitro, that digitoxin inhibits NFκB-driven TGFBR2 expression, as well as Vimentin, while elevating E-cadherin expression. Digitoxin also significantly reduces HSPB1 mRNA and the HSPB1/RBFOX2 mRNA ratio in PC3 cells. In vivo, in a syngeneic, immune competent rat model of metastatic CRPC, we show that digitoxin also suppresses Tgfbr2 expression, as well as expression of other genes classically driven by NFκB, and of multiple EMT genes associated with metastasis. Concurrently, digitoxin suppresses tumor growth and metastasis in these animals, and prolongs survival. Gross tumor recurrence following tumor resection also appears prevented in ca 30% of cases. While the existence of a collaboration between NFκB and TGFß to drive EMT and metastasis has previously been appreciated, we show here, for the first time, that chronic, low concentrations of digitoxin are able to block CRPC tumor progression, EMT and the ensuing metastatic disease.

Regulation of mRNA turnover in cystic fibrosis lung disease.

Cystic fibrosis (CF) is an autosomal recessive disease due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, F508del-CFTR being the most frequent mutation. The CF lung is characterized by a hyperinflammatory phenotype and is regulated by multiple factors that coordinate its pathophysiology. In CF the expression of CFTR as well as proinflammatory genes are regulated at the level of messenger RNA (mRNA) stability, which subsequently affect translation. These mechanisms are mediated by inflammatory RNA-binding proteins as well as small endogenous noncoding microRNAs, in coordination with cellular signaling pathways. These regulatory factors exhibit altered expression and function in vivo in the CF lung, and play a key role in the pathophysiology of CF lung disease. In this review, we have described the role of mRNA stability and associated regulatory mechanisms in CF lung disease. WIREs RNA 2017, 8:e1408. doi: 10.1002/wrna.1408 For further resources related to this article, please visit the WIREs website.

Loss of miR-449a in ERG-associated prostate cancer promotes the invasive phenotype by inducing SIRT1.

Epigenetic regulation by SIRT1, a multifaceted NAD+-dependent protein deacetylase, is one of the most common factors modulating cellular processes in a broad range of diseases, including prostate cancer (CaP). SIRT1 is over-expressed in CaP cells, however the associated mechanism is not well understood. To identify whether specific microRNAs might mediate this linkage, we have screened a miRNA library for differential expression in ERG-associated CaP tissues. Of 20 differentially and significantly expressed miRNAs that distinguish ERG-positive tumors from ERG-negative tumors, we find miR-449a is highly suppressed in ERG-positive tumors. We establish that SIRT1 is a direct target of miR-449a and is also induced by ERG in ERG-associated CaP. Our data suggest that attenuation of miR-449a promotes the invasive phenotype of the ERG-positive CaP in part by inducing the expression of SIRT1 in prostate cancer cells. Furthermore, we also find that suppression of SIRT1 results in a significant reduction in ERG expression in ERG-positive CaP cells, indicating a feed-back regulatory loop associated with ERG, miR-449a and SIRT1. We also report that ERG suppresses p53 acetylation perhaps through miR-449a-SIRT1 axis in CaP cells. Our findings provide new insight into the function of miRNAs in regulating ERG-associated CaP. Thus, miR-449a activation or SIRT1 suppression may represent new therapeutic opportunity for ERG-associated CaP.

Gamma-Tocotrienol Modulates Radiation-Induced MicroRNA Expression in Mouse Spleen.

Ionizing radiation causes depletion of hematopoietic cells and enhances the risk of developing secondary hematopoietic malignancies. Vitamin E analog gamma-tocotrienol (GT3), which has anticancer properties, promotes postirradiation hematopoietic cell recovery by enhancing spleen colony-forming capacity, and provides protection against radiation-induced lethality in mice. However, the underlying molecular mechanism involved in GT3-mediated postirradiation survival is not clearly understood. Recent studies have shown that natural dietary products including vitamin E provide a benefit to biological systems by modulating microRNA (miR) expression. In this study, we show that GT3 differentially modulates the miR footprint in the spleen of irradiated mice compared to controls at early times (day 1), as well as later times (day 4 and 15) after total-body irradiation. We observed that miR expression was altered in a dose- and time-dependent manner in GT3-pretreated spleen tissues from total-body irradiated mice. GT3 appeared to affect the expression of a number of radiation-modulated miRs known to be involved in hematopoiesis and lymphogenesis. Moreover, GT3 pretreatment also suppressed the upregulation of radiation-induced p53, suggesting the function of GT3 in the prevention of radiation-induced damage to the spleen. In addition, we have shown that GT3 significantly reduced serum levels of Flt3L, a biomarker of radiation-induced bone marrow aplasia. Further in silico analyses of the effect of GT3 implied the association of p38 MAPK, ERK and insulin signaling pathways. Our study provides initial insight into the mechanism by which GT3 mediates protection of spleen after total-body irradiation.

Differential regulation of inflammation by inflammatory mediators in cystic fibrosis lung epithelial cells.

Cystic fibrosis (CF) is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which cause a massively proinflammatory phenotype in the CF airway. The chemical basis of the inflammation is hyperproduction of interleukin-8 (IL-8) by CF airway epithelial cells, based on both an intrinsic mutation-dependent mechanism and by infection. In infection-free, cultured CF lung epithelial cells, high levels of the microRNA (miR), miR-155, is responsible for hyperexpression of IL-8. However, whether infection-induced IL-8 expression in CF cells is also mediated by miR-155 is not known. We have hypothesized that miR-155 might be a general mediator of enhanced IL-8 expression in CF cells, either in response to other cytokine/chemokine mediators of inflammation, or after exposure to infectious agents. Here we find that a reduction in miR-155 accompanies suppression of IL-8 by either the anti-inflammatory cytokine IL-10 or by inhibition of ambient IL-1ß with a neutralizing antibody. However, attempts to elevate IL-8 levels with either intact bacteria [viz. a mucoid strain of Pseudomonas aeruginosa (PA)], or lipopolysaccharide were unable to elevate miR-155 above its intrinsically high level in the absence of these agents. Instead, in response to PA infection, the CF cells modestly suppress the expression of miR-155, and express a novel set of miRs, including miR-215. We find that ex vivo CF lung epithelial cells also express high levels of both miR-155 and miR-215. The predicted module of infection-induced mRNA targets focuses on activation of the NFκB-signaling pathway, and on the proapoptotic p53-signaling pathway. We interpret these data to suggest that that CF lung epithelial cells respond to PA or bacterial cell products with a novel miR program that may carry with it serious challenges to survival.

Assessment of the N-PCR assay in diagnosis of pleural tuberculosis: detection of M. tuberculosis in pleural fluid and sputum collected in tandem.

BACKGROUND: The nonspecific clinical presentation and paucibacillary nature of tuberculous pleuritis remains a challenge for diagnosis. Diagnosis of tuberculous pleural effusion depends on the demonstration of the presence of tubercle bacilli in the sputum, pleural fluid, or pleural biopsy specimen, or demonstration of granuloma in pleura by histological examination. We examined the clinical utility of the diagnosis of pleural tuberculosis using the in house N-PCR assay, AFB smear microscopy and culture. Besides pleural fluid the inclusion of sputum in the efficacy of diagnosis of pleural tuberculosis was scrutinized. METHODOLOGY/PRINCIPAL FINDINGS: Pleural fluid and sputum samples of 58 tuberculous and 42 non-tuberculous pleural effusion patients were processed for AFB smear microscopy, culture and the N-PCR assay. Mycobacteria were detected exclusively in tuberculous pleural effusion samples. None of the non-tuberculous pleural effusion samples were positive for mycobacteria. Comparative analysis showed that the N-PCR assay had the highest sensitivity. Inclusion of sputum along with pleural fluid increased N-PCR sensitivity from 51.7 to 70.6% (p<0.0001).This improved sensitivity was reflected in AFB smear microscopy and isolation by culture. The sensitivity enhanced on inclusion of sputum from 3.4 (p = 0.50) to 10.3% (p = 0.038) for AFB smear microscopy and for isolation of mycobacteria from 10.3(p = 0.03) to 22.4% (p = 0.0005). Thirteen isolates were obtained from 58 pleural tuberculosis patients. Eleven mycobacterial isolates were identified as M. tuberculosis and two as M. fortuitum and M. chelonae. Complete concordance was seen between the biochemical identification of isolates and the N-PCR identification of mycobacterial species prior to isolation. CONCLUSIONS/SIGNIFICANCE: To the best of our knowledge this is the first PCR based report on utility of sputum for diagnosis of pleural tuberculosis. The present study demonstrates that a combination of pleural fluid with sputum sample and N-PCR improved the diagnosis of pleural tuberculosis.

Visual format for detection of Mycobacterium tuberculosis and M. bovis in clinical samples using molecular beacons.

A real-time polymerase chain reaction (PCR) assay for the direct identification of Mycobacterium tuberculosis and M. bovis using molecular beacons was developed. The assay was modified for use in regular thermal cyclers. Molecular beacons that were specific for M. tuberculosis (Tb-B) and M. bovis (Bo-B) were designed. The fluorescence of the target PCR product-molecular beacon probe complex was detected visually using a transilluminator. The results were then compared with those of conventional multiplex PCR (CM-PCR) assays and biochemical identification. The detection limit of Tb-B and Bo-B beacons was 500 fg and 50 fg by the visual format and real-time PCR assay, respectively, compared with 5 pg by CM-PCR assay. Pulmonary and extrapulmonary samples were examined. The agreement between culture and the two assays was very good in sputum samples and fair in extrapulmonary samples. The agreement between clinical diagnoses with the two assays was moderate in extrapulmonary samples. There was very good agreement between CM-PCR and visual format assays for all samples used in the study. Concordance in the identification of isolates by the visual, CM-PCR assay, and biochemical identification was seen. Hence, the use of molecular beacon detection of M. tuberculosis and M. bovis in clinical samples is feasible by setting up two asymmetric PCRs concurrently. The assay is sensitive, specific, simple to interpret, and takes less than 3 hours to complete.