Investigating miR-9 as a mediator in laryngeal cancer health disparities.

Background: For several decades, Black patients have carried a higher burden of laryngeal cancer among all races. Even when accounting for sociodemographics, a disparity remains. Differentially expressed microRNAs have been linked to racially disparate clinical outcomes in breast and prostate cancers, yet an association in laryngeal cancer has not been addressed. In this study, we present our computational analysis of differentially expressed miRNAs in Black compared with White laryngeal cancer and further validate microRNA-9-5p (miR-9-5p) as a potential mediator of cancer phenotype and chemoresistance. Methods: Bioinformatic analysis of 111 (92 Whites, 19 Black) laryngeal squamous cell carcinoma (LSCC) specimens from the TCGA revealed miRNAs were significantly differentially expressed in Black compared with White LSCC. We focused on miR-9-5 p which had a significant 4-fold lower expression in Black compared with White LSCC (p<0.05). After transient transfection with either miR-9 mimic or inhibitor in cell lines derived from Black (UM-SCC-12) or White LSCC patients (UM-SCC-10A), cellular migration and cell proliferation was assessed. Alterations in cisplatin sensitivity was evaluated in transient transfected cells via IC50 analysis. qPCR was performed on transfected cells to evaluate miR-9 targets and chemoresistance predictors, ABCC1 and MAP1B. Results: Northern blot analysis revealed mature miR-9-5p was inherently lower in cell line UM-SCC-12 compared with UM-SCC-10A. UM -SCC-12 had baseline increase in cellular migration (p < 0.01), proliferation (p < 0.0001) and chemosensitivity (p < 0.01) compared to UM-SCC-10A. Increasing miR-9 in UM-SCC-12 cells resulted in decreased cellular migration (p < 0.05), decreased proliferation (p < 0.0001) and increased sensitivity to cisplatin (p < 0.001). Reducing miR-9 in UM-SCC-10A cells resulted in increased cellular migration (p < 0.05), increased proliferation (p < 0.05) and decreased sensitivity to cisplatin (p < 0.01). A significant inverse relationship in ABCC1 and MAP1B gene expression was observed when miR-9 levels were transiently elevated or reduced in either UM-SCC-12 or UM-SCC-10A cell lines, respectively, suggesting modulation by miR-9. Conclusion: Collectively, these studies introduce differential miRNA expression in LSCC cancer health disparities and propose a role for low miR-9-5p as a mediator in LSCC tumorigenesis and chemoresistance.

Development of Human Adrenocortical Adenoma (HAA1) Cell Line from Zona Reticularis.

The human adrenal cortex is composed of distinct zones that are the main source of steroid hormone production. The mechanism of adrenocortical cell differentiation into several functionally organized populations with distinctive identities remains poorly understood. Human adrenal disease has been difficult to study, in part due to the absence of cultured cell lines that faithfully represent adrenal cell precursors in the early stages of transformation. Here, Human Adrenocortical Adenoma (HAA1) cell line derived from a patient's macronodular adrenocortical hyperplasia and was treated with histone deacetylase inhibitors (HDACis) and gene expression was examined. We describe a patient-derived HAA1 cell line derived from the zona reticularis, the innermost zone of the adrenal cortex. The HAA1 cell line is unique in its ability to exit a latent state and respond with steroidogenic gene expression upon treatment with histone deacetylase inhibitors. The gene expression pattern of differentiated HAA1 cells partially recreates the roster of genes in the adrenal layer that they have been derived from. Gene ontology analysis of whole genome RNA-seq corroborated increased expression of steroidogenic genes upon HDAC inhibition. Surprisingly, HDACi treatment induced broad activation of the Tumor Necrosis Factor (TNF) alpha pathway. This novel cell line we developed will hopefully be instrumental in understanding the molecular and biochemical mechanisms controlling adrenocortical differentiation and steroidogenesis.

Sequencing of Argonaute-bound microRNA/mRNA hybrids reveals regulation of the unfolded protein response by microRNA-320a.

MicroRNAs (miRNA) are short non-coding RNAs widely implicated in gene regulation. Most metazoan miRNAs utilize the RNase III enzymes Drosha and Dicer for biogenesis. One notable exception is the RNA polymerase II transcription start sites (TSS) miRNAs whose biogenesis does not require Drosha. The functional importance of the TSS-miRNA biogenesis is uncertain. To better understand the function of TSS-miRNAs, we applied a modified Crosslinking, Ligation, and Sequencing of Hybrids on Argonaute (AGO-qCLASH) to identify the targets for TSS-miRNAs in HCT116 colorectal cancer cells with or without DROSHA knockout. We observed that miR-320a hybrids dominate in TSS-miRNA hybrids identified by AGO-qCLASH. Targets for miR-320a are enriched for the eIF2 signaling pathway, a downstream component of the unfolded protein response. Consistently, in miR-320a mimic- and antagomir- transfected cells, differentially expressed gene products are associated with eIF2 signaling. Within the AGO-qCLASH data, we identified the endoplasmic reticulum (ER) chaperone calnexin as a direct miR-320a down-regulated target, thus connecting miR-320a to the unfolded protein response. During ER stress, but not amino acid deprivation, miR-320a up-regulates ATF4, a critical transcription factor for resolving ER stress. In summary, our study investigates the targetome of the TSS-miRNAs in colorectal cancer cells and establishes miR-320a as a regulator of unfolded protein response.

Systematic identification of A-to-I editing associated regulators from multiple human cancers.

A-to-I editing is the most common editing type in humans that is catalyzed by ADAR family members (ADARs), ADAR1 and ADAR2. Although millions of A-to-I editing sites have recently been discovered, the regulation mechanisms of the RNA editing process are still not clear. Herein, we developed a two-step logistic regression model to identify genes that are potentially involved in the RNA editing process in four human cancers. In the first step, we tested the association of each editing site with known enzymes. To validate the logistic regression model, we collected 10 genes with 168 editing sites from multiple published studies and obtained a nearly 100% validation rate. ADAR1 was identified as the enzyme associated with the majority of the A-to-I editing sites. Thus, ADAR1 was taken as a control gene in the second step to identify genes that have a stronger regulation effect on editing sites than ADAR1. Using our advanced method, we successfully found a set of genes that were significantly positively or negatively associated (PA or NA) with specific sets of RNA editing sites. 51 of these genes had been reported in at least one previous study. We highlighted two genes: 1), SRSF5, supported by three previous studies, and 2) MIR22HG, supported by one previous study and two of our cancer datasets. The PA and NA genes were cancer-specific but shared common pathways. Interestingly, the PA genes from kidney cancer were enriched for survival-associated genes while the NA genes were not, indicating that the PA genes may play more important roles in kidney cancer progression.

Author Correction: Integrating multi-platform genomic datasets for kidney renal clear cell carcinoma subtyping using stacked denoising autoencoders.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Integrating multi-platform genomic datasets for kidney renal clear cell carcinoma subtyping using stacked denoising autoencoders.

Clear cell renal cell carcinoma (ccRCC) is highly heterogeneous and is the most lethal cancer of all urologic cancers. We developed an unsupervised deep learning method, stacked denoising autoencoders (SdA), by integrating multi-platform genomic data for subtyping ccRCC with the goal of assisting diagnosis, personalized treatments and prognosis. We successfully found two subtypes of ccRCC using five genomics datasets for Kidney Renal Clear Cell Carcinoma (KIRC) from The Cancer Genome Atlas (TCGA). Correlation analysis between the last reconstructed input and the original input data showed that all the five types of genomic data positively contribute to the identification of the subtypes. The first subtype of patients had significantly lower survival probability, higher grade on neoplasm histology and higher stage on pathology than the other subtype of patients. Furthermore, we identified a set of genes, proteins and miRNAs that were differential expressed (DE) between the two subtypes. The function annotation of the DE genes from pathway analysis matches the clinical features. Importantly, we applied the model learned from KIRC as a pre-trained model to two independent datasets from TCGA, Lung Adenocarcinoma (LUAD) dataset and Low Grade Glioma (LGG), and the model stratified the LUAD and LGG patients into clinical associated subtypes. The successful application of our method to independent groups of patients supports that the SdA method and the model learned from KIRC are effective on subtyping cancer patients and most likely can be used on other similar tasks. We supplied the source code and the models to assist similar studies at https://github.com/tjgu/cancer_subtyping.

Clinical Relevance of Noncoding Adenosine-to-Inosine RNA Editing in Multiple Human Cancers.

PURPOSE: RNA editing is a post-transcriptional process that alters the nucleotide sequences of certain transcripts, in vertebrate most often converting adenosines to inosines. Multiple studies have recently implicated RNA editing in cancer development; however, most studies have focused on recoding RNA editing events. The function and clinical relevance of noncoding RNA (ncRNA) editing events in cancers have not been systematically examined. PATIENTS AND METHODS: We improved our previously published pipeline to identify ncRNA editing sites from four human cancers: liver hepatocellular carcinoma, lung adenocarcinoma, kidney renal clear-cell carcinoma, and thyroid carcinoma. We then developed multiple advanced statistical models to identify significantly differential edited (DE) sites between tumor and normal samples and clinical relevance ncRNA editing sites, as well as to investigate the association between gene expression, ncRNA editing, and microRNAs. Finally, we validated computational results with experiments. RESULTS: We identified 3,788 ncRNA editing sites of high confidence from the four cancers. We found thousands of DE sites which had distinct profiles across the four cancers. In kidney cancer, which had the largest uncensored survival data among the four cancers, 80 DE sites were significantly associated with patient survival. We identified 3' untranslated region (UTR) RNA editing sites that can affect gene expression, either independent of or by working with microRNAs. We validated that the 3'UTR RNA editing sites in CWF19L1 and F11R genes resulted in increased protein levels and that alterations of the expression of the two genes affected the proliferation of human embryonic kidney cells. CONCLUSION: On the basis of our computational and experimental results, we hypothesize that 3'UTR editing sites may affect their host gene expression, thereby affecting cell proliferation.

Sex-specific airway hyperreactivity and sex-specific transcriptome remodeling in neonatal piglets challenged with intra-airway acid.

Acute airway acidification is a potent stimulus of sensory nerves and occurs commonly with gastroesophageal reflux disease, cystic fibrosis, and asthma. In infants and adults, airway acidification can acutely precipitate asthma-like symptoms, and treatment-resistant asthma can be associated with gastroesophageal reflux disease. Airway protective behaviors, such as mucus secretion and airway smooth muscle contraction, are often exaggerated in asthma. These behaviors are manifested through activation of neural circuits. In some populations, the neural response to acid might be particularly important. For example, the immune response in infants is relatively immature compared with adults. Infants also have a high frequency of gastroesophageal reflux. Thus, in the current study, we compared the transcriptomes of an airway-nervous system circuit (e.g., tracheal epithelia, nodose ganglia, and brain stem) in neonatal piglets challenged with intra-airway acid. We hypothesized that the identification of parallel changes in the transcriptomes of two neutrally connected tissues might reveal the circuit response, and, hence, molecules important for the manifestation of asthma-like features. Intra-airway acid induced airway hyperreactivity and airway obstruction in male piglets. In contrast, female piglets displayed airway obstruction without airway hyperreactivity. Pairwise comparisons revealed parallel changes in genes directly implicated in airway hyperreactivity ( scn10a) in male acid-challenged piglets, whereas acid-challenged females exhibited parallel changes in genes associated with mild asthma ( stat 1 and isg15). These findings reveal sex-specific responses to acute airway acidification and highlight distinct molecules within a neural circuit that might be critical for the manifestation of asthma-like symptoms in pediatric populations.

GSR is not essential for the maintenance of antioxidant defenses in mouse cochlea: Possible role of the thioredoxin system as a functional backup for GSR.

Glutathione reductase (GSR), a key member of the glutathione antioxidant defense system, converts oxidized glutathione (GSSG) to reduced glutathione (GSH) and maintains the intracellular glutathione redox state to protect the cells from oxidative damage. Previous reports have shown that Gsr deficiency results in defects in host defense against bacterial infection, while diquat induces renal injury in Gsr hypomorphic mice. In flies, overexpression of GSR extended lifespan under hyperoxia. In the current study, we investigated the roles of GSR in cochlear antioxidant defense using Gsr homozygous knockout mice that were backcrossed onto the CBA/CaJ mouse strain, a normal-hearing strain that does not carry a specific Cdh23 mutation that causes progressive hair cell degeneration and early onset of hearing loss. Gsr-/- mice displayed a significant decrease in GSR activity and GSH/GSSG ratios in the cytosol of the inner ears. However, Gsr deficiency did not affect ABR (auditory brainstem response) hearing thresholds, wave I amplitudes or wave I latencies in young mice. No histological abnormalities were observed in the cochlea of Gsr-/- mice. Furthermore, there were no differences in the activities of cytosolic glutathione-related enzymes, including glutathione peroxidase and glutamate-cysteine ligase, or the levels of oxidative damage markers in the inner ears between WT and Gsr-/- mice. In contrast, Gsr deficiency resulted in increased activities of cytosolic thioredoxin and thioredoxin reductase in the inner ears. Therefore, under normal physiological conditions, GSR is not essential for the maintenance of antioxidant defenses in mouse cochlea. Given that the thioredoxin system is known to reduce GSSG to GSH in multiple species, our findings suggest that the thioredoxin system can support GSSG reduction in the mouse peripheral auditory system.

Genetic Architectures of Quantitative Variation in RNA Editing Pathways.

RNA editing refers to post-transcriptional processes that alter the base sequence of RNA. Recently, hundreds of new RNA editing targets have been reported. However, the mechanisms that determine the specificity and degree of editing are not well understood. We examined quantitative variation of site-specific editing in a genetically diverse multiparent population, Diversity Outbred mice, and mapped polymorphic loci that alter editing ratios globally for C-to-U editing and at specific sites for A-to-I editing. An allelic series in the C-to-U editing enzyme Apobec1 influences the editing efficiency of Apob and 58 additional C-to-U editing targets. We identified 49 A-to-I editing sites with polymorphisms in the edited transcript that alter editing efficiency. In contrast to the shared genetic control of C-to-U editing, most of the variable A-to-I editing sites were determined by local nucleotide polymorphisms in proximity to the editing site in the RNA secondary structure. Our results indicate that RNA editing is a quantitative trait subject to genetic variation and that evolutionary constraints have given rise to distinct genetic architectures in the two canonical types of RNA editing.

Pla2g12b and Hpn are genes identified by mouse ENU mutagenesis that affect HDL cholesterol.

Despite considerable progress understanding genes that affect the HDL particle, its function, and cholesterol content, genes identified to date explain only a small percentage of the genetic variation. We used N-ethyl-N-nitrosourea mutagenesis in mice to discover novel genes that affect HDL cholesterol levels. Two mutant lines (Hlb218 and Hlb320) with low HDL cholesterol levels were established. Causal mutations in these lines were mapped using linkage analysis: for line Hlb218 within a 12 Mbp region on Chr 10; and for line Hlb320 within a 21 Mbp region on Chr 7. High-throughput sequencing of Hlb218 liver RNA identified a mutation in Pla2g12b. The transition of G to A leads to a cysteine to tyrosine change and most likely causes a loss of a disulfide bridge. Microarray analysis of Hlb320 liver RNA showed a 7-fold downregulation of Hpn; sequencing identified a mutation in the 3' splice site of exon 8. Northern blot confirmed lower mRNA expression level in Hlb320 and did not show a difference in splicing, suggesting that the mutation only affects the splicing rate. In addition to affecting HDL cholesterol, the mutated genes also lead to reduction in serum non-HDL cholesterol and triglyceride levels. Despite low HDL cholesterol levels, the mice from both mutant lines show similar atherosclerotic lesion sizes compared to control mice. These new mutant mouse models are valuable tools to further study the role of these genes, their affect on HDL cholesterol levels, and metabolism.