Regulation of UGT2B Expression and Activity by miR-216b-5p in Liver Cancer Cell Lines.

The UDP-glucuronosyltransferase (UGT) 2B enzymes are important in the detoxification of a variety of endogenous and exogenous compounds, including many hormones, drugs, and carcinogens. Identifying novel mechanisms governing their expression is important in understanding patient-specific response to drugs and cancer risk factors. In silico prediction algorithm programs were used to screen for microRNAs (miRNAs) as potential regulators of UGT2B enzymes, with miR-216b-5p identified as a potential candidate. Luciferase data suggested the presence of a functional miR-216b-5p binding motif within the 3' untranslated regions of UGTs 2B7, 2B4, and 2B10. Overexpression of miR-216b-5p mimics significantly repressed UGT2B7 (P < 0.001) and UGT2B10 (P = 0.0018) mRNA levels in HuH-7 cells and UGT2B4 (P < 0.001) and UGT2B10 (P = 0.018) mRNA in Hep3B cells. UGT2B7 protein levels were repressed in both HuH-7 and Hep3B cells in the presence of increasing miR-216b-5p concentrations, corresponding with significant (P < 0.001 and P = 0.011, respectively) decreases in glucuronidation activity against the UGT2B7-specific substrate epirubicin. Inhibition of endogenous miR-216b-5p levels significantly increased UGT2B7 mRNA levels in HuH-7 (P = 0.021) and Hep3B (P = 0.0068) cells, and increased epirubicin glucuronidation by 85% (P = 0.057) and 50% (P = 0.012) for HuH-7 and Hep3B cells, respectively. UGT2B4 activity against codeine and UGT2B10 activity against nicotine were significantly decreased in both HuH-7 and Hep3B cells (P < 0.001 and P = 0.0048, and P = 0.017 and P = 0.043, respectively) after overexpression of miR-216b-5p mimic. This is the first evidence that miRNAs regulate UGT 2B7, 2B4, and 2B10 expression, and that miR-216b-5p regulation of UGT2B proteins may be important in regulating the metabolism of UGT2B substrates.

MicroRNA regulation of the major drug-metabolizing enzymes and related transcription factors.

Identifying novel mechanisms contributing to patient variability of drug response is a major goal of personalized medicine. Epigenetic regulation of gene expression by microRNA (miRNA) impacts a broad range of cellular processes, but knowledge of its regulation of drug-metabolizing enzymes (DMEs) is more limited. This review provides an introduction to miRNA and their functionality and summarizes known miRNA regulation of DME families, including the cytochrome P450s, UDP-glucuronoslytransferases, glutathione-S-transferases, sulfotransferases and aldo-keto reductases, and the transcription factors known to be involved in DME regulation.

Regulation of UDP-glucuronosyltransferase 1A1 expression and activity by microRNA 491-3p.

The UDP-glucuronosyltransferase (UGT) 1A enzymes are involved in the phase II metabolism of many important endogenous and exogenous compounds. The nine UGT1A isoforms exhibit high interindividual differences in expression, but their epigenetic regulation is not well understood. The purpose of the present study was to examine microRNA (miRNA) regulation of hepatic UGT1A enzymes and determine whether or not that regulation impacts enzymatic activity. In silico analysis identified miRNA 491-3p (miR-491-3p) as a potential regulator of the UGT1A gene family via binding to the shared UGT1A 3'-untranslated region common to all UGT1A enzymes. Transfection of miR-491-3p mimic into HuH-7 cells significantly repressed UGT1A1 (P < 0.001), UGT1A3 (P < 0.05), and UGT1A6 (P < 0.05) mRNA levels. For UGT1A1, this repression correlated with significantly reduced metabolism of raloxifene into raloxifene-6-glucuronide (ral-6-gluc; P < 0.01) and raloxifene-4'-glucuronide (ral-4'-gluc; P < 0.01). In HuH-7 cells with repressed miR-491-3p expression, there was a significant increase (~80%; P < 0.01) in UGT1A1 mRNA and a corresponding increase in glucuronidation of raloxifene into ral-6-gluc (50%; P < 0.05) and ral-4'-gluc (22%; P < 0.01). Knockdown of endogenous miR-491-3p in HepG2 cells did not significantly alter UGT1A1 mRNA levels but did increase the formation of ral-6-gluc (50%; P < 0.05) and ral-4'-gluc (34%; P < 0.001). A significant inverse correlation between miR-491-3p expression and both UGT1A3 (P < 0.05) and UGT1A6 (P < 0.01) mRNA levels was observed in a panel of normal human liver specimens, with a significant (P < 0.05) increase in UGT1A3 and UGT1A6 mRNA levels observed in miR-491-3p nonexpressing versus expressing liver specimens. These results suggest that miR-491-3p is an important factor in regulating the expression of UGT1A enzymes in vivo.

Importance of UDP-glucuronosyltransferases 2A2 and 2A3 in tobacco carcinogen metabolism.

UDP-glucuronosyltransferase A1 (UGT2A1) is expressed in the lung and exhibits activity against polycyclic aromatic hydrocarbons (PAHs), suggesting UGT2A1 involvement in the local metabolism of PAH tobacco carcinogens. The goal of the present study was to investigate the importance of two additional UGT2A enzymes, UGT2A2 and UGT2A3, in tobacco carcinogen metabolism. Real-time polymerase chain reaction suggested that wild-type UGT2A2 had the highest expression in the breast, followed by trachea > larynx > kidney. A novel splice variant of UGT2A2 lacking exon 3 (termed UGT2A2Δexon3) was investigated, with UGT2A2Δexon3 expression determined to be 25-50% that of wild-type UGT2A2 in all tissues examined. UGT2A3 was determined to be well expressed in the liver and colon, followed by pancreas > kidney > lung > tonsil > trachea > larynx. Cell homogenates prepared from human embryonic kidney (HEK)293 cells overexpressing wild-type UGT2A2 (termed UGT2A2_i1) exhibited glucuronidation activity, as observed by reverse-phase ultra-pressure liquid chromatography, against 1-hydroxy-(OH)-pyrene, 1-naphthol, and hydroxylated benzo(a)pyrene metabolites, whereas homogenates prepared from HEK293 cells overexpressing UGT2A3 only showed activity against simple PAHs like 1-OH-pyrene and 1-naphthol. Activity assays showed the UGT2A2Δexon3 protein (termed UGT2A2_i2) exhibited no detectable glucuronidation activity against all substrates examined; however, coexpression studies suggested that UGT2A2_i2 negatively modulates UGT2A2_i1 activity. Both UGT2A2 and UGT2A3 exhibited no detectable activity against complex PAH proximate carcinogens, tobacco-specific nitrosamines, or heterocyclic amines. These data suggest that, although UGT2A1 is the only UGT2A enzyme active against PAH proximate carcinogens (including PAH diols), both UGTs 2A1 and 2A2 play an important role in the local detoxification of procarcinogenic monohydroxylated PAH metabolites.

A nonparametric alternative to the Cochran-Armitage trend test in genetic case-control association studies: The Jonckheere-Terpstra trend test.

Identifications of novel genetic signals conferring susceptibility to human complex diseases is pivotal to the disease diagnosis, prevention, and treatment. Genetic association study is a powerful tool to discover candidate genetic signals that contribute to diseases, through statistical tests for correlation between the disease status and genetic variations in study samples. In such studies with a case-control design, a standard practice is to perform the Cochran-Armitage (CA) trend test under an additive genetic model, which suffers from power loss when the model assumption is wrong. The Jonckheere-Terpstra (JT) trend test is an alternative method to evaluate association in a nonparametric way. This study compares the power of the JT trend test and the CA trend test in various scenarios, including different sample sizes (200-2000), minor allele frequencies (0.05-0.4), and underlying modes of inheritance (dominant genetic model to recessive genetic model). By simulation and real data analysis, it is shown that in general the JT trend test has higher, similar, and lower power than the CA trend test when the underlying mode of inheritance is dominant, additive, and recessive, respectively; when the sample size is small and the minor allele frequency is low, the JT trend test outperforms the CA trend test across the spectrum of genetic models. In sum, the JT trend test is a valuable alternative to the CA trend test under certain circumstances with higher statistical power, which could lead to better detection of genetic signals to human diseases and finer dissection of their genetic architecture.

Angiotensin II Increases Oxidative Stress and Inflammation in Female, But Not Male, Endothelial Cells.

Introduction: Women are at elevated risk for certain cardiovascular diseases, including pulmonary arterial hypertension, Alzheimer's disease, and vascular complications of diabetes. Angiotensin II (AngII), a circulating stress hormone, is elevated in cardiovascular disease; however, our knowledge of sex differences in the vascular effects of AngII are limited. We therefore analyzed sex differences in human endothelial cell response to AngII treatment. Methods: Male and female endothelial cells were treated with AngII for 24 h and analyzed by RNA sequencing. We then used endothelial and mesenchymal markers, inflammation assays, and oxidative stress indicators to measure female and male endothelial cell functional changes in response to AngII. Results: Our data show that female and male endothelial cells are transcriptomically distinct. Female endothelial cells treated with AngII had widespread gene expression changes related to inflammatory and oxidative stress pathways, while male endothelial cells had few gene expression changes. While both female and male endothelial cells maintained their endothelial phenotype with AngII treatment, female endothelial cells showed increased release of the inflammatory cytokine interleukin-6 and increased white blood cell adhesion following AngII treatment concurrent with a second inflammatory cytokine. Additionally, female endothelial cells had elevated reactive oxygen species production compared to male endothelial cells after AngII treatment, which may be partially due to nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX2) escape from X-chromosome inactivation. Conclusions: These data suggest that endothelial cells have sexually dimorphic responses to AngII, which could contribute to increased prevalence of some cardiovascular diseases in women. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00762-2.

Atherosclerotic Aortic Calcification-Associated Polymorphism in HDAC9 and Associations with Mortality, Cardiovascular Disease, and Kidney Disease.

Histone deacetylase 9 (HDAC9) has recently been demonstrated as a key regulator of vascular smooth muscle cell (VSMC) phenotype and is associated with abdominal aortic calcification, myocardial infarction, and ischemic stroke. It is uncertain whether HDAC9 is also implicated in other VSMC-driven diseases. Our objective was to assess associations between abdominal aortic calcification-associated genetic variation in HDAC9 and VSMC-associated phenotypes. In this prospective population study of 335,146 adults enrolled in the UK Biobank, the abdominal aortic calcification-associated risk allele of a genetic variant in HDAC9 was associated with increased risk of systolic hypertension, non-ST segment elevation myocardial infarction, and ischemic stroke. There was a suggestive protective association with kidney disease outcomes that did not reach experiment-wise significance. These genetic results lend further support for HDAC9 as a potential therapeutic target for arterial stenotic and calcific disease.

MicroRNA-1253 Regulation of WASF2 (WAVE2) and its Relevance to Racial Health Disparities.

The prevalence of hypertension among African Americans (AAs) in the US is among the highest of any demographic and affects over two-thirds of AA women. Previous data from our laboratory suggest substantial differential gene expression (DGE) of mRNAs and microRNAs (miRNAs) exists within peripheral blood mononuclear cells (PBMCs) isolated from AA and white women with or without hypertension. We hypothesized that DGE by race may contribute to racial differences in hypertension. In a reanalysis of our previous dataset, we found that the Wiskott-Aldrich syndrome protein Verprolin-homologous protein 2 (WASF2 (also known as WAVE2)) is differentially expressed in AA women with hypertension, along with several other members of the actin cytoskeleton signaling pathway that plays a role in cell shape and branching of actin filaments. We performed an in silico miRNA target prediction analysis that suggested miRNA miR-1253 regulates WASF2. Transfection of miR-1253 mimics into human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) significantly repressed WASF2 mRNA and protein levels (p < 0.05), and a luciferase reporter assay confirmed that miR-1253 regulates the WASF2 3' UTR (p < 0.01). miR-1253 overexpression in HUVECs significantly increased HUVEC lamellipodia formation (p < 0.01), suggesting the miR-1253-WASF2 interaction may play a role in cell shape and actin cytoskeleton function. Together, we have identified novel roles for miR-1253 and WASF2 in a hypertension-related disparities context. This may ultimately lead to the discovery of additional actin-related genes which are important in the vascular-related complications of hypertension and influence the disproportionate susceptibility to hypertension among AAs in general and AA women in particular.

The association between poverty and gene expression within peripheral blood mononuclear cells in a diverse Baltimore City cohort.

Socioeconomic status (SES), living in poverty, and other social determinants of health contribute to health disparities in the United States. African American (AA) men living below poverty in Baltimore City have a higher incidence of mortality when compared to either white males or AA females living below poverty. Previous studies in our laboratory and elsewhere suggest that environmental conditions are associated with differential gene expression (DGE) patterns in peripheral blood mononuclear cells (PBMCs). DGE have also been associated with hypertension and cardiovascular disease (CVD) and correlate with race and sex. However, no studies have investigated how poverty status associates with DGE between male and female AAs and whites living in Baltimore City. We examined DGE in 52 AA and white participants of the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) cohort, who were living above or below 125% of the 2004 federal poverty line at time of sample collection. We performed a microarray to assess DGE patterns in PBMCs from these participants. AA males and females living in poverty had the most genes differentially-expressed compared with above poverty controls. Gene ontology (GO) analysis identified unique and overlapping pathways related to the endosome, single-stranded RNA binding, long-chain fatty-acyl-CoA biosynthesis, toll-like receptor signaling, and others within AA males and females living in poverty and compared with their above poverty controls. We performed RT-qPCR to validate top differentially-expressed genes in AA males. We found that KLF6, DUSP2, RBM34, and CD19 are expressed at significantly lower levels in AA males in poverty and KCTD12 is higher compared to above poverty controls. This study serves as an additional link to better understand the gene expression response in peripheral blood mononuclear cells in those living in poverty.

Extracellular RNA profiles with human age.

Circulating extracellular RNAs (exRNAs) are potential biomarkers of disease. We thus hypothesized that age-related changes in exRNAs can identify age-related processes. We profiled both large and small RNAs in human serum to investigate changes associated with normal aging. exRNA was sequenced in 13 young (30-32 years) and 10 old (80-85 years) African American women to identify all RNA transcripts present in serum. We identified age-related differences in several RNA biotypes, including mitochondrial transfer RNAs, mitochondrial ribosomal RNA, and unprocessed pseudogenes. Age-related differences in unique RNA transcripts were further validated in an expanded cohort. Pathway analysis revealed that EIF2 signaling, oxidative phosphorylation, and mitochondrial dysfunction were among the top pathways shared between young and old. Protein interaction networks revealed distinct clusters of functionally-related protein-coding genes in both age groups. These data provide timely and relevant insight into the exRNA repertoire in serum and its change with aging.

Extracellular RNA in aging.

Since the discovery of extracellular RNA (exRNA) in circulation and other bodily fluids, there has been considerable effort to catalog and assess whether exRNAs can be used as markers for health and disease. A variety of exRNA species have been identified including messenger RNA and noncoding RNA such as microRNA (miRNA), small nucleolar RNA, transfer RNA, and long noncoding RNA. Age-related changes in exRNA abundance have been observed, and it is likely that some of these transcripts play a role in aging. In this review, we summarize the current state of exRNA profiling in various body fluids and discuss age-related changes in exRNA abundance that have been identified in humans and other model organisms. miRNAs, in particular, are a major focus of current research and we will highlight and discuss the potential role that specific miRNAs might play in age-related phenotypes and disease. We will also review challenges facing this emerging field and various strategies that can be used for the validation and future use of exRNAs as markers of aging and age-related disease. WIREs RNA 2017, 8:e1385. doi: 10.1002/wrna.1385 For further resources related to this article, please visit the WIREs website.

MicroRNAs Modulate Oxidative Stress in Hypertension through PARP-1 Regulation.

Oxidative stress is thought to contribute to aging and age-related diseases, such as cardiovascular and neurodegenerative diseases, and is a risk factor for systemic arterial hypertension. Previously, we reported differential mRNA and microRNA (miRNA) expression between African American (AA) and white women with hypertension. Here, we found that the poly-(ADP-ribose) polymerase 1 (PARP-1), a DNA damage sensor protein involved in DNA repair and other cellular processes, is upregulated in AA women with hypertension. To explore this mechanism, we identified two miRNAs, miR-103a-2-5p and miR-585-5p, that are differentially expressed with hypertension and were predicted to target PARP1. Through overexpression of each miRNA-downregulated PARP-1 mRNA and protein levels and using heterologous luciferase reporter assays, we demonstrate that miR-103a-2-5p and miR-585-5p regulate PARP1 through binding within the coding region. Given the important role of PARP-1 in DNA repair, we assessed whether overexpression of miR-103a-2-5p or miR-585-5p affected DNA damage and cell survival. Overexpression of these miRNAs enhanced DNA damage and decreased both cell survival and colony formation. These findings highlight the role for PARP-1 in regulating oxidative DNA damage in hypertension and identify important new miRNA regulators of PARP-1 expression. These insights may provide additional avenues to understand hypertension health disparities.

Metformin-mediated increase in DICER1 regulates microRNA expression and cellular senescence.

Metformin, an oral hypoglycemic agent, has been used for decades to treat type 2 diabetes mellitus. Recent studies indicate that mice treated with metformin live longer and have fewer manifestations of age-related chronic disease. However, the molecular mechanisms underlying this phenotype are unknown. Here, we show that metformin treatment increases the levels of the microRNA-processing protein DICER1 in mice and in humans with diabetes mellitus. Our results indicate that metformin upregulates DICER1 through a post-transcriptional mechanism involving the RNA-binding protein AUF1. Treatment with metformin altered the subcellular localization of AUF1, disrupting its interaction with DICER1 mRNA and rendering DICER1 mRNA stable, allowing DICER1 to accumulate. Consistent with the role of DICER1 in the biogenesis of microRNAs, we found differential patterns of microRNA expression in mice treated with metformin or caloric restriction, two proven life-extending interventions. Interestingly, several microRNAs previously associated with senescence and aging, including miR-20a, miR-34a, miR-130a, miR-106b, miR-125, and let-7c, were found elevated. In agreement with these findings, treatment with metformin decreased cellular senescence in several senescence models in a DICER1-dependent manner. Metformin lowered p16 and p21 protein levels and the abundance of inflammatory cytokines and oncogenes that are hallmarks of the senescence-associated secretory phenotype (SASP). These data lead us to hypothesize that changes in DICER1 levels may be important for organismal aging and to propose that interventions that upregulate DICER1 expression (e.g., metformin) may offer new pharmacotherapeutic approaches for age-related disease.

Racial differences in microRNA and gene expression in hypertensive women.

Systemic arterial hypertension is an important cause of cardiovascular disease morbidity and mortality. African Americans are disproportionately affected by hypertension, in fact the incidence, prevalence, and severity of hypertension is highest among African American (AA) women. Previous data suggests that differential gene expression influences individual susceptibility to selected diseases and we hypothesized that this phenomena may affect health disparities in hypertension. Transcriptional profiling of peripheral blood mononuclear cells from AA or white, normotensive or hypertensive females identified thousands of mRNAs differentially-expressed by race and/or hypertension. Predominant gene expression differences were observed in AA hypertensive females compared to AA normotensives or white hypertensives. Since microRNAs play important roles in regulating gene expression, we profiled global microRNA expression and observed differentially-expressed microRNAs by race and/or hypertension. We identified novel mRNA-microRNA pairs potentially involved in hypertension-related pathways and differently-expressed, including MCL1/miR-20a-5p, APOL3/miR-4763-5p, PLD1/miR-4717-3p, and PLD1/miR-4709-3p. We validated gene expression levels via RT-qPCR and microRNA target validation was performed in primary endothelial cells. Altogether, we identified significant gene expression differences between AA and white female hypertensives and pinpointed novel mRNA-microRNA pairs differentially-expressed by hypertension and race. These differences may contribute to the known disparities in hypertension and may be potential targets for intervention.

Posttranscriptional Regulation of the Inflammatory Marker C-Reactive Protein by the RNA-Binding Protein HuR and MicroRNA 637.

C-reactive protein (CRP), an acute-phase plasma protein, is a major component of inflammatory reactions functioning as a mediator of innate immunity. It has been widely used as a validated clinical biomarker of the inflammatory state in trauma, infection, and age-associated chronic diseases, including cancer and cardiovascular disease (CVD). Despite this, the molecular mechanisms that regulate CRP expression are not well understood. Given that the CRP 3' untranslated region (UTR) is long and AU rich, we hypothesized that CRP may be regulated posttranscriptionally by RNA-binding proteins (RBPs) and by microRNAs. Here, we found that the RBP HuR bound directly to the CRP 3' UTR and affected CRP mRNA levels. Through this interaction, HuR selectively increased CRP mRNA stability and promoted CRP translation. Interestingly, treatment with the age-associated inflammatory cytokine interleukin-6 (IL-6) increased binding of HuR to CRP mRNA, and conversely, HuR was required for IL-6-mediated upregulation of CRP expression. In addition, we identified microRNA 637 (miR-637) as a microRNA that potently inhibited CRP expression in competition with HuR. Taken together, we have uncovered an important posttranscriptional mechanism that modulates the expression of the inflammatory marker CRP, which may be utilized in the development of treatments for inflammatory processes that cause CVD and age-related diseases.

Glucuronidation genotypes and nicotine metabolic phenotypes: importance of functional UGT2B10 and UGT2B17 polymorphisms.

Glucuronidation is an important pathway in the metabolism of nicotine, with previous studies suggesting that ∼22% of urinary nicotine metabolites are in the form of glucuronidated compounds. Recent in vitro studies have suggested that the UDP-glucuronosyltransferases (UGT) 2B10 and 2B17 play major roles in nicotine glucuronidation with polymorphisms in both enzymes shown to significantly alter the levels of nicotine-glucuronide, cotinine-glucuronide, and trans-3'-hydroxycotinine (3HC)-glucuronide in human liver microsomes in vitro. In the present study, the relationship between the levels of urinary nicotine metabolites and functional polymorphisms in UGTs 2B10 and 2B17 was analyzed in urine specimens from 104 Caucasian smokers. Based on their percentage of total urinary nicotine metabolites, the levels of nicotine-glucuronide and cotinine-glucuronide were 42% (P < 0.0005) and 48% (P < 0.0001), respectively, lower in the urine from smokers exhibiting the UGT2B10 (*1/*2) genotype and 95% (P < 0.05) and 98% (P < 0.05), respectively, lower in the urine from smokers with the UGT2B10 (*2/*2) genotype compared with the urinary levels in smokers having the wild-type UGT2B10 (*1/*1) genotype. The level of 3HC-glucuronide was 42% (P < 0.001) lower in the urine from smokers exhibiting the homozygous UGT2B17 (*2/*2) deletion genotype compared with the levels in urine from wild-type UGT2B17 subjects. These data suggest that UGTs 2B10 and 2B17 play important roles in the glucuronidation of nicotine, cotinine, and 3HC and suggest that the UGT2B10 codon 67 SNP and the UGT2B17 gene deletion significantly reduce overall glucuronidation rates of nicotine and its major metabolites in smokers.