Dicer Expression Research Articles

UVB radiation suppresses Dicer expression through β-catenin.

Ultraviolet (UV) rays prompt a natural response in epidermal cells, particularly within melanocytes. The changes in gene expression and related signaling pathways in melanocytes following exposure to UVR are still not entirely understood. Our findings reveal that UVB irradiation suppresses the expression of Dicer. This repression is intricately linked to the activation of the PI3K, RSK, and WNT/β-catenin signaling pathways and is directly associated with transcriptional repression by β-catenin. Notably, we have identified specific binding sites for the LEF/β-catenin complex in the Dicer promoter. Collectively, these results emphasize the significance of the UV-induced pathway involving LEF/β-catenin, which impacts Dicer expression. UV radiation also reduced the levels of specific miRNAs known to be important in the biology of melanocytes. This pathway holds potential importance in governing melanocyte physiology.

SARS-CoV-2 N protein-induced Dicer, XPO5, SRSF3, and hnRNPA3 downregulation causes pneumonia

Though RNAi and RNA-splicing machineries are involved in regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, their precise roles in coronavirus disease 2019 (COVID-19) pathogenesis remain unclear. Herein, we show that decreased RNAi component (Dicer and XPO5) and splicing factor (SRSF3 and hnRNPA3) expression correlate with increased COVID-19 severity. SARS-CoV-2 N protein induces the autophagic degradation of Dicer, XPO5, SRSF3, and hnRNPA3, inhibiting miRNA biogenesis and RNA splicing and triggering DNA damage, proteotoxic stress, and pneumonia. Dicer, XPO5, SRSF3, and hnRNPA3 knockdown increases, while their overexpression decreases, N protein-induced pneumonia’s severity. Older mice show lower expression of Dicer, XPO5, SRSF3, and hnRNPA3 in their lung tissues and exhibit more severe N protein-induced pneumonia than younger mice. PJ34, a poly(ADP-ribose) polymerase inhibitor, or anastrozole, an aromatase inhibitor, ameliorates N protein- or SARS-CoV-2-induced pneumonia by restoring Dicer, XPO5, SRSF3, and hnRNPA3 expression. These findings will aid in developing improved treatments for SARS-CoV-2-associated pneumonia.

Inhibiting essential elements of the DNA methylation pathway negatively impacts the miRNA pathway and fitness of the whitefly Bemisia tabaci

DNA methylation is an epigenetic process that involves the chemical modification of DNA, leading to the regulation of its transcriptional activity. It is primarily known for the addition of methyl groups to cytosine in DNA. The whitefly Bemisia tabaci is a polyphagous pest insect and a vector that is responsible for transmitting numerous plant viruses, resulting in significant economic losses in agricultural crops globally. In our study, we characterized the expression of two key DNA methylation genes, the DNA methyltransferases Dnmt1 and Dnmt3, in B. tabaci. Additionally, we explored the impact of inhibiting DNMTs on the miRNA pathway and fitness of whitefly. To investigate the role of the DNA methylation pathway in B. tabaci, we found that the expression of Dnmt1 and Dnmt3 varied across different tissues and developmental stages of B. tabaci. We employed azacytidine (5-AZA) treatment of adults to inhibit DNMTs (DNMT1 and DNMT3). Administration of 5-AZA affected the survival and reproduction of this pest. Moreover, inhibition of DNMTs led to a decrease in the expression of the miRNA pathway core genes Dicer1 and Argonaute1, which subsequently resulted in reduced expression of Let-7 and miR-184 which are essential microRNAs in the physiology and biology of insects. The study suggests that DNA methyltransferases could be targeted for developing an inhibition strategy to control this pest and vector insect.

Evaluation of expression levels of microRNA processing elements in patients with sudden sensorineural hearing loss

BackgroundMicroRNAs have a significant role in the function and development of the hearing system. Idiopathic sudden sensorineural hearing loss (SSNHL) is a complicated disorder with no long-established reason. Since microRNAs play imperative roles in every aspect of the neural system, their dysregulation may contribute to the onset of SSNHL. The current study aimed to assess the expression patterns of microRNA processing elements (DROSHA, DICER, and DGCR-8) as the vital factors in microRNA biology that can affect the expression levels of microRNA. This study assessed DROSHA, DICER, and DGCR-8 mRNA expression levels in the peripheral blood mononuclear cells (PBMC) of 50 patients with SSNHL and 50 matched controls. After the isolation of PBMC, total RNA was extracted, and the expression levels of DROSHA, DICER, and DGCR-8 genes were evaluated using quantitative real-time PCR.ResultsThe results illustrated significant up-regulation of DICER and DGCR-8 genes in SSNHL patients at the mRNA level. Furthermore, despite no significant change in DROSHA level, DICER and DGCR-8 were significantly correlated with SSNHL. However, there was no significant correlation between these gene expressions and the clinicopathological features of patients.ConclusionThis study verified for the first time that the DGCR_8 and DICER mRNA expression levels were significantly up-regulated in patients with SSNHL, proposing that microRNAs and their processing pathways play key roles in the progression and development of SSNHL.

AMPK-upregulated microRNA-708 plays as a suppressor of cellular senescence and aging via downregulating disabled-2 and mTORC1 activation.

Senescence-associated microRNAs (SA-miRNAs) are important molecules for aging regulation. While many aging-promoting SA-miRNAs have been identified, confirmed aging-suppressive SA-miRNAs are rare, that impeded our full understanding on aging regulation. In this study, we verified that miR-708 expression is decreased in senescent cells and aged tissues and revealed that miR-708 overexpression can alleviate cellular senescence and aging performance. About the molecular cascade carrying the aging suppressive action of miR-708, we unraveled that miR-708 directly targets the 3'UTR of the disabled 2 (Dab2) gene and inhibits the expression of DAB2. Interestingly, miR-708-caused DAB2 downregulation blocks the aberrant mammalian target of rapamycin complex 1 (mTORC1) activation, a driving metabolic event for senescence progression, and restores the impaired autophagy, a downstream event of aberrant mTORC1 activation. We also found thatAMP-activated protein kinase (AMPK) activation can upregulate miR-708 via the elevation of DICER expression, and miR-708 inhibitor is able to blunt the antiaging effect of AMPK. In summary, this study characterized miR-708 as an aging-suppressive SA-miRNA for the first time and uncovered a new signaling cascade, in which miR-708 links the DAB2/mTOR axis and AMPK/DICER axis together. These findings not only demonstrate the potential role of miR-708 in aging regulation, but also expand the signaling network connecting AMPK and mTORC1.

Environmental arsenic pollution induced liver oxidative stress injury by regulating miR-155 through inhibition of AUF1

Arsenic (As), a common environmental pollutant, has become a hot topic in recent years due to its potentially harmful effects. Liver damage being a central clinical feature of chronic arsenic poisoning. However, the underlying mechanisms remain unclear. We demonstrated that arsenic can lead to oxidative stress in the liver and result in structural and functional liver damage, significantly correlated with the expression of AUF1, Dicer1, and miR-155 in the liver. Interestingly, knockdown AUF1 promoted the up-regulatory effects of arsenic on Dicer1 and miR-155 and the inhibitory effects on SOD1, which exacerbated oxidative damage in rat liver. However, overexpression of AUF1 reversed the up-regulatory effects of arsenic on Dicer1 and miR-155, restored arsenic-induced SOD1 depletion, and attenuated liver oxidative stress injury. Further, we verified the mechanism and targets of miR-155 in regulating SOD1 by knockdown/overexpression of miR-155 and nonsense mutant SOD1 3’UTR experiments. In conclusion, these results powerfully demonstrate that arsenic inhibits AUF1 protein expression, which in turn reduces the inhibitory effect on Dicer1 expression, which promotes miR-155 to act on the SOD1 3’UTR region after high expression, thus inhibiting SOD1 protein expression and enzyme activity, and inducing liver injury. This finding provides a new perspective for the mechanism research and targeted prevention of arsenic poisoning, as well as scientific evidence for formulating strategies to prevent and control environmental arsenic pollution.

MiRNome targeting NF-κB signaling orchestrates macrophage-triggered cancer metastasis and recurrence

Whether and how tumor intrinsic signature determines macrophage-elicited metastasis remain elusive. Here, we show, in detailed studies of data regarding 7,477 patients of 20 types of human cancers, that only 13.8%± 2.6%/27.9%± 3.03% of patients with high macrophage infiltration index exhibit early recurrence/vascular invasion. In parallel, although macrophages enhance the motility of various hepatoma cells, their enhancement intensity is significantly heterogeneous. We identify that the expression of malignant Dicer, a ribonuclease that cleaves miRNA precursors into mature miRNAs, determines macrophage-elicited metastasis. Mechanistically, the downregulation of Dicer in cancer cells leads to defects in miRNome targeting NF-κB signaling, which in turn enhances the ability of cancer cells to respond to macrophage-related inflammatory signals and ultimately promotes metastasis. Importantly, transporting miR-26b-5p, the most potential miRNA targeting NF-κB signaling in hepatocellular carcinoma, can effectively reverse macrophage-elicited metastasis of hepatoma invivo. Our results provide insights into the crosstalk between Dicer-elicited miRNome and cancer immune microenvironments and suggest that strategies to remodel malignant cell miRNome may overcome pro-tumorigenic activities of inflammatory cells.

Resveratrol modulates miRNA machinery proteins in different types of colon cancer cells

Abstract Objectives Resveratrol (RSV) is a stilbenoid compound that shows anticancer activity in many cancer cells. Exosomes might affect carcinogenesis and the development of colorectal cancer by affecting communication between tumor cells in the tumor microenvironment via their cargo content miRNA. The aim of this study is to determine the effect of RSV on the expression of Dicer, Ago2, eIf2α, CD-9, CD-63, and exosomal miRNA levels in COLO320 and COLO741 colon cancer cell lines. Methods The MTT method was used for cell growth and cytotoxicity in both COLO320 and COLO741 cell lines. Dicer, Ago2, eIF2α, CD-9, and CD-63 antibodies were used for the immunocytochemical evaluation. Total miRNA analysis was performed using a miRCURY Exosome Isolation Kit. Results As a result of immunocytochemical staining, increased CD-63 immunoreactivity was observed in RSV-treated COLO320 cells vs. RSV-treated COLO-741 cells. Dicer immunoreactivity increased after the RSV treatment in COLO320 cells. Higher eIF2α immunoreactivity was observed in RSV-treated COLO741 cells compared to both COLO741 control cells and RSV-treated COLO320 cells. Non-significant decreases were observed in miRNA concentration in RSV-treated COLO320 and COLO741 cells compared to control group cells. Conclusions RSV could increase miRNA biogenesis in COLO320 cancer cells and decrease it in COLO741 cancer cells.

RNA-seq–based miRNA signature as an independent predictor of relapse in pediatric B-cell acute lymphoblastic leukemia

AbstractAberrant micro-RNA (miRNA) expression profiles have been associated with disease progression and clinical outcome in pediatric cancers. However, few studies have analyzed genome-wide dysregulation of miRNAs and messenger RNAs (mRNAs) in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). To identify novel prognostic factors, we comprehensively investigated miRNA and mRNA sequencing (miRNA-seq and mRNA-seq) data in pediatric BCP-ALL samples with poor outcome. We analyzed 180 patients, including 43 matched pairs at diagnosis and relapse. Consensus clustering of miRNA expression data revealed a distinct profile characterized by mainly downregulation of miRNAs (referred to as an miR-low cluster [MLC]). The MLC profile was not associated with any known genetic subgroups. Intriguingly, patients classified as MLC had significantly shorter event-free survival (median 21 vs 33 months; log-rank P = 3 ×10−5). Furthermore, this poor prognosis was retained even in hyperdiploid ALL. This poor prognostic MLC profiling was confirmed in the validation cohort. Notably, non-MLC profiling at diagnosis (n = 9 of 23; Fisher exact test, P = .039) often changed into MLC profiling at relapse for the same patient. Integrated analysis of miRNA-seq and mRNA-seq data revealed that the transcriptional profile of MLC was characterized by enrichment of MYC target and oxidative phosphorylation genes, reduced intron retention, and low expression of DICER1. Thus, our miRNA-mRNA integration approach yielded a truly unbiased molecular stratification of pediatric BCP-ALL cases based on a novel prognostic miRNA signature, which may lead to better clinical outcomes.

Silencing of Dicer enhances dacarbazine resistance in melanoma cells by inhibiting ADSL expression.

Dacarbazine (DTIC) is the primary first-line treatment for advanced-stage metastatic melanoma; thus, DTIC resistance is poses a major challenge. Therefore, investigating the mechanism underlying DTIC resistance must be investigated. Dicer, a type III cytoplasmic endoribonuclease, plays a pivotal role in the maturation of miRNAs. Aberrant Dicer expression may contribute to tumor progression, clinical aggressiveness, and poor prognosis in various tumors. Dicer inhibition led to a reduction in DTIC sensitivity and an augmentation in stemness in melanoma cells. Clinical analyses indicated a low Dicer expression level as a predictor of poor prognosis factor. Metabolic alterations in tumor cells may interfere with drug response. Adenylosuccinate lyase (ADSL) is a crucial enzyme in the purine metabolism pathway. An imbalance in ADSL may interfere with the therapeutic efficacy of drugs. We discovered that DTIC treatment enhanced ADSL expression and that Dicer silencing significantly reduced ADSL expression in melanoma cells. Furthermore, ADSL overexpression reversed Dicer silencing induced DTIC resistance and cancer stemness. These findings indicate that Dicer-mediated ADSL regulation influences DTIC sensitivity and stemness in melanoma cells.

USP7 reduces the level of nuclear DICER, impairing DNA damage response and promoting cancer progression.

Endoribonuclease DICER is an RNase III enzyme that mainly processes microRNAs in the cytoplasm but also participates in nuclear functions such as chromatin remodelling, epigenetic modification and DNA damage repair. The expression of nuclear DICER is low in most human cancers, suggesting a tight regulation mechanism that is not well understood. Here, we found that ubiquitin carboxyl-terminal hydrolase 7 (USP7), a deubiquitinase, bounded to DICER and reduced its nuclear protein level by promoting its ubiquitination and degradation through MDM2, a newly identified E3 ubiquitin-protein ligase for DICER. This USP7-MDM2-DICER axis impaired histone γ-H2AX signalling and the recruitment of DNA damage response (DDR) factors, possibly by influencing the processing of small DDR noncoding RNAs. We also showed that this negative regulation of DICER by USP7 via MDM2 was relevant to human tumours using cellular and clinical data. Our findings revealed a new way to understand the role of DICER in malignant tumour development and may offer new insights into the diagnosis, treatment and prognosis of cancers.

Unraveling The Effects of DICER1 Overexpression on Immune-Related Genes Expression in Mesenchymal Stromal/Stem Cells: Insights for Therapeutic Applications.

The immunoregulatory properties of mesenchymal stromal/stem cells (MSCs) bring a promise for the treatment of inflammatory diseases. However, their ability to suppress the immune system is unstable. To enhance their effectiveness against immune responses, it may be necessary to manipulate MSCs. Although some dsRNA transcripts come from invading viruses, the majority of dsRNA has an endogenous origin and is known as endo-siRNA. DICER1 is a ribonuclease protein that can generate small RNAs to modulate gene expression at the post-transcriptional level. We aimed to evaluate the expression of several immune-related genes at mRNA and protein levels in MSCs overexpressing DICER1 exogenously. In this comparative transcriptomic experimental study, the adipose-derived MSCs (Ad-MSCs) were transfected using the pCAGGS-Flag-hsDicer vector for the DICER1 overexpression. Following the RNA extraction, mRNA expression level of DICER1 and several inflammatory cytokines were examined. We performed a relative real-time polymerase chain reaction (PCR) assay and transcriptome analysis between two groups including DICER1- transfected MSCs and control MSCs. Moreover, media from the transfected MSCs were evaluated for various interferon response factors by ELISA. The overexpression of DICER1 is associated with a significant increase in the mRNA expression level of COX-2, DDX-58, IFIH1, MYD88, RNase L, TLR3/4, and TDO2 genes and a downregulation of the TSG-6 gene in MSCs. Moreover, the expression levels of IL-1, 6, 8, 17, 18, CCL2, INF-γ, TGF-β, and TNF-α were higher in the DICER1-transfected MSCs group. It seems that the ectopic expression of DICER1 in Ad-MSCs is linked to alterations in the expression level of immune-related genes. It is suggested that the manipulation of immune-related pathways in MSCs via the Dicer1 overexpression could facilitate the development of MSCs with distinct immunoregulatory phenotypes.

Nuclear receptor RXRα binds the precursor of miR-103 to inhibit its maturation

BackgroundThe maturation of microRNAs (miRNAs) successively undergoes Drosha, Dicer, and Argonaute ˗mediated processing, however, the intricate regulations of the individual miRNA maturation are largely unknown. Retinoid x receptor alpha (RXRα) belongs to nuclear receptors that regulate gene transcription by binding to DNA elements, however, whether RXRα binds to miRNAs to exert physiological functions is not known.ResultsIn this work, we found that RXRα directly binds to the precursor of miR-103 (pre-miR-103a-2) via its DNA-binding domain with a preferred binding sequence of AGGUCA. The binding of RXRα inhibits the processing of miR-103 maturation from pre-miR-103a-2. Mechanistically, RXRα prevents the nuclear export of pre-miR-103a-2 for further processing by inhibiting the association of exportin-5 with pre-miR-103a-2. Pathophysiologically, the negative effect of RXRα on miR-103 maturation correlates to the positive effects of RXRα on the expression of Dicer, a target of miR-103, and on the inhibition of breast cancer.ConclusionsOur findings unravel an unexpected role of transcription factor RXRα in specific miRNA maturation at post-transcriptional level through pre-miRNA binding, and present a mechanistic insight regarding RXRα role in breast cancer progression.

MicroRNA expression as a diagnostic parameter in early endometrial cancer

ObjectivesMicroRNAs (miRNAs) have emerged as biomarkers that showed strong diagnostic potential in various diseases, including cancer. This study aimed to estimate the expression and diagnostic potential of miRNAs (miR-200a, miR-21,...

Cold water immersion in recovery following a single bout resistance exercise suppresses mechanisms of miRNA nuclear export and maturation.

Cold water immersion (CWI) following intense exercise is a common athletic recovery practice. However, CWI impacts muscle adaptations to exercise training, with attenuated muscle hypertrophy and increased angiogenesis. Tissue temperature modulates the abundance of specific miRNA species and thus CWI may affect muscle adaptations via modulating miRNA expression following a bout of exercise. The current study focused on the regulatory mechanisms involved in cleavage and nuclear export of mature miRNA, including DROSHA, EXPORTIN-5, and DICER. Muscle biopsies were obtained from the vastus lateralis of young males (n = 9) at rest and at 2, 4, and 48 h of recovery from an acute bout of resistance exercise, followed by either 10 min of active recovery (ACT) at ambient temperature or CWI at 10°C. The abundance of key miRNA species in the regulation of intracellular anabolic signaling (miR-1 and miR-133a) and angiogenesis (miR-15a and miR-126) were measured, along with several gene targets implicated in satellite cell dynamics (NCAM and PAX7) and angiogenesis (VEGF and SPRED-1). When compared to ACT, CWI suppressed mRNA expression of DROSHA (24 h p = 0.025 and 48 h p = 0.017), EXPORTIN-5 (24 h p = 0.008), and DICER (24 h p = 0.0034). Of the analyzed miRNA species, miR-133a (24 h p < 0.001 and 48 h p = 0.007) and miR-126 (24 h p < 0.001 and 48 h p < 0.001) remained elevated at 24 h post-exercise in the CWI trial only. Potential gene targets of these miRNA, however, did not differ between trials. CWI may therefore impact miRNA abundance in skeletal muscle, although the precise physiological relevance needs further investigation.

Phosphorylated nuclear DICER1 promotes open chromatin state and lineage plasticity of AT2 tumor cells in lung adenocarcinomas.

KRAS/ERK pathway phosphorylates DICER1, causing its nuclear translocation, and phosphomimetic Dicer1 contributes to tumorigenesis in mice. Mechanisms through which phospho-DICER1 regulates tumor progression remain undefined. While DICER1 canonically regulates microRNAs (miRNA) and epithelial-to-mesenchymal transition (EMT), we found that phosphorylated nuclear DICER1 (phospho-nuclear DICER1) promotes late-stage tumor progression in mice with oncogenic Kras, independent of miRNAs and EMT. Instead, we observe that the murine AT2 tumor cells exhibit altered chromatin compaction, and cells from disorganized advanced tumors, but not localized tumors, express gastric genes. Collectively, this results in subpopulations of tumor cells transitioning from a restricted alveolar to a broader endodermal lineage state. In human LUADs, we observed expression of phospho-nuclear DICER1 in advanced tumors together with the expression of gastric genes. We define a multimeric chromatin-DICER1 complex composed of the Mediator complex subunit 12, CBX1, MACROH2A.1, and transcriptional regulators supporting the model that phospho-nuclear DICER1 leads to lineage reprogramming of AT2 tumor cells to mediate lung cancer progression.

Epstein–Barr virus microRNA miR-BART2-5p accelerates nasopharyngeal carcinoma metastasis by suppressing RNase Ⅲ endonuclease DICER1

The development and progression of nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. NPC is usually asymptomatic until it spreads to other sites, and more than 70% of cases are classified as locally advanced disease at diagnosis. EBV-positive nasopharyngeal cancer tissues express only limited viral latent proteins, but express high levels of the EBV-encoded BamHI-A rightward transcript (BART) miRNA molecules. Here, we report that EBV-miRNA-BART2-5p (BART2-5p) promotes NPC cell invasion and metastasis invivo and invitro but has no effect on NPC cell proliferation and apoptosis. In addition, BART2-5p altered the mRNA and miRNA expression profiles of NPC cells. The development of human tumors has been reported to be associated with altered miRNAs expression, and overall miRNAs expression is reduced in many types of tumors. We found that BART2-5p downregulated the expression of several miRNAs that could exert oncogenic functions. Mechanistically, BART2-5p directly targets the RNase III endonuclease DICER1, inhibiting its function of cleaving double-stranded stem-loop RNA into short double-stranded RNA, which in turn causes altered expression of a series of key epithelial-mesenchymal transition molecules, and reverting DICER1 expression can rescue this phenotype. Furthermore, analysis from clinical samples showed a negative correlation between BART2-5p and DICER1 expression. According to our study, high expression of BART2-5p in tissues and plasma of patients with NPC is associated with poor prognosis. Our results suggest that, BART2-5p can accelerate NPC metastasis through modulating miRNA profiles which are mediated by DICER1, implying a novel role of EBV miRNAs in the pathogenesis of NPC.

The Impact of Variations in the Expression of Dicer on Pediatric Acute Lymphoblastic Leukemia

Background &amp; Objective: Dicer a member of the RNase III endonucleases has a crucial function in the development of miRNAs that affects gene expression in different kinds of cancers. The present research focused on examining the expression level of Dicer and its relation with clinicopathological features in children with Acute Lymphoblastic Leukemia (ALL) among the population in the northwest region of Iran.&#x0D; Materials &amp; Methods: In a case-control study, 140 samples (70 patients and 70 healthy controls of similar age and sex rates) from 2019 to 2021 were included in the Dicer mRNA expression levels assessment. Quantitative Real-Time PCR analyses were utilized to determine expression levels of Dicer in samples and the demographic and clinicopathologic features and the relationship of these variables were evaluated.&#x0D; Results: Average Dicer expression ct values were 2.8267 and up-regulated compared to healthy samples (P=0.000). No significant relationships have been found among gene expression, demographical and clinicopathological parameters. Also, demographical and clinicopathological factors were not correlated with the mRNA expression level of the gene except age (r=0.148, P=0.001).&#x0D; Conclusion: The current research shows that the expression of Dicer is significantly upregulated in ALL patients, which suggests that it could have a vital function in ALL pathology. It will also theoretically be seen in the potential as a new intervention objective for ALL cases.

Regulation of MicroRNA Expression in Scleroderma and Idiopathic Pulmonary Fibrosis: A Research Study

Introduction: Scleroderma (SSc) is an autoimmune disorder with the hallmark of fibrosis of the skin, vasculature and internal organs. Patients with SSc and undifferentiated connective tissue disease (UCTD) are susceptible to interstitial lung disease (ILD), leading to decreased lung function and death. Idiopathic pulmonary fibrosis (IPF) is a form of ILD that is not associated with extrapulmonary manifestations. In this study, lung involvement of SSc was studied by observing how disease progression and pathogenesis differ among patients with SSc, UCTD, and ILD compared to healthy controls and patients with IPF. Our group has previously identified disease targets through microRNA sequencing, including the DICER enzyme, which works closely with the protein DGCR8 and the enzyme DROSHA in the RNA interference pathway. The canonical pathway stipulates that DICER processes microRNAs in the cytosol while DGCR8 and DROSHA process microRNAs in the nucleus. DICER, DROSHA, and DGCR8 are hypothesized to contribute to ILD progression. Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from voluntary participants, including healthy controls. PBMCs were subsequently lysed with subcellular fractionation buffer. Western blotting was done on the resulting cytosolic and nucleic fractions for DICER, DROSHA, and DGCR8 protein expression. The cytosolic fractions were normalized to GAPDH, while the nucleic fractions were normalized to B2M. Nonparametric Kruskal‐Wallis tests were used for statistical analysis. Results: The medians were significantly higher for healthy controls for DICER in the nucleus with a p-value of 0.0302, and DROSHA in the cytosol with a p-value of 0.0406 compared to patients with SSc, UCTD, and IPF. Discussion: Differences in expression were found for DROSHA in the cytosol and DICER in the nucleus, suggesting dysregulation of the non-canonical RNA interference pathways in SSc, UCTD, and IPF patients. Variability of disease progression within the groups could lead to variable enzyme and protein levels within the same disease status. With larger sample sizes, statistically insignificant differences would become significant. Lipid nanoparticle technology could be used to deliver deficient microRNAs to silence mRNA in patients. Conclusion: Due to dysregulation of the RNA interference pathway, microRNAs may be inadequately processed in the patient groups.

Peptidylarginine deiminase 2 regulates expression of DGCR8 affecting miRNA biogenesis in gonadotrope cells.

DGCR8 microprocessor complex, which is important for miRNA biogenesis, is regulated by peptidylarginine deiminase 2 and expression fluctuates in gonadotrope cells across the mouse estrous cycle. Canonical miRNA biogenesis requires DGCR8 microprocessor complex subunit, which helps cleave pri-miRNAs into pre-miRNAs. Previous studies found that inhibiting peptidylarginine deiminase (PAD) enzyme activity results in increased DGCR8 expression. PADs are expressed in mouse gonadotrope cells, which play a central role in reproduction by synthesizing and secreting the luteinizing and follicle stimulating hormones. Given this, we tested whether inhibiting PADs alters expression of DGCR8, DROSHA, and DICER in the gonadotrope-derived LβT2 cell line. To test this, LβT2 cells were treated with vehicle or 1 µM pan-PAD inhibitor for 12 h. Our results show that PAD inhibition leads to an increase in DGCR8 mRNA and protein. To corroborate our results, dispersed mouse pituitaries were also treated with 1 µM pan-PAD inhibitor for 12 h which increases DGCR8 expression in gonadotropes. Since PADs epigenetically regulate gene expression, we hypothesized that histone citrullination alters Dgcr8 expression thereby affecting miRNA biogenesis. LβT2 samples were subjected to ChIP using an antibody to citrullinated histone H3, which shows that citrullinated histones are directly associated with Dgcr8. Next, we found that when DGCR8 expression is elevated in LβT2 cells, pri-miR-132 and -212 are reduced, while mature miR-132 and -212 are increased suggesting heightened miRNA biogenesis. In mouse gonadotropes, DGCR8 expression is higher in diestrus as compared to estrus, which is the inverse of PAD2 expression. Supporting this idea, treatment of ovariectomized mice with 17β-estradiol results in an increase in PAD2 expression in gonadotropes with a corresponding decrease in DGCR8. Collectively, our work suggests that PADs regulate DGCR8 expression leading to changes in miRNA biogenesis in gonadotropes.