miR-200 Family Research Articles

Association of miR-181c/d gene locus rs8108402 C/T polymorphism with susceptibility to Kawasaki disease in Chinese children.

BackgroundKawasaki disease (KD) is an acute systemic vasculitis of unknown etiology. The rs8108402 C/T single nucleotide polymorphism (SNP) is located in the promoter region of miR-181-c/d gene and the intron of Nanos3 gene. The miR-181 family contributes to the pathogenesis of cardiovascular and inflammatory disorders, while Nanos3 is involved in DNA transcription regulation and cell proliferation. However, no studies have examined the association between miR-181c/d and Nanos3 polymorphisms and the susceptibility and progression of KD.ObjectiveThe purpose of our study is to examine the association of miR-181c/miR-181d/Nanos3 gene locus rs8108402 C/T polymorphism with KD susceptibility, intravenous immunoglobulin (IVIG) responsiveness, and the development of coronary artery lesions (CAL).MethodsPeripheral blood specimens from 100 children with KD and 100 healthy children were collected. The polymorphism of rs8108402 C/T was detected using polymerase chain reaction-sequencing-based typing technique.ResultsThere were statistically significant differences in C and T allele frequency distributions between the KD group and healthy controls for the polymorphic site rs8108402 C/T (P = 0.002). The distribution of the genotypes CC, CT, and TT also presented statistical significant difference between the KD and control groups (P = 0.003). Compared to the rs8108402 C allele, the T allele was associated with increased KD susceptibility (OR = 2.080, 95% CI = 1.317∼3.283). However, there were no significant associations discovered between the rs8108402 C/T polymorphism and CAL formation or IVIG unresponsiveness in the study.ConclusionSNP rs8108402 C/T located in the miR-181c/d promoter and Nanos3 intronic region is associated with susceptibility to Kawasaki disease but not with the development of coronary artery lesions or IVIG unresponsiveness in Chinese children.

The miR164-TaNAC14 module regulates root development and abiotic-stress tolerance in wheat seedlings

Previous studies have revealed the miR164 family and the miR164-targeted NAC transcription factor genes in rice (Oryza sativa) and Arabidopsis that play versatile roles in developmental processes and stress responses. In wheat (Triticum aestivum L.), we found nine genetic loci of tae-miR164 (tae-MIR164 a to i) producing two mature sequences that down-regulate the expression of three newly identified target genes of TaNACs (TaNAC1, TaNAC11, and TaNAC14) by the cleavage of the respective mRNAs. Overexpression of tae-miR164 or one of its target genes (TaNAC14) demonstrated that the miR164-TaNAC14 module greatly affects root growth and development and stress (drought and salinity) tolerance in wheat seedlings, and TaNAC14 promotes root growth and development in wheat seedlings and enhances drought tolerance, while tae-miR164 inhibits root development and reduces drought and salinity tolerance by down-regulating the expression of TaNAC14. These findings identify the miR164-TaNAC14 module as well as other tae-miR164-regulated genes which can serve as new genetic resources for stress-resistance wheat breeding.

The developmentally timed decay of an essential microRNA family is seed-sequence dependent.

SUMMARYMicroRNA (miRNA) abundance is tightly controlled by regulation of biogenesis and decay. Here, we show that the mir-35 miRNA family undergoes selective decay at the transition from embryonic to larval development in C. elegans. The seed sequence of the miRNA is necessary and largely sufficient for this regulation. Sequences outside the seed (3′ end) regulate mir-35 abundance in the embryo but are not necessary for sharp decay at the transition to larval development. Enzymatic modifications of the miRNA 3′ end are neither prevalent nor correlated with changes in decay, suggesting that miRNA 3′ end display is not a core feature of this mechanism and further supporting a seed-driven decay model. Our findings demonstrate that seed-sequence-specific decay can selectively and coherently regulate all redundant members of a miRNA seed family, a class of mechanism that has great biological and therapeutic potential for dynamic regulation of a miRNA family’s target repertoire.

Evolutionary conservation of nested MIR159 structural microRNA genes and their promoter characterization in Arabidopsis thaliana.

MicroRNAs (miRNAs) are endogenous small RNAs, that are vital for gene expression regulation in eukaryotes. Whenever a pri-miRNA precursor includes another miRNA precursor, and both of these precursors may generate independent, non-overlapping mature miRNAs, we named them nested miRNAs. However, the extent of nested miR159 structural evolutionary conservation and its promoter characterization remains unknown. In this study, the sequence alignment and phylogenetic analysis reveal that the MIR159 family is ancient, and its nested miR159 structures are evolutionary conserved in different plant species. The overexpression of ath-MIR159a, including the 1.2 kb downstream region, has no effect on rescuing the mir159ab phenotype. The promoter truncation results revealed that the 1.0 kb promoter of ath-MIR159a is sufficient for rescuing the mir159ab phenotype. The cis-regulatory elements in the ath-miR159a promoters indicated functions related to different phytohormones, abiotic stresses, and transcriptional activation. While the MybSt1 motif-containing region is not responsible for activating the regulation of the miR159a promoter. The qRT-PCR results showed that overexpression of ath-MIR159a led to high expression levels of miR159a.1–5 and miR159a.1–3 and complemented the growth defect of mir159ab via downregulation of MYB33 and MYB65. Furthermore, continuously higher expression of the miR159a.2 duplex in transgenic lines with the curly leaf phenotype indicates that miR159a.2 is functional in Arabidopsis and suggests that it is possible for a miRNA precursor to encode several regulatory small RNAs in plants. Taken together, our study demonstrates that the nested miR159 structure is evolutionary conserved and miRNA-mediated gene regulation is more complex than previously thought.

Integrated bioinformatic analysis of dysregulated microRNA-mRNA co-expression network in ovarian endometriosis.

Ovarian endometriosis is a frequently occurring gynecological disease with large socioeconomic impact. Accumulating evidence has suggested that aberrant miRNA-mRNA interactions are involved in the pathogenesis and progression of ovarian endometriosis. This study aims to identify key miRNAs in ovarian endometriosis by using integrated bioinformatic analysis of a dysregulated miRNA-mRNA co-expression network. Expression profiling of miRNA and mRNA in three normal endometria and five pairs of ectopic/eutopic endometria from patients with ovarian endometriosis was determined by high-throughput sequencing techniques. The data were then integrated with the public sequencing datasets (GSE105764 and GSE105765) using a non-biased approach and a miRNA-mRNA co-expression regulatory network was constructed by in-depth bioinformatic analysis. The constructed miRNA-mRNA network included 87 functionally DEMs, 482 target mRNAs and 1850 paired miRNA-mRNA regulatory interactions. Specifically, five miRNAs (miR-141-3p, miR-363-3p, miR-577, miR-767-5p, miR-96-5p) were gradually decreased and two miRNAs (miR-493-5p, miR-592) were gradually increased from normal endometria to eutopic endometria, and then ectopic endometria tissues. Importantly, miR-141-3p, miR-363-3p and miR-96-5p belonged to the miR-200 family, miR-106a-363 cluster and miR-183/96/182 cluster, respectively. Their target mRNAs were mainly associated with cell adhesion, locomotion and binding, which are suggested to play vital regulatory roles in the pathogenesis of ovarian endometriosis. Integrated bioinformatic analysis of the miRNA-mRNA co-expression network defines the crucial roles of the miR-200 family, miR-106a-363 cluster and miR-183/96/182 cluster in the pathogenesis of ovarian endometriosis. Further in-depth functional studies are needed to unveil the molecular mechanisms of these miRNAs, and may provide clues for the optimization of therapeutic strategies for ovarian endometriosis.

MiR-15b-5p REGULATES THE NLRP3 INFLAMMASOME SIGNAL THROUGH TARGETING SIRT3 TO REGULATE HYPOXIA/REOXYGENATION-INDUCED CARDIOMYOCYTE PYROPTOSIS PROCESS.

Hypoxia/reoxygenation (H/R) induces pyroptosis in the setting of acute myocardial infarction (AMI). Previous studies have shown that the expression of the miR-15 family is stimulated in myocardial ischemia-reperfusion injury or H/R-induced cardiomyocyte injury, and miR-15 is a promoter of cardiac ischemia-reperfusion or H/R injury. However, whether miR-15b-5p regulates H/R injury and cardiomyocyte pyroptosis and its mechanism still need to be further clarified. Bioinformatics analysis elicited that SIRT3 was the downstream regulatory target gene of miR-15b-5p. SIRT3 has been shown to participate in the regulation of pyroptosis by negatively regulating the NLRP3 inflammasome pathway. Therefore, we hypothesized that miR-15b-5p targets SIRT3 and activated the NLRP3 inflammasome pathway to promote H/R-induced cardiomyocyte pyroptosis. We first show that H/R increases miR-15b-5p in rat cardiomyocytes H9C2. Next, we tested the effects of inhibition of miR-15b-5p or overexpression of SIRT3. We found that miR-15b-5p downregulation or SIRT3 overexpression could reverse the H/R-induced pyroptosis. Furthermore, silencing SIRT3 antagonized the protective effect of miR-15b-5p downregulation on H9C2 cells. NLRP3 inhibitor MCC950 annulled the previously mentioned antagonistic effect of silencing SIRT3 on the protection of miR-15b-5p downregulation against pyroptosis. We then used a rat AMI model to analyze myocardial infarction area by triphenyl tetrazolium chloride staining and assess serum cardiac troponin T level by ELISA and found that miR-15b-5p silencing reduced AMI injury in rats. Collectively, these results suggest that miR-15b-5p increase H/R-induced pyroptosis in cardiomyocytes by targeting SIRT3 and activating the NLRP3 inflammasome.

Liver-specific deletion of miR-181ab1 reduces liver tumour progression via upregulation of CBX7

MiR-181 expression levels increased in hepatocellular carcinoma (HCC) compared to non-cancerous tissues. MiR-181 has been widely reported as a possible driver of tumourigenesis but also acts as a tumour suppressor. In addition, the miR-181 family regulates the development and function of immune and vascular cells, which play vital roles in the progression of tumours. More complicatedly, many genes have been identified as miR-181 targets to mediate the effects of miR-181. However, the role of miR-181 in the development of primary tumours remains largely unexplored. We aimed to examine the function of miR-181 and its vital mediators in the progression of diethylnitrosamine-induced primary liver cancers in mice. The size of liver tumours was significantly reduced by 90% in global (GKO) or liver-specific (LKO) 181ab1 knockout mice but not in hematopoietic and endothelial lineage-specific knockout mice, compared to WT mice. In addition, the number of tumours was significantly reduced by 50% in GKO mice. Whole-genome RNA-seq analysis and immunohistochemistry showed that epithelial-mesenchymal transition was partially reversed in GKO tumours compared to WT tumours. The expression of CBX7, a confirmed miR-181 target, was up-regulated in GKO compared to WT tumours. Stable CBX7 expression was achieved with an AAV/Transposase Hybrid-Vector System and up-regulated CBX7 expression inhibited liver tumour progression in WT mice. Hepatic CBX7 deletion restored the progression of LKO liver tumours. MiR-181a expression was the lowest and CBX7 expression the highest in iClust2 and 3 subclasses of human HCC compared to iClust1. Gene expression profiles of GKO tumours overlapped with low-proliferative peri-portal-type HCCs. Liver-specific loss of miR-181ab1 inhibited primary liver tumour progression via up-regulating CBX7 expression, but tumour induction requires both hepatic and non-hepatic miR-181. Also, miR-181ab1-deficient liver tumours may resemble low-proliferative periportal-type human HCC.Graphical abstractmiR-181 was increased with liver tumour growth. More miR-181, darker colour and higher shape. CBX7 was very low in pericentral hepatocytes, increased in early liver tumours, but reduced in advanced liver tumours. Its levels were maintained in miR-181 KO liver tumours. In tumours (T), brown (darker is more) represents miR-181, the blue circle (thicker is more) represents CBX7.

Identification of Antitumor miR-30e-5p Controlled Genes; Diagnostic and Prognostic Biomarkers for Head and Neck Squamous Cell Carcinoma.

Analysis of microRNA (miRNA) expression signatures in head and neck squamous cell carcinoma (HNSCC) has revealed that the miR-30 family is frequently downregulated in cancer tissues. The Cancer Genome Atlas (TCGA) database confirms that all members of the miR-30 family (except miR-30c-5p) are downregulated in HNSCC tissues. Moreover, low expression of miR-30e-5p and miR-30c-1-3p significantly predicts shorter survival of HNSCC patients (p = 0.0081 and p = 0.0224, respectively). In this study, we focused on miR-30e-5p to investigate its tumor-suppressive roles and its control of oncogenic genes in HNSCC cells. Transient expression of miR-30e-5p significantly attenuated cancer cell migration and invasive abilities in HNSCC cells. Nine genes (DDIT4, FOXD1, FXR1, FZD2, HMGB3, MINPP1, PAWR, PFN2, and RTN4R) were identified as putative targets of miR-30e-5p control. Their expression levels significantly predicted shorter survival of HNSCC patients (p < 0.05). Among those targets, FOXD1 expression appeared to be an independent factor predicting patient survival according to multivariate Cox regression analysis (p = 0.049). Knockdown assays using siRNAs corresponding to FOXD1 showed that malignant phenotypes (e.g., cell proliferation, migration, and invasive abilities) of HNSCC cells were significantly suppressed. Overexpression of FOXD1 was confirmed by immunostaining of HNSCC clinical specimens. Our miRNA-based approach is an effective strategy for the identification of prognostic markers and therapeutic target molecules in HNSCC. Moreover, these findings led to insights into the molecular pathogenesis of HNSCC.

MicroRNA-466 and microRNA-200 increase endothelial permeability in hyperglycemia by targeting Claudin-5

Endothelial cell (EC) permeability is essential to vascular homeostasis in diabetes. MicroRNAs are critical gene regulators whose roles in the EC permeability have yet to be characterized. This study aims to examine the change in cell permeability induced by miR-200 and miR-466 in ECs. Human aortic ECs and dermal microvascular ECs from healthy subjects and type 2 diabetic patients were used. Our in vitro experiments unveiled higher expressions of miR-200 family members and miR-466 in diabetic ECs and in healthy ECs when exposed to high glucose. Overexpression of both miR-200 and miR-466 significantly increased EC permeability through transcriptional suppression of Claudin-5, the cell tight junction protein, by directly binding to its 3′ untranslated region. In a mouse model of chronic hyperglycemia mimicking type 2 diabetes in humans (db/db mice), the delayed closure rate of a full-thickness excisional wound was partly rescued by topical application of the miR-200 inhibitor. The topical application of both miR-200 and miR-466 inhibitors exhibited improved efficacy in accelerating wound closure compared with the topical application of miR-200 inhibitor alone. Our study demonstrated the potentially effective approach of miR-200/miR-466 cocktail inhibition to restore vascular integrity and tissue repair in hyperglycemia.

Diagnosis accuracy of the miR-200 family tumor marker series in ovarian cancer: a systematic review and meta-analysis.

BackgroundTo evaluate the diagnosis value of the microRNA-200 family in ovarian cancer patients. However, there is much controversy regarding the diagnosis of miR-200. Therefore, it is necessary to use meta method to further confirm the significance of diagnosis role of the miR-200 family tumor marker series in ovarian cancer.MethodsWe performed a careful literature search of the PubMed, EMBASE, and Web of Science databases and search language is English for articles related to ovarian cancer diagnose and the miR-200 family. The retrieval period was from the date of establishment of the database until September 20, 2021. The search keywords included microR-200, microR-200a, microR-200b, microR-200c, ovarian cancer, ovarian carcinoma, ovarian tumor, and sensitivity (SEN), specificity (SPE), area under the curve (AUC) were then calculated to estimate the diagnostic accuracy of the miR-200 family, and meta-analysis was performed using Stata 15.0 software.ResultsFive articles were included in the meta-analysis. The diagnostic value of miR-200a in epithelial ovarian cancer (EOC) was expressed as 0.76 (95% CI: 0.67–0.84) by SEN; the combined SPE was 0.71 (95% CI: 0.49–0.86); the pooled AUC was 0.79 (95% CI: 0.76–0.83). The diagnostic value of miR-200b in EOC was expressed by SEN of 0.84 (95% CI: 0.76–0.90) and SPE of 0.73 (95% CI: 0.48–0.88). Combined AUC was 0.86 (95% CI: 0.83–0.89). The diagnostic value of miR-200c in EOC was 0.90 (95% CI:0.69–0.97), and the SPE was 0.87 (95% CI: 0.37–0.99). Combined AUC was 0.94 (95% CI: 0.92–0.96).DiscussionThe miR-200 family may be a marker for the diagnosis evaluation of ovarian cancer patients.

Brassica napus miR169 regulates BnaNF-YA in salinity, drought and ABA responses

The NF-YA transcription factor in plants is a subunit of the NF-Y (NUCLEAR FACTOR-Y) complex, which plays important roles in plant stress responses and development. The transcripts of genes encoding NF-YA are predicted to be cleaved by members of the miR169 family. We previously identified several miR169s from rapeseed (Brassica napus) based on high-throughput sequencing. In this study, by using 5′ RACE and transient expression in Nicotiana benthamiana, we established that Bna-MIR169m, Bna-MIR169n, Bna-MIR169t (newly identified in this study) cleave target BnaNF-YAs. Quantitative PCR analysis revealed that most Bna-MIR169s detected in the leaves and roots were responsive to salinity, drought, and abscisic acid (ABA) treatment. To examine that these regulations are delivered to downstream of NF-YA, Bna-MI169n and BnaNF-YA9 were heterologously expressed in Arabidopsis thaliana plants. Bna-MI169n exhibited delayed seed germination and root elongation, oppositely, those expressing BnaNF-YA9 had higher rates of seed germination and root elongation under abiotic stress compared to untransformed Arabidopsis. Furthermore, through digital gene expression profiling, we identified several downstream genes co-regulated by Bna-miR169n and BnaNF-YA9. All these results point out that Bna-miR169n and BnaNF-YA9 form a cascade regulates plant abiotic stress responses.

P595: DISCOVERY OF NOVEL CIRCULAR RNAS (CIRCRNAS) OF THE APOPTOSIS-RELATED BAX AND BCL2L12 GENES WITH A MULTIFACETED ROLE IN CHRONIC LYMPHOCYTIC LEUKEMIA, USING CUTTING-EDGE NANOPORE SEQUENCING TECHNOLOGY

Background: Circular RNAs (circRNAs), a novel RNA type generated by back-splicing, are key indirect regulators of gene expression, with deregulated expression and established involvement in several human malignancies. Many circRNAs have been shown to bind microRNAs (miRNAs), thus making miRNAs unavailable to regulate protein-coding gene expression. Moreover, some of them have been described to interact with chromatin and affecting its structure. Despite their pivotal roles in the development and progression of cancer, only three studies have investigated their expression and role(s) in chronic lymphocytic leukemia (CLL), so far. The products of BCL2 and its homologues, including BAX and BCL2-like 12 (BCL2L12), are implicated in CLL, as apoptosis regulators. Moreover, expression levels of both BAX and BCL2L12 mRNAs are deregulated in CLL and are associated with patients’ response to therapy and overall survival. However, to the best of our knowledge, nothing is known about circRNAs produced by these two genes and their regulatory potential. Aims: We sought to further elucidate the contribution of both BAX and BCL2L12 in CLL by unraveling the identity of their circRNAs in a B-cell leukemic cell line (EHEB) and by studying in silico their potential interactions with miRNAs having an already established role in CLL. Methods: EHEB cells were propagated according to ATCC instructions. After total RNA extraction, 2μg of total RNA were reverse-transcribed using random hexamers. Next, nested PCR with divergent primers was performed, starting from each exon of BAX and BCL2L12 genes; thus, only cDNAs from BAX and BCL2L12 circRNAs were amplified, respectively. After having mixed and purified all nested-PCR products, nanopore sequencing libraries were built according to an optimized protocol. Following nanopore sequencing and basecalling in a MinION Mk1C 3rd-generation sequencer, sequencing reads were aligned and corrected using Minimap2 and TranscriptClean, respectively. circRNA sequences were identified using an in-house–built, PERL-based algorithm. A single-molecule resolution fluorescent in situ hybridization (FISH) method called circFISH was used to visualize the circRNA distribution in the cells. Lastly, miRDB was used to predict interactions of the most abundant BAX and BCL2L12 circRNAs with miRNAs. Results: We discovered 18 and 21 novel circRNAs produced by BAX and BCL2L12 genes, respectively, in this leukemic cell line. The exon structure of these novel circRNAs showed remarkable diversity, also comprising genomic regions that have previously been considered as intronic, as they are not included in messenger RNAs (mRNAs). Interestingly, one of the two most abundant BAX circRNAs is composed of a single exon, also spanning two introns. Similar intron retentions have been observed in a few BCL2L12 circRNAs. The gamut of miRNAs predicted to be bound by novel circRNAs includes miR-484, miR-181a-2-3p and miR-214-3p. Particularly, the inhibition of miR-214-3p is associated with elevated apoptosis in leukemic B-cells. Summary/Conclusion: Alternative splicing and back-splicing mechanisms of the primary BAX and BCL2L12 transcripts produce multiple distinct circRNAs in this B-cell CLL cell line. Interestingly, one of the two most abundant circular transcripts of BAX is a circRNA spanning three exons and two intervening introns of this gene. Lastly, several miRNAs are predicted to be sequestered by circRNAs of either of these apoptosis-related genes, including miR-181 family members that have been associated with chemosensitivity. Our data suggest a multifaceted role of BAX and BCL2L12 circRNAs in B-cell CLL.

Enhanced Cognition and Neurogenesis in miR-146b Deficient Mice.

The miR-146 family consists of two microRNAs (miRNAs), miR-146a and miR-146b, which are both known to suppress a variety of immune responses. Here in this study, we show that miR-146b is abundantly expressed in neuronal cells, while miR-146a is mainly expressed in microglia and astroglia of adult mice. Accordingly, miR-146b deficient (Mir146b-/-) mice exhibited anxiety-like behaviors and enhanced cognition. Characterization of cellular composition of Mir146b-/- mice using flow cytometry revealed an increased number of neurons and a decreased abundancy of astroglia in the hippocampus and frontal cortex, whereas microglia abundancy remained unchanged. Immunohistochemistry showed a higher density of neurons in the frontal cortex of Mir146b-/- mice, enhanced hippocampal neurogenesis as evidenced by an increased proliferation, and survival of newly generated cells with enhanced maturation into neuronal phenotype. No microglial activation or signs of neuroinflammation were observed in Mir146b-/- mice. Further analysis demonstrated that miR-146b deficiency is associated with elevated expression of glial cell line-derived neurotrophic factor (Gdnf) mRNA in the hippocampus, which might be at least in part responsible for the observed neuronal expansion and the behavioral phenotype. This hypothesis is partially supported by the positive correlation between performance of mice in the object recognition test and Gdnf mRNA expression in Mir146b-/- mice. Together, these results show the distinct function of miR-146b in controlling behaviors and provide new insights in understanding cell-specific function of miR-146b in the neuronal and astroglial organization of the mouse brain.

Characterization and bioinformatics analysis of ptc-miR396g-5p in response to drought stress of Paeonia ostii.

Drought is one of the main abiotic stress factors affecting yield of Paeonia ostii. In this study, we conducted bioinformatics and differential expression analyses of P. ostii ‘Feng Dan’ ptc-miR396g-5p in leaf samples under different drought stress. ptc-miR396g-5p belongs to the miR396 family. Among the 271 plant species registered in the miRBase database, at least one miR396 member was found in 48 Angiospermae species, 3 in Gymnospermae species, and 1 in Pteridophy. Mature sequence alignment showed that P. ostii ‘Feng Dan’ ptc-miR396g-5p had high sequence similarity with miR396 from other species. Secondary structure prediction showed that the precursor sequence of ‘Feng Dan’ ptc-miR396g-5p could form a stable stem-loop structure, and the mature sequence was located on the 5′ arm of the secondary structure. Phylogenetic tree analysis showed that ‘Feng Dan’ was closely related to 20 species such as Glycine max, Medicago truncatula, Populus trichocarpa, Citrus sinensis, Vitis vinifera, and Theobroma cacao. The predicted target gene of the ‘Feng Dan’ ptc-miR396g-5p encodes a Signal Transducer and Activator of Transcription (STAT) transcription factor. The negative correlation of expression between the miRNA and its target gene was confirmed by qRT-PCR. Our data indicate that ‘Feng Dan’ ptc-miR396g-5p′s expression decreases under drought, leading to an expression increase of the STAT transcription factor.

Metabolic imprinting induced by a high-sugar diet: effects on microRNA expression and insulin resistance in young rats.

Metabolic imprinting describes associations between nutritional experiences of early life and the development of diseases later in life. The goal of this study was to evaluate the metabolic imprinting induced by a high-sugar diet (HSD) and its effects on microRNA (miRNA) expression and insulin resistance (IR) in young rats. We assessed the effects of expression of adipogenic (miR-200c) and metabolic (miR-126a) miRNAs in retroperitoneal white adipose tissue (rWAT) on IR development. Weaned male Wistar rats (N = 6) were fed a standard chow diet or HSD (68% carbohydrates) for 4-, 8-, or 12-weeks. Serum samples were collected to measure triacylglycerol and VLDL-cholesterol, and we assessed glucometabolic parameters (glucose, insulin, HOMA-IR, and QUICKI). rWAT was collected for microRNA analysis (N = 3). The HSD resulted in body fat accretion and IR after 8-weeks, which resolved by 12-weeks. Moreover, the HSD had a time-dependent effect on miRNA relative expression, downregulating rno-miR-200c-3p at week 8 and rno-miR-126a-3p at week 12. MiR-200 family dysregulation has been related to IR, and miR-126a downregulation could be associated with the improvement in IR observed after a 12-week HSD feeding period. This is the first time that excessive sugar intake post-weaning has been associated with miRNA production by rWAT with an impact on IR development.

The microRNA-29 family: role in metabolism and metabolic disease.

The microRNA-29 family members miR-29a-3p, miR-29b-3p, and miR-29c-3p are ubiquitously expressed and consistently increased in various tissues and cell types in conditions of metabolic disease, obesity, insulin resistance, and type 2 diabetes. In pancreatic β cells, miR-29a is required for normal exocytosis, but increased levels are associated with impaired β-cell function. Similarly, in liver, miR-29 species are higher in models of insulin resistance and type 2 diabetes, and either knock-out or depletion using a microRNA inhibitor improves hepatic insulin resistance. In skeletal muscle, miR-29 family upregulation is associated with insulin resistance and altered substrate oxidation, and similarly, in adipocytes, overexpression of miR-29a leads to insulin resistance. Blocking miR-29a using nucleic acid antisense therapeutics show promising results in preclinical animal models of obesity and type 2 diabetes, although the widespread expression pattern of miR-29 family members complicates the exploration of single target tissues. However, in fibrotic diseases, such as in late complications of diabetes and metabolic disease (diabetic kidney disease, nonalcoholic steatohepatitis), miR-29 species expression is suppressed by TGF-β allowing increased extracellular matrix collagen to form. In the clinical setting, circulating levels of miR-29a and miR-29b are consistently increased in type 2 diabetes and in gestational diabetes and are also possible prognostic markers for deterioration of glucose tolerance. In conclusion, miR-29 family miRNAs play an essential role in various organs relevant to intermediary metabolism and its upregulation contributes to impaired glucose metabolism, whereas it suppresses fibrosis development. Thus, a correct balance of levels of miR-29 family miRNA seems important for cellular and organ homeostasis in metabolism.

Abstract 3872: Overexpression of miR-200 in cancer-associated fibroblasts suppresses the migration and invasion of cancer cells in co-culture systems

Abstract Cancer-associated fibroblasts (CAFs) promote cancer progression and metastasis in the tumor microenvironment. CAFs are regulated by diverse processes including regulation by microRNAs (miRNAs). To profile miRNAs involved in the regulation of CAF activation, we performed the NanoString nCounter® assay in mouse CAFs and normal lung fibroblasts (LFs). Among the miRNAs differentially expressed between CAFs and LFs, miR-200 family members were significantly downregulated in CAFs. Overexpression of miR-200 in CAFs suppressed the migration and invasion of lung cancer cells in co-culture systems (transwell migration and spheroid invasion assays). Next, we tried to identify novel miR-200 target genes based on RNA sequencing data obtained from control and miR-200-overexpressed CAFs, lung adenocarcinoma TCGA data, and TargetScan. We are now studying on these candidates to see whether they are involved in the interaction between CAFs and cancer cells.On the basis of these findings, we conclude that miR-200 suppresses the interaction between CAFs and lung cancer cells in the tumor microenvironment. Keywords: cancer-associated fibroblasts, miR-200, lung cancer, tumor microenvironment, invasion Citation Format: Inyoung Cheon, Sieun Lee, Young-Ho Ahn. Overexpression of miR-200 in cancer-associated fibroblasts suppresses the migration and invasion of cancer cells in co-culture systems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3872.

Abstract 1458: Single cell resolution chromatin accessibility and transcriptomic atlas of genetic ancestry mapped healthy breast tissues

Abstract Single cell mRNA atlases of multiple organs have been reported using scRNA-seq. We had previously reported single cell mRNA atlas of the breast. In this study, we performed scATAC-seq and scRNA-seq of breast tissues from 22 women of Ashkenazi (A) ancestry (19,935 nuclei), 20 women of European (non-A) ancestry (19,090 nuclei), six BRCA1 (6,261 nuclei) and five BRCA2 (3,400 nuclei) mutation carriers to correlate cell type-specific gene expression with chromatin accessibility. An integrated analysis revealed 17 cell clusters including basal, luminal progenitor, and mature luminal epithelial cells, two endothelial subtypes and two fibroblast/mesenchymal stem-like cell types. Basal cells could be further subdivided into two subclusters that differentially expressed genes such as TSHZ2, POSTN and ETV6. Similarly, there were four luminal progenitor subclusters that differentially expressed genes such as MALAT1, SOX13, and MDM4. Although ESR1 expression was observed predominantly in mature luminal cells, binding sites for ESR1 were accessible in multiple epithelial and non-epithelial cells with the exception of basal cells. FOXA1 expression and binding site enrichment were observed mostly in mature luminal cells. GATA3 expression was observed in all epithelial cell types with highest expression in mature luminal cells but binding sites for GATA3 were accessible in multiple cell types. Expression and binding site enrichment of alveolar differentiation-associated EHF were predominant in luminal progenitor cells. TP63 expression and binding site enrichment were restricted to basal cells, which is consistent with the proposed role of TP63 in maintaining stem cells among the basal cell populations. EMT-associated ZEB1 showed an intriguing pattern. Similar to our published immunohistochemistry-based analysis of breast tissues, ZEB1 expression was detected in fibroblasts/mesenchymal cells, endothelial cells and adipocytes but not in epithelial cells. However, binding sites for this transcription factor were enriched in epithelial cell types and in T-cells. ZEB1 mRNA stability is tightly controlled by the epithelial cell-enriched miR-200 family of microRNAs, and it is possible that ZEB1 transcribed in epithelial cells is immediately targeted for degradation through miR-200. To gain insight into this possibility, we examined the chromatin accessibility status of ZEB1 regulatory regions in fibroblasts/mesenchymal cells and in epithelial cells. ZEB1 regulatory regions displayed open chromatin status in both epithelial cells and fibroblasts. With the exception of a few genes such as SIGLEC1 in macrophages and IL7R in T cells, cell type-specific expression of many genes is not controlled at the level of chromatin accessibility of their regulatory regions. Collectively, these results reveal complexities in gene expression in different cell types of the breast. Citation Format: Poornima Bhat-Nakshatri, Duojiao Chen, Hongyu Gao, Yunlong Liu, Anna Maria Storniolo, Harikrishna Nakshatri. Single cell resolution chromatin accessibility and transcriptomic atlas of genetic ancestry mapped healthy breast tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1458.

Revisiting the miR-200 Family: A Clan of Five Siblings with Essential Roles in Development and Disease

Over two decades of studies on small noncoding RNA molecules illustrate the significance of microRNAs (miRNAs/miRs) in controlling multiple physiological and pathological functions through post-transcriptional and spatiotemporal gene expression. Among the plethora of miRs that are essential during animal embryonic development, in this review, we elaborate the indispensable role of the miR-200 family (comprising miR-200a, -200b, 200c, -141, and -429) in governing the cellular functions associated with epithelial homeostasis, such as epithelial differentiation and neurogenesis. Additionally, in pathological contexts, miR-200 family members are primarily involved in tumor-suppressive roles, including the reversal of the cancer-associated epithelial–mesenchymal transition dedifferentiation process, and are dysregulated during organ fibrosis. Moreover, recent eminent studies have elucidated the crucial roles of miR-200s in the pathophysiology of multiple neurodegenerative diseases and tissue fibrosis. Lastly, we summarize the key studies that have recognized the potential use of miR-200 members as biomarkers for the diagnosis and prognosis of cancers, elaborating the application of these small biomolecules in aiding early cancer detection and intervention.

Transcriptome analysis reveals the spinal expression profiles of non-coding RNAs involved in anorectal malformations in rat fetuses

BackgroundDespite improvements in anorectal malformation (ARM) therapy, patients might still experience post-operative problems such as fecal incontinence, constipation, and soiling. In particular, the dysplasia of the lumbosacral spinal cord in ARM patients is a major disorder that affects fecal function post-operation. However, the pathological mechanisms involved are still unclear. MethodsThe non-coding RNAs (ncRNAs) in the lumbosacral spinal cord of fetal rats with ethylenethiourea-induced ARM were identified using RNA sequencing (RNA-seq) and examined to determine their potential function. The lumbosacral spinal cord was isolated on embryonic day 17 for subsequent RNA extraction and RNA-seq. The transcriptome data was analyzed using bioinformatics analysis, followed by validation using quantitative reverse transcription PCR. ResultsCompared to the control group, 26 differentially expressed microRNAs (DEMs; 22 upregulated, 4 downregulated) and 112 differentially expressed long non-coding RNAs (63 upregulated, 49 downregulated) were identified in the ARM group. Several DEMs related to development, namely miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-200a-5p, and miR-429, were selected for further analysis. Notably, compared to the control, the relative expression of miR-200 family members was highly upregulated in ARM fetal rats. Furthermore, GO and KEGG enrichment and miRNA-transcription factor-lncRNA/mRNA network analysis was explored to show molecular mechanism underlying DEMs. ConclusionsOur findings suggest the involvement of ncRNAs, especially the miR-200 family members, in the pathogenesis of lumbosacral spinal cord dysplasia in ARM fetal rats.