Degradation Of Target mRNAs Research Articles

Post-transcriptional regulation of aromatic amino acid metabolism by GcvB small RNA in Escherichia coli.

Escherichia coli synthesizes aromatic amino acids (AAAs) through the common pathway to produce the precursor, chorismate, and the three terminal pathways to convert chorismate into Phe, Tyr, and Trp. E. coli also imports exogenous AAAs through five transporters. GcvB small RNA post-transcriptionally regulates more than 50 genes involved in amino acid uptake and biosynthesis in E. coli, but the full extent of GcvB regulon is still underestimated. This study examined all genes involved in AAA biosynthesis and transport using translation reporter assay and qRT-PCR analysis. In addition to previously verified targets, aroC, aroP, and trpE, we identified new target genes that were significantly repressed by GcvB primarily via the R1 seed region. Exceptionally, GcvB strongly inhibits the expression of aroG, which encodes the major isozyme of the first reaction in the common pathway, through direct base pairing between the aroG translation initiation region and the GcvB R3 seed sequence. RNase E mediates the degradation of target mRNAs except aroC and aroP via its C-terminal domain. GcvB overexpression prolongs the lag phase and reduces the growth rate in minimal media supplemented with AAAs and confers resistance to an antibiotic compound, azaserine, by repressing AAA transporters.IMPORTANCEE. coli strains have been genetically modified in relevant transcription factors and biosynthetic enzymes for industrial use in the fermentative production of aromatic amino acids (AAAs) and their derivative compounds. This study focuses on GcvB small RNA, a global regulator of amino acid metabolism in E. coli, and identifies new GcvB targets involved in AAA biosynthesis and uptake. GcvB represses the expression of the first and last enzymes of the common pathway and the first enzymes of Trp and Phe terminal pathways. GcvB also limits import of AAAs. This paper documents the impact of RNA-mediated regulation on AAA metabolism in E. coli.

Impact of Exogenous dsRNA on miRNA Composition in Arabidopsis thaliana.

The application of double-stranded RNAs (dsRNAs) to plant surfaces has emerged as a promising tool for manipulating gene expression in plants and pathogens, offering new opportunities for crop improvement. While research has shown the capability of exogenous dsRNAs to silence genes, the full spectrum of their impact, particularly on the intricate network of microRNAs (miRNAs), remains largely unexplored. Here, we show that the exogenous application of chalcone synthase (CHS)-encoding dsRNA to the rosette leaves of Arabidopsis thaliana induced extensive alterations in the miRNA profile, while non-specific bacterial neomycin phosphotransferase II (NPTII) dsRNA had a minimal effect. Two days after treatment, we detected 60 differentially expressed miRNAs among the 428 miRNAs found in the A. thaliana genome. A total of 59 miRNAs were significantly changed after AtCHS-dsRNA treatment compared with water and NPTII-dsRNA, and 1 miRNA was significantly changed after AtCHS-dsRNA and NPTII-dsRNA compared with the water control. A comprehensive functional enrichment analysis revealed 17 major GO categories enriched among the genes potentially targeted by the up- and downregulated miRNAs. These categories included processes such as aromatic compound biosynthesis (a pathway directly related to CHS activity), heterocycle biosynthesis, RNA metabolism and biosynthesis, DNA transcription, and plant development. Several predicted targets of upregulated and downregulated miRNAs, including APETALA2, SCL27, SOD1, GRF1, AGO2, PHB, and PHV, were verified by qRT-PCR. The analysis showed a negative correlation between the expression of miRNAs and the expression of their predicted targets. Thus, exogenous plant gene-specific dsRNAs induce substantial changes in the plant miRNA composition, ultimately affecting the expression of a wide range of genes. These findings have profound implications for our understanding of the effects of exogenously induced RNA interference, which can have broader effects beyond targeted mRNA degradation, affecting the expression of other genes through miRNA regulation.

Genes Related to Frontonasal Malformations Are Regulated by miR-338-5p, miR-653-5p, and miR-374-5p in O9-1 Cells.

Frontonasal malformations are caused by a failure in the growth of the frontonasal prominence during development. Although genetic studies have identified genes that are crucial for frontonasal development, it remains largely unknown how these genes are regulated during this process. Here, we show that microRNAs, which are short non-coding RNAs capable of targeting their target mRNAs for degradation or silencing their expression, play a crucial role in the regulation of genes related to frontonasal development in mice. Using the Mouse Genome Informatics (MGI) database, we curated a total of 25 mouse genes related to frontonasal malformations, including frontonasal hypoplasia, frontonasal dysplasia, and hypotelorism. MicroRNAs regulating the expression of these genes were predicted through bioinformatic analysis. We then experimentally evaluated the top three candidate miRNAs (miR-338-5p, miR-653-5p, and miR-374c-5p) for their effect on cell proliferation and target gene regulation in O9-1 cells, a neural crest cell line. Overexpression of these miRNAs significantly inhibited cell proliferation, and the genes related to frontonasal malformations (Alx1, Lrp2, and Sirt1 for miR-338-5p; Alx1, Cdc42, Sirt1, and Zic2 for miR-374c-5p; and Fgfr2, Pgap1, Rdh10, Sirt1, and Zic2 for miR-653-5p) were directly regulated by these miRNAs in a dose-dependent manner. Taken together, our results highlight miR-338-5p, miR-653-5p, and miR-374c-5p as pathogenic miRNAs related to the development of frontonasal malformations.

Recent advances in understanding of the mechanisms of RNA interference in insects.

We highlight the recent 5 years of research that contributed to our understanding of the mechanisms of RNA interference (RNAi) in insects. Since its first discovery, RNAi has contributed enormously as a reverse genetic tool for functional genomic studies. RNAi is also being used in therapeutics, as well as agricultural crop and livestock production and protection. Yet, for the wider application of RNAi, improvement of its potency and delivery technologies is needed. A mechanistic understanding of every step of RNAi, from cellular uptake of RNAi trigger molecules to targeted mRNA degradation, is key for developing an efficient strategy to improve RNAi technology. Insects provide an excellent model for studying the mechanism of RNAi due to species-specific variations in RNAi efficiency. This allows us to perform comparative studies in insect species with different RNAi sensitivity. Understanding the mechanisms of RNAi in different insects can lead to the development of better strategies to improve RNAi and its application to manage agriculturally and medically important insects.

Long Noncoding RNA MSL3P1 Regulates CUL3 mRNA Cytoplasmic Transport and Stability and Promotes Lung Adenocarcinoma Metastasis.

Lung adenocarcinoma (LUAD) is the most prevalent histological type of lung cancer. Previous studies have reported that specific long noncoding RNAs (lncRNA) are involved in cancer development and progression. The phenotype and mechanism of ENST00000440028, named MSL3P1, an lncRNA referred to as a cancer-testis gene with potential roles in tumorigenesis and progression, have not been reported. MSL3P1 is overexpressed in LUAD tumor tissues, which is significantly associated with clinical characteristics, metastasis, and poor clinical prognosis. MSL3P1 promotes the metastasis of LUAD in vitro and in vivo. The enhancer reprogramming in LUAD tumor tissue is the major driver of the aberrant expression of MSL3P1. Mechanistically, owing to the competitive binding to CUL3 mRNA with ZFC3H1 protein (a protein involved in targeting polyadenylated RNA to exosomes and promoting the degradation of target mRNA), MSL3P1 can prevent the ZFC3H1-mediated RNA degradation of CUL3 mRNA and transport it to the cytoplasm. This activates the downstream epithelial-to-mesenchymal transition signaling pathway and promotes tumor invasion and metastasis. Implications: This study indicates that lncRNA MSL3P1 regulates CUL3 mRNA stability and promotes metastasis and holds potential as a prognostic biomarker and therapeutic target in LUAD.

Participation of miR165a in the Phytochrome Signal Transduction in Maize (Zea mays L.) Leaves under Changing Light Conditions.

The involvement of the microRNA miR165a in the light-dependent mechanisms of regulation of target genes in maize (Zea mays) has been studied. The light-induced change in the content of free miR165a was associated with its binding by the AGO10 protein and not with a change in the rate of its synthesis from the precursor. The use of knockout Arabidopsis plants for the phytochrome A and B genes demonstrated that the presence of an active form of phytochrome B causes an increase in the level of the RNA-induced silencing miR165a complex, which triggers the degradation of target mRNAs. The two fractions of vesicles from maize leaves, P40 and P100 that bind miR165a, were isolated by ultracentrifugation. The P40 fraction consisted of larger vesicles of the size >0.170 µm, while the P100 fraction vesicles were <0.147 µm. Based on the quantitative PCR data, the predominant location of miR165a on the surface of extracellular vesicles of both fractions was established. The formation of the active form of phytochrome upon the irradiation of maize plants with red light led to a redistribution of miR165a, resulting in an increase in its proportion inside P40 vesicles and a decrease in P100 vesicles.

Molecular Mechanisms of Tumorgenesis and Metastasis of Long Non-coding RNA (lncRNA) NEAT1 in Human Solid Tumors; An Update.

The expression of the nuclear paraspeckle assembly transcript 1 (NEAT1), as a well-known long non-coding RNA (lncRNA), is often upregulated in varied types of cancers and associated with poor survival outcomes in patients suffering from tumors. NEAT1 promotes the tumors growth by influencing the various genes' expression profile that regulate various aspects of tumor cell behavior, in particular tumor growth, metastasis and drug resistance. This suggests that NEAT1 are capable of serving as a new diagnostic biomarker and target for therapeutic intervention. Through interrelation with enhancer of zeste homolog 2 (EZH2), NEAT1 acts as a scaffold RNA molecule, and thus regulating the expression EZH2-associated genes. Additionally, by perform as miRNA sponge, it constrains suppressing the interactions between miRNAs-mediated degradation of target mRNAs. In light of this, NEAT1 inhibition by small interfering RNA (siRNA) hampers tumorgenesis. We summarize recent findings about the expression, biological functions, and regulatory process of NEAT1 in human tumors. It specifically emphasizes the clinical significance of NEAT1 as a novel diagnostic biomarker and a promising therapeutic mark for many types of cancers.

The Growing Class of Novel RNAi Therapeutics.

The clinical use of RNA interference (RNAi) molecular mechanisms has introduced a novel, growing class of RNA therapeutics capable of treating diseases by controlling target gene expression at the posttranscriptional level. With the newly approved nedosiran (Rivfloza), there are now six RNAi-based therapeutics approved by the United States Food and Drug Administration (FDA). Interestingly, five of the six FDA-approved small interfering RNA (siRNA) therapeutics [patisiran (Onpattro), lumasiran (Oxlumo), inclisiran (Leqvio), vutrisiran (Amvuttra), and nedosiran] were revealed to act on the 3'-untranslated regions of target mRNAs, instead of coding sequences, thereby following the common mechanistic action of genome-derived microRNAs (miRNA). Furthermore, three of the FDA-approved siRNA therapeutics [patisiran, givosiran (Givlaari), and nedosiran] induce target mRNA degradation or cleavage via near-complete rather than complete base-pair complementarity. These features along with previous findings confound the currently held characteristics to distinguish siRNAs and miRNAs or biosimilars, of which all converge in the RNAi regulatory pathway action. Herein, we discuss the RNAi mechanism of action and current criteria for distinguishing between miRNAs and siRNAs while summarizing the common and unique chemistry and molecular pharmacology of the six FDA-approved siRNA therapeutics. The term "RNAi" therapeutics, as used previously, provides a coherently unified nomenclature for broader RNAi forms as well as the growing number of therapeutic siRNAs and miRNAs or biosimilars that best aligns with current pharmacological nomenclature by mechanism of action. SIGNIFICANCE STATEMENT: The common and unique chemistry and molecular pharmacology of six FDA-approved siRNA therapeutics are summarized, in which nedosiran is newly approved. We point out rather a surprisingly mechanistic action as miRNAs for five siRNA therapeutics and discuss the differences and similarities between siRNAs and miRNAs that supports using a general and unified term "RNAi" therapeutics to align with current drug nomenclature criteria in pharmacology based on mechanism of action and embraces broader forms and growing number of novel RNAi therapeutics.

Potential Role of Selected miRNAs in the Pathogenesis of Autoimmune Thyroid Diseases in Children and Adolescents.

Many epigenetic factors, including microRNAs, are involved in the process of changing gene expressions. Small non-coding RNA molecules, called miRNAs, are responsible for regulating gene translation by silencing or degrading target mRNAs. It is acknowledged that for many diseases, they may be novel diagnostic and prognostic biomarkers. Patients with autoimmune thyroid diseases are more likely to develop nodules in the thyroid tissue, and Hashimoto's thyroiditis and Graves' disease predispose patients to thyroid cancer. We evaluated the concentrations of microRNA molecules (miR-15a-5p, miR-126-3p, miR-142-5p, miR-21-5p, miR-150-5p) in the blood of children with thyroid disorders. In addition, we wished to identify molecules whose change in concentration predisposes to the development of thyroid cancer. The aim of this study is to evaluate selected epigenetic elements by analyzing the levels of miR-15a-5p, miR-126-3p, miR-142-5p, miR-150-5p and miR-21-5p in the blood of pediatric patients with Graves' disease (n = 25), Hashimoto's thyroiditis (n = 26) and thyroid nodular disease (n = 20) compared to a control group of healthy children (n = 17). The study consists of groups of children and adolescents aged 10-18 years with autoimmune thyroid disease, with thyroid nodular disease compared to a control group. The miR-15a-5p, miR-126-3p, miR-142-5p, miR-21-5p and miR-150-5p molecules were determined through an immunoenzymatic assay using BioVendor reagents. There is a statistically significant decrease in the expression of the miR-15a-5p in children with Graves' disease (21.61 vs. 50.22 amol/μL, p = 0.03) and in patients with thyroid nodular disease compared to controls (20.23 vs. 50.22 amol/μL, p = 0.04). Higher levels of the miR-142-5p molecule are found in patients with thyroid disease (with GD-3.8 vs. 3.14 amol/μL, p = 0.01; with HT-3.7 vs. 3.14 amol/μL, p = NS, with thyroid nodular disease-4.16 vs. 3.14 amol/μL, p = 0.04). Lower levels of miR-126-3p were noted in the GD group compared to the control group (7.09 vs. 7.24 amol/μL, p = 0.02). No statistically significant changes in the expressions of miR-150-5p and miR-21-5p molecules were observed in the study groups. 1. The overexpression of the miR-142-5p molecule occurs in children and adolescents with thyroid diseases. 2. Decreased blood levels of miR-15a-5p predispose patients to the formation of focal lesions in the thyroid gland. 3. Identifying a lower expression of the miR-126-3p molecule in the blood of children with GD requires careful follow-up for the development of focal lesions in the thyroid gland and evaluation for their potential malignancy.

Abstract 3402: Integration of microRNAs and transcriptome signatures identifies the persistent infection of hepatitis C virus-induced hepatocellular carcinoma

Abstract MicroRNAs (miRNAs) are short non-coding RNAs (ncRNAs) of ~22 nucleotides that mediate gene expression through various functions, including gene silencing, which can lead to translation repression and degradation of target mRNAs. Dysregulation of miRNA function has been associated with numerous diseases, particularly cancer, including hepatocellular carcinoma (HCC). During the progression of HCV-HCC, the function of miRNAs may be altered thereby altering gene expression to favor HCC. Therefore, the involved miRNA-mRNA interaction needs to be further studied. In addition, there are many clinical studies have shown that HCV patients who do not adequately control their diet which are prone to fatty liver accumulation (steatosis), it may increase the possibility of liver cirrhosis. In the current study, we aim to investigate the function and effects of persistent HCV-induced miRNAs expression in liver hepatocellular carcinoma progression and gene expression and to explore the mechanisms and regulatory genes of liver steatosis caused by HCV patients in a high-fat environment. We used the Huh751 cell line and the infected with the HCV JFH1 replicon of low-viral load S2 cells. We used an HCV miRNA array for initial identification of potential candidates. These miRNAs were analyzed for KEGG pathways via DIANA. Next, we identified miRNA-mRNA targets in 3 databases (miRDB; miRPathDB; TargetScan) and analyzed the targets for KEGG pathways using ShinyGO. The target miRNA-mRNA candidate genes were then compared with long-term JFH1 replicon infectious cell (L-HCV) NGS mRNA data to integrate miRNA-mRNA. The identified genes were analyzed in The Cancer Genome Atlas (TCGA) for survival, biological process, and expression in LIHC Big Data. Hsa-miR-215, hsa-mir-10b, and hsa-let-7a of L-HCV miRNAs group showed high significance for LIHC overall survival with a hazard ratio &amp;gt;1.0. Moreover, miRNAs associated with L-HCV survival were LIHC-specific but not in other cancers. Through systematic integration of these findings, we identified HMOX1 and BMF genes as being specifically associated with HCC and regulated by hsa-miR-215-5p, hsa-miR-10b-5p, and hsa-miR-7a-5p. In addition, we also found that the expression of the adipogenesis genes, FABP4 and adipogenesis gene, SREBP1, as well as the viral non-structural protein, NS3, was significantly increased in the HCV-high-fat group; and the expression of the gene IRS1, which is involved in hyperinsulinemia, showed a decreasing trend. The main cause of HCC is that HCV inhibits the expression of hsa-miR-215-5p, hsa-miR-10b-5p, and hsa-miR-7a-5p, leading to alter the expression of target genes, these have a significant effect on survival, suggesting that an essential role in virus-related liver cancer. In the future, we will further explore the regulatory mechanisms between these genes and HCV in a high-fat environment. Citation Format: Wen-Hsiu (Vivian) Su, Ciniso Sylvester Shabangu, Shu-Chi Wang. Integration of microRNAs and transcriptome signatures identifies the persistent infection of hepatitis C virus-induced hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3402.

MiR-142-3p/5p role in cancer: From epigenetic regulation to immunomodulation.

MicroRNAs (miRNAs) play critical roles in cancer pathobiology, acting as regulators of gene expression and pivotal drivers of tumorigenesis. It is believed that miRNAs act through canonical mechanisms, involving the binding of mature miRNAs to target messenger RNAs (mRNAs) and subsequent repression of protein translation or degradation of target mRNAs. miR-142-3p/5p has been extensively studied and established as a key regulator in various malignancies. Recent discoveries have revealed miR-142-3p/5p serve as either oncogene or tumor suppressor in cancer. By targeting epigenetic factor and cancer-related signaling pathway, miR-142-3p/5p can regulate wide range of downstream genes. The immune modulatory role of miR-142-3p/5p has been shown in various cancers, which provides significant insight into immunosuppression and tumor escape from the immune response. Exosomes with miR-142-3p/5p facilitate cell communication and can affect cancer cell behavior, offering potential therapeutic, and diagnosis applications in cancer therapy. In this review, for the first time, we comprehensively summarize the current knowledge regarding mentioned functions of miR-142-3p/5p in cancer pathobiology.

Androgen receptor modulatory miR-1271-5p can promote hormone sensitive prostate cancer cell growth.

In most patients with advanced prostate cancer treated with hormonal therapy, androgen independence eventually emerges, leading to death. Androgen receptor signalling remains an important prostate cancer driver, even in the advanced disease stage. MicroRNAs (miRs), non-coding RNAs that regulate gene expression by inhibiting translation and/or promoting degradation of target mRNAs, can act as tumour suppressors or "oncomiRs" and modulate tumour growth. Because of their stability in tissues and in circulation, and their specificity, microRNAs have emerged as potential biomarkers, as well as therapeutic targets in cancer. We identified miR-1271-5p as an androgen receptor modulatory microRNA and we show it can promote hormone sensitive prostate cancer cell growth. Inhibition or overexpression of miR-1271-5p levels affects prostate cancer cell growth, apoptosis and expression of both androgen receptor target genes and other genes that are likely direct targets, dependent on androgen receptor status, and tumour stage. We conclude that miR-1271-5p has the potential to drive progression of hormone-dependent disease and that the use of specific inhibitors of miR-1271-5p may have therapeutic potential in prostate cancer.

IGF2BP1 Stabilizes and Inhibits Degradation of Pro-Oncogenic Transcripts in ETV6-RUNX1 Positive B-Cell Acute Lymphoblastic Leukemia

Insulin like growth factor 2 mRNA binding protein 1(IGF2BP1) belongs to a family of oncofetal proteins expressed in embryonic life followed by re-emergence during malignant transformation. It is an RNA binding protein known to be dysregulated and overexpressed in multiple malignancies. We have demonstrated its specific overexpression in ETV6-RUNX1 translocated B-cell Acute Lymphoblastic Leukemia (B-ALL). RNA immunoprecipitation using anti-IGF2BP1 antibody, followed by sequencing (RIP-Seq) in Reh cell line (ETV6-RUNX1 positive) identified multiple oncogenic transcripts belonging to the non-canonical NF-κB and PI3K signaling pathways. Overexpression of genes from these pathways (IL6ST, GADD45A, NFAT5, MDM2, CDK6, FGFR1) was also observed in our B-ALL patient samples specifically in the ETV6-RUNX1 positive patients (Total n=111; ETV6-RUNX1 positive: 39) in comparison to MACS sorted, peripheral blood CD19 positive B-cells from healthy volunteers as well as in comparison to non ETV6-RUNX1 translocated B-Acute lymphoblastic leukemia patients. This was also corroborated by public B-ALL gene expression data from the TARGET database in the cBioportal. However, knockout of IGF2BP1 in Reh cell line using CRISPR-Cas9 technology led to significant downregulation of only some of these targets. This implied that the expression of some of the mRNA targets binding to IGF2BP1 was not influenced by its knockout. To better elucidate the role played by IGF2BP1 in the stability of its binding mRNA targets, we decided to study the rate of degradation of target transcripts. mRNA stability was assessed to quantify the rate of degradation of target mRNA after inhibition of transcription using actinomycin. Since actinomycin causes global transcriptional block, we first quantified the effect of actinomycin on the expression of multiple (n=7) housekeeping genes against each other, and selected 3 ( HGPRT, B2M and PPIA) which showed least change after actinomycin treatment. These 3 housekeeping genes were used for further quantification of target transcripts. Dual specificity phosphatase 1( DUSP1) a transcript known to be rapidly degraded, was used to validate the transcriptional inhibition by actinomycin. We quantified the rate of degradation of target transcripts in Reh cells using BTYNB, a small molecule inhibitor of IGF2BP1. BTYNB binds to IGF2BP1 and prevents binding to target transcripts which leads to their destabilization and reduced half-life. We used a combination of BTYNB and actinomycin to study rate of degradation after IGF2BP1 inhibition. Effect of IGF2BP1 on the stability of three target mRNAs involved in non-canonical NF-κB signalling pathway was quantified by comparing their rate of degradation in BTYNB treated cells against the DMSO (vehicle) treated cells. Rate of degradation was measured by actinomycin treatment of both these groups followed by hourly RNA isolation and qPCR over 12 hours. MYC is a known mRNA binding target of IGF2BP1. BTYNB was also shown to decrease the expression of MYC mRNA by accelerating its degradation. Simultaneously there was an increase in the expression of MYC pre-mRNA indicating a possible for IGF2BP1 in splicing of MYC RNA. In the NF-κB signalling pathway, we had previously identified that NFAT5 expression was not altered significantly after IGF2BP1 knockout even though it binds to IGF2BP1. BTYNB followed by actinomycin treatment demonstrated a dramatic increase in the rate of degradation of NFAT5 implying that IGF2BP1 prevents the degradation of NFAT5 transcript and directly influences its stability. Interestingly, the rates of degradation of other targets like IL6ST and GADD45 were not altered. These findings suggest a heterogeneous influence of IGF2BP1 on the stability of its target transcripts. Some of the transcripts seem to be directly stabilised by IGF2BP1 while others may be influenced indirectly. Taken together, these findings suggest a role of IGF2BP1 in multiple phases of the RNA synthesis and degradation warranting further studies to elucidate role of IGF2BP1 in each of these steps. IGF2BP1 appears to have a critical role in preventing the degradation and stabilizing MYC and NFAT5 transcripts.

Mechanistic Pharmacokinetics and Pharmacodynamics of GalNAc-siRNA: Translational Model Involving Competitive Receptor-Mediated Disposition and RISC-Dependent Gene Silencing Applied to Givosiran

Triantennary N-acetyl-D galactosamine (GalNAc)3-conjugated small interfering RNA (siRNA) have majorly advanced the development of RNA-based therapeutics. Chemically stabilized GalNAc-siRNAs exhibit extensive albeit capacity-limited (nonlinear) distribution into hepatocytes with additional complexities in intracellular liver disposition and pharmacology. A mechanism-based pharmacokinetic-pharmacodynamic (PK-PD) model of GalNAc-siRNA was developed to i) quantitate ASGPR-mediated disposition and downstream RNA-induced silencing complex (RISC)-dependent pharmacology following intravenous (IV) and subcutaneous (SC) dosing, ii) assess the kinetics of formed active metabolite, iii) leverage, as an example, published experimental data for givosiran, and iv) demonstrate PK translation across two preclinical species (rat and monkey) with subsequent prediction of human plasma PK. The structural model is based on competition between parent and formed active metabolite for occupancy and uptake via ASGPR into hepatocytes, intracellular sequestration and degradation, and downstream engagement of RNA-induced silencing complex (RISC) governing target mRNA degradation. The model jointly and accurately captured available concentration-time profiles of givosiran and/or AS(N-1)3′ givosiran in rat and/or monkey plasma, liver, and/or kidney following givosiran administered both IV and SC. RISC-dependent gene silencing of ALAS1 mRNA was well-characterized. The model estimated an in vivo affinity (KD) value of 27.7 nM for GalNAc-ASGPR and weight-based allometric exponents of −0.27 and −0.24 for SC absorption and intracellular (endolysosomal) degradation rate constants. The model well-predicted reported givosiran plasma PK profiles in humans. PK simulations revealed net-shifts in liver-to-kidney distribution ratios with increasing IV and SC dose. Importantly, decreases in the relative liver uptake efficiency were demonstrated following IV and, to a lesser extent, following SC dosing explained by differential ASGPR occupancy profiles over time.

LncRNA miR663AHG represses the development of colon cancer in a miR663a-dependent manner

The MIR663AHG gene encodes both miR663AHG and miR663a. While miR663a contributes to the defense of host cells against inflammation and inhibits colon cancer development, the biological function of lncRNA miR663AHG has not been previously reported. In this study, the subcellular localization of lncRNA miR663AHG was determined by RNA-FISH. miR663AHG and miR663a were measured by qRT-PCR. The effects of miR663AHG on the growth and metastasis of colon cancer cells were investigated in vitro and in vivo. CRISPR/Cas9, RNA pulldown, and other biological assays were used to explore the underlying mechanism of miR663AHG. We found that miR663AHG was mainly distributed in the nucleus of Caco2 and HCT116 cells and the cytoplasm of SW480 cells. The expression level of miR663AHG was positively correlated with the level of miR663a (r = 0.179, P = 0.015) and significantly downregulated in colon cancer tissues relative to paired normal tissues from 119 patients (P < 0.008). Colon cancers with low miR663AHG expression were associated with advanced pTNM stage (P = 0.021), lymph metastasis (P = 0.041), and shorter overall survival (hazard ratio = 2.026; P = 0.021). Experimentally, miR663AHG inhibited colon cancer cell proliferation, migration, and invasion. The growth of xenografts from RKO cells overexpressing miR663AHG was slower than that of xenografts from vector control cells in BALB/c nude mice (P = 0.007). Interestingly, either RNA-interfering or resveratrol-inducing expression changes of miR663AHG or miR663a can trigger negative feedback regulation of transcription of the MIR663AHG gene. Mechanistically, miR663AHG could bind to miR663a and its precursor pre-miR663a, and prevent the degradation of miR663a target mRNAs. Disruption of the negative feedback by knockout of the MIR663AHG promoter, exon-1, and pri-miR663A-coding sequence entirely blocked these effects of miR663AHG, which was restored in cells transfected with miR663a expression vector in rescue experiment. In conclusion, miR663AHG functions as a tumor suppressor that inhibits the development of colon cancer through its cis-binding to miR663a/pre-miR663a. The cross talk between miR663AHG and miR663a expression may play dominant roles in maintaining the functions of miR663AHG in colon cancer development.

Integrated Osteoinductive Factors─Exosome@MicroRNA-26a Hydrogel Enhances Bone Regeneration.

MicroRNAs (miRNAs) are a new therapeutic tool that can target multiple genes by inducing translation repression and target mRNA degradation. Although miRNAs have gained significant attention in oncology and in work on genetic disorders and autoimmune diseases, their application in tissue regeneration remains hindered by several challenges, such as miRNA degradation. Here, we reported Exosome@MicroRNA-26a (Exo@miR-26a), an osteoinductive factor that can be substituted for routinely used growth factors, which was constructed using bone marrow stem cell (BMSC)-derived exosomes and microRNA-26a (miR-26a). Exo@miR-26a-integrated hydrogels significantly promoted bone regeneration when implanted into defect sites; as the exosome stimulated angiogenesis, miR-26a promoted osteogenesis while the hydrogel enabled a site-directed release. Moreover, BMSC-derived exosomes further facilitated healthy bone regeneration by repressing osteoclast differentiation-related genes rather than damaging osteoclasts. Taken together, our findings demonstrate the promising potential of Exo@miR-26a for bone regeneration and provide a new strategy for the application of miRNA therapy in tissue engineering.

MicroRNA-593-5p contributes to cell death following exposure to 1-methyl-4-phenylpyridinium by targeting PTEN-induced putative kinase 1

Neurodegenerative diseases are characterized by a decline in neuronal function and structure, leading to neuronal death. Understanding the molecular mechanisms of neuronal death is crucial for developing therapeutics. MiRs are small noncoding RNAs that regulate gene expression by degrading target mRNAs or inhibiting translation. MiR dysregulation has been linked to many neurodegenerative diseases, but the underlying mechanisms are not well understood. As mitochondrial dysfunction is one of the common molecular mechanisms leading to neuronal death in many neurodegenerative diseases, here we studied miRs that modulate neuronal death caused by 1-methyl-4-phenylpyridinium (MPP+), an inhibitor of complex I in mitochondria. We identified miR-593-5p, levels of which were increased in SH-SY5Y human neuronal cells, after exposure to MPP+. We found that intracellular Ca2+, but not of reactive oxygen species, mediated this miR-593-5p increase. Furthermore, we found the increase in miR-593-5p was due to enhanced stability, not increased transcription or miR processing. Importantly, we show the increase in miR-593-5p contributed to MPP+-induced cell death. Our data revealed that miR-593-5p inhibits a signaling pathway involving PTEN-induced putative kinase 1 (PINK1) and Parkin, two proteins responsible for the removal of damaged mitochondria from cells, by targeting the coding sequence of PINK1 mRNA. Our findings suggest that miR-593-5p contributes to neuronal death resulting from MPP+ toxicity, in part, by impeding the PINK1/Parkin-mediated pathway that facilitates the clearance of damaged mitochondria. Taken together, our observations highlight the potential significance of inhibiting miR-593-5p as a therapeutic approach for neurodegenerative diseases.

Screening of Drosophila microRNA-degradation sequences reveals Argonaute1 mRNA’s role in regulating miR-999

MicroRNAs (miRNA) load onto AGO proteins to target mRNAs for translational repression or degradation. However, miRNA degradation can be triggered when extensively base-paired with target RNAs, which induces confirmational change of AGO and recruitment of ZSWIM8 ubiquitin ligase to mark AGO for proteasomal degradation. This target RNA-directed miRNA degradation (TDMD) mechanism appears to be evolutionarily conserved, but recent studies have focused on mammalian systems. Here, we performed AGO1-CLASH in Drosophila S2 cells, with Dora (ortholog of vertebrate ZSWIM8) knockout mediated by CRISPR-Cas9 to identify five TDMD triggers (sequences that can induce miRNA degradation). Interestingly, one trigger in the 3′ UTR of AGO1 mRNA induces miR-999 degradation. CRISPR-Cas9 knockout of the AGO1 trigger in S2 cells and in Drosophila specifically elevates miR-999, with concurrent repression of the miR-999 targets. AGO1 trigger knockout flies respond poorly to hydrogen peroxide-induced stress, demonstrating the physiological importance of this TDMD event.

Phosphorylated Upstream Frameshift 1-dependent Nonsense-mediated μ-Opioid Receptor mRNA Decay in the Spinal Cord Contributes to the Development of Neuropathic Allodynia-like Behavior in Rats.

Nonsense-mediated messenger RNA (mRNA) decay increases targeted mRNA degradation and has been implicated in the regulation of gene expression in neurons. The authors hypothesized that nonsense-mediated μ-opioid receptor mRNA decay in the spinal cord is involved in the development of neuropathic allodynia-like behavior in rats. Adult Sprague-Dawley rats of both sexes received spinal nerve ligation to induce neuropathic allodynia-like behavior. The mRNA and protein expression contents in the dorsal horn of animals were measured by biochemical analyses. Nociceptive behaviors were evaluated by the von Frey test and the burrow test. On Day 7, spinal nerve ligation significantly increased phosphorylated upstream frameshift 1 (UPF1) expression in the dorsal horn (mean ± SD; 0.34 ± 0.19 in the sham ipsilateral group vs. 0.88 ± 0.15 in the nerve ligation ipsilateral group; P < 0.001; data in arbitrary units) and drove allodynia-like behaviors in rats (10.58 ± 1.72 g in the sham ipsilateral group vs. 1.19 ± 0.31 g in the nerve ligation ipsilateral group, P < 0.001). No sex-based differences were found in either Western blotting or behavior tests in rats. Eukaryotic translation initiation factor 4A3 (eIF4A3) triggered SMG1 kinase (0.06 ± 0.02 in the sham group vs. 0.20 ± 0.08 in the nerve ligation group, P = 0.005, data in arbitrary units)-mediated UPF1 phosphorylation, leading to increased nonsense-mediated mRNA decay factor SMG7 binding and µ-opioid receptor mRNA degradation (0.87 ± 0.11-fold in the sham group vs. 0.50 ± 0.11-fold in the nerve ligation group, P = 0.002) in the dorsal horn of the spinal cord after spinal nerve ligation. Pharmacologic or genetic inhibition of this signaling pathway in vivo ameliorated allodynia-like behaviors after spinal nerve ligation. This study suggests that phosphorylated UPF1-dependent nonsense-mediated μ-opioid receptor mRNA decay is involved in the pathogenesis of neuropathic pain.

SRNA expedites polycistronic mRNA decay in Escherichia coli.

In bacteria, most small RNA (sRNA) elicits RNase E-mediated target mRNA degradation by binding near the translation initiation site at the 5' end of the target mRNA. Spot 42 is an sRNA that binds in the middle of the gal operon near the translation initiation site of galK, the third gene of four, but it is not clear whether this binding causes degradation of gal mRNA. In this study, we measured the decay rate of gal mRNA using Northern blot and found that Spot 42 binding caused degradation of only a specific group of gal mRNA that shares their 3' end with full-length mRNA. The results showed that in the MG1655Δspf strain in which the Spot 42 gene was removed, the half-life of each gal mRNA in the group increased by about 200% compared to the wild type. Since these mRNA species are intermediate mRNA molecules created by the decay process of the full-length gal mRNA, these results suggest that sRNA accelerates the mRNA decaying processes that normally operate, thus revealing an unprecedented role of sRNA in mRNA biology.