mRNA Interactions Research Articles

Enhancing mRNA Interactions by Engineering the Arc Protein with Nucleocapsid Domain.

Activity-regulated cytoskeleton-associated protein (Arc) forms virus-like capsids for mRNA transport between neurons. Unlike HIV-1 Group-specific Antigen (Gag), which uses its Nucleocapsid (NC) domain to bind HIV-1 genomic mRNA, mammalian Arc lacks the NC domain, and their direct mRNA binding interactions remain underexplored. This study examined rat Arc's binding to rat Arc 5' UTR (A5U), HIV-1 5' UTR (H5U), and GFP mRNAs, revealing weak binding with no significant preference. Adding the HIV-1 NC domain to rArc's C-terminus significantly improved binding to H5U, while also showing substantial binding to A5U at about 60% of its H5U level and exhibiting twice the affinity for A5U over GFP mRNA. Importantly, rArc-NC binds 3.4 times more A5U and H5U than GST-NC, indicating that rArc NTD-CA aids mRNA binding by HIV-1 NC. These findings suggest a conserved Gag protein-mRNA interaction mechanism, highlighting the potential for developing mRNA delivery systems that combine endogenous Gag NTD-CA with retroviral NC and UTRs.

Systems analysis of miR-199a/b-5p and multiple miR-199a/b-5p targets during chondrogenesis

Changes in chondrocyte gene expression can contribute to the development of osteoarthritis (OA), and so recognition of the regulative processes during chondrogenesis can lead to a better understanding of OA. microRNAs (miRNAs) are key regulators of gene expression in chondrocytes/OA, and we have used a combined experimental, bioinformatic, and systems biology approach to explore the multiple miRNA–mRNA interactions that regulate chondrogenesis. A longitudinal chondrogenesis bioinformatic analysis identified paralogues miR-199a-5p and miR-199b-5p as pro-chondrogenic regulators. Experimental work in human cells demonstrated alteration of miR-199a-5p or miR-199b-5p expression led to significant inverse modulation of key chondrogenic genes and extracellular matrix production. miR-199a/b-5p targets FZD6, ITGA3 and CAV1 were identified by inhibition experiments and verified as direct targets by luciferase assay. The experimental work was used to generate and parameterise a multi-miRNA 14-day chondrogenesis kinetic model to be used as a repository for the experimental work and as a resource for further investigation of this system. This is the first multi-miRNA model of a chondrogenesis-based system, and highlights the complex relationships between regulatory miRNAs, and their target mRNAs.

A Combined mRNA and microRNA Transcriptome Analysis of B. oleracea Response to Plasmodiophora brassicae Infection

Clubroot disease, caused by the pathogen Plasmodiophora brassicae, is a serious disease that poses a critical threat to cabbage production. However, the molecular mechanism of the microRNAs (miRNAs) involved in the cabbage’s response to P. brassicae infection remains to be elucidated. Here, the mRNA and miRNA expression profiles of cabbage in response to a P. brassicae infection were analyzed. In the transcriptome analysis, 2217 and 5552 differentially expressed genes (DEGs) were identified at 7d and 21d after inoculation, which were enriched in MAPK signaling, plant–pathogen interaction, plant hormone signal transduction, and phenylpropanoid biosynthesis pathways. BolC02g057640.2J, BolC09g006890.2J, BolC02g013230.2J, BolC06g006490.2J, BolC03g052660.2J, BolC07g052580.2J, and BolC04g044910.2J were predicted to be significantly involved in the defense response or plant–pathogen interaction through co-expression network analysis. Small RNA data analysis identified 164 miRNAs belonging to 51 families. miR1515, miR166, miR159, and miR9563 had the greatest number of members among the miRNA families. Integrated analysis revealed 23 miRNA–mRNA interactions related to a P. brassicae infection. The target genes of differentially expressed miRNAs (DEMs) revealed the NAC, ARF, TCP, and SPL transcription factor members that probably participate in the defense response. This study provided new insights into the miRNA-involved regulatory system during the process of disease infection with P. brassicae in cabbage.

Profiling the lncRNA–miRNA–mRNA interaction network in the cold-resistant exercise period of grape (Vitis amurensis Rupr.)

BackgroundGrape is a plant that is sensitive to low temperature and vulnerable to low-temperature damage. However, little is known about the roles of lncRNAs, miRNAs and mRNAs in regulating the hypothermia response mechanism in Vitis amurensis Rupr.MethodsIn this study, the expression and regulatory network of low-temperature response genes were studied in the phloem of grape under different low-temperature stress.ResultsHere, we performed analyses related to RNA-seq and miRNA-seq on grape phloem tissues from five periods of cold resistance campaigns. Three RNAs (lncRNAs, miRNAs and mRNAs) obtained by KEGG and GO analyses were used to identify starch and sucrose metabolism associated with cold resistance, and specific changes in BP, CC, and MF were identified in four comparisons. Venn diagrams, thermograms and pathway maps were used to analyze the differentially expressed genes (DEGs), and their specific gene expression during the cold exercise were obtained. The six DEGs finally selected were used for qRT-PCR to verify the RNA-seq data. In addition, we found that the regulatory networks of miRNAs and lncRNAs correspond to the six DEGs. This study will contribute to further experimental studies to elucidate the cold resistance mechanism of Vitis amurensis Rupr.ConclusionsThe low-temperature response genes of grape are mainly enriched in the starch and sucrose metabolism, and they are regulated by miRNAs and lncRNAs. The conclusions will provide basic information for further understanding of the cold resistance mechanism of grape in the future.Graphical

Long Non-Coding RNA CRNDE, LINC00957, and AC072061.1 as a Promising Diagnostic and Prognostic Biomarker in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is one of the most invasive types of brain cancer. LncRNAs can be considered a new prognostic and diagnostic biomarker in GBM. This study comprehensively explored the interaction of lncRNAs with mRNAs in the TCGA database and proposed a novel promising biomarker with favorable diagnostic and prognostic values. The public data of RNA-seq and related clinical data were downloaded from the TCGA database. Differential expression analysis was conducted in R. GO and KEGG signaling pathways were used for enrichment. The STRING database was used for PPI analysis. CE-network was constructed by STAR database. Kaplan-Meier survival analysis and ROC curve analysis to indicate the biomarkers' diagnostic and prognostic values. Differentially expressed data illustrated that 4428 mRNAs were differentially expressed in GBM. The GO and KEGG pathway analysis showed that the differentially expressed mRNAs were enriched in critical biological processes. The PPI showed that WEE1, BARD1, and CDK6 were the important PPI hubs. The ceRNA network data demonstrated critical lncRNAs. The data revealed that the lncRNA CRNDE, LINC00957, AC072061.1, AC068888.1, and DBH-AS1 are potential diagnostic prognostic biomarkers in the GBM patients. Altogether, we demonstrated lncRNA, and mRNA interaction and mentioned regulatory networks, considered a therapeutic option in GBM. In addition, we proposed potential diagnostic and prognostic biomarkers for the patients.

Licochalcone A decreases cancer cell proliferation and enhances ferroptosis in acute myeloid leukemia through suppressing the IGF2BP3/MDM2 cascade.

Licochalcone A (Lico A), a naturally bioactive flavonoid, has shown antitumor activity in several types of cancers. However, few studies have focused on its effect on acute myeloid leukemia (AML). Cell viability and colony formation potential were detected by CCK-8 assay and colony formation assay, respectively. Cell cycle distribution and apoptosis were assessed by flow cytometry. Ferroptosis was assessed by measuring reactive oxygen species (ROS), lipid ROS, malondialdehyde (MDA), and glutathione (GSH). Protein expression levels were determined by immunoblotting and immunohistochemistry (IHC), and mRNA expression was detected by real-time qPCR. The m6A modification of MDM2 mRNA was verified by methylated RNA immunoprecipitation (MeRIP) assay, and the interaction of IGF2BP3 and MDM2 mRNA was analyzed by RIP assay. Actinomycin D was used to evaluate mRNA stability. The efficacy of Lico A in vivo was examined by a murine xenograft model. Lico A suppressed cell proliferation and induced ferroptosis in MOLM-13 and U-937 in vitro, and slowed the growth of xenograft tumors in vivo. IGF2BP3 was highly expressed in human AML specimens and cells, and Lico A suppressed IGF2BP3 expression in AML cells. Lico A exerted the anti-proliferative and pro-ferroptosis effects by downregulating IGF2BP3. Moreover, IGF2BP3 enhanced the stability and expression of MDM2 mRNA through an m6A-dependent manner. Downregulation of IGF2BP3 impeded AML cell proliferation and enhanced ferroptosis via repressing MDM2. Furthermore, Lico A could affect the MDM2/p53 pathway by downregulating IGF2BP3 expression. Lico A exerts the anti-proliferative and pro-ferroptosis activity in AML cells by affecting the IGF2BP3/MDM2/p53 pathway, providing new evidence for Lico A as a promising agent for the treatment of AML.

Initial Evaluation of lncRNA A2M-AS1 Gene Expression in Multiple Sclerosis Patients

Background: Multiple sclerosis (MS) is one of the three leading neurodegenerative diseases worldwide. Gene expression profile studies play an important role in recognizing and preventing disease. Considering the inherent ability of biomarkers to diagnose and prognose the occurrence of a disease, with the aim of gene therapy and changing gene expression, it can be helped to treat it. In this study, by examining the gene interaction and expression of non-coding genes in patients with MS, using bioinformatics analyses, laboratory research and potential non-coding diagnostic biomarkers of MS were selected for further investigations. Materials and Methods: First, by using micro-array data analysis of the GEO database, the expression status of the long non-coding ribonucleic acid (RNA) (lncRNA) A2M-AS1 gene was investigated in patients with MS. lncRNA–mRNA interaction analysis was performed in the lncRRisearch database. After sample collection, the total RNA extracted using the RNA extraction kit from 20 patient samples and 20 healthy samples was synthesized into cDNA with the synthesis kit. The quantitative reverse transcriptase polymerase chain reaction experiment was performed for the final validation of expression change. Results: Based on bioinformatic and laboratory analysis, the expression of the A2M-AS1 gene in MS samples showed a significant decrease in expression compared to healthy samples. Also, based on the receiver operating characteristic analysis, lncRNA A2M-AS1 can be introduced as an acceptable diagnostic biomarker to distinguish MS samples from healthy samples. Conclusion: lncRNA A2M-AS1, by reducing its expression as an acceptable diagnostic biomarker, can increase the risk of developing MS.

LncRNA-536 and RNA Binding Protein RBM25 Interactions in Pulmonary Arterial Hypertension.

Hyperproliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the essential features of the maladaptive inward remodeling of the pulmonary arteries in pulmonary arterial hypertension (PAH). In this study, we define the mechanistic association between long-noncoding RNA: ENST00000495536 (Lnc-536) and anti-proliferative HOXB13 in mediating smooth muscle hyperplasia. Antisense oligonucleotide-based GapmeRs or plasmid overexpressing lnc-536 were used to evaluate the role of lnc-536 in mediating hyperproliferation of PDGF-treated or idiopathic PAH (IPAH) PASMCs. Further, we pulled down lnc536 to identify the proteins directly interacting with lnc536. The in-vivo role of lnc-536 was determined in Sugen-hypoxia and HIV-transgenic pulmonary hypertensive rats. Increased levels of lnc-536 in PDGF-treated or IPAH PASMCs promote hyperproliferative phenotype by downregulating the HOXB13 expression. Knockdown of lnc-536 in-vivo prevented increased RVSP, Fulton Index, and pulmonary vascular remodeling in Sugen-Hypoxia rats. The lncRNA-536 pull-down assay demonstrated the interactions of RNA binding protein: RBM25 with SFPQ, a transcriptional regulator that has a binding motif on HOXB13 exon Further, The RNA-IP experiment using the SFPQ antibody showed direct interaction of RBM25 with SFPQ and knockdown of RBM25 resulted in increased interactions of SFPQ and HOXB13 mRNA while attenuating PASMC proliferation. Finally, we examined the role of lnc-536 and HOXB13 axis in the PASMCs exposed to the dual hit of HIV and a stimulant: cocaine as well. lnc-536 acts as a decoy for RBM25, which in turn sequesters SFPQ, leading to the decrease in HOXB13 expression and hyperproliferation of smooth muscle cells associated with PAH development.

Comparative Transcriptome Analysis of mRNA and miRNA during the Development of Longissimus Dorsi Muscle of Gannan Yak and Tianzhu White Yak.

The Gannan yak, a superior livestock breed found on the Tibetan Plateau, exhibits significantly enhanced body size, weight, and growth performance in comparison to the Tianzhu white yak. MiRNAs play a pivotal role in regulating muscle growth by negatively modulating target genes. In this study, we found the average diameter, area, and length of myofibers in Gannan yaks were significantly higher than those of Tianzhu white yaks. Further, we focused on analyzing the longissimus dorsi muscle from both Gannan yaks and Tianzhu white yaks through transcriptome sequencing to identify differentially expressed (DE)miRNAs that influence skeletal muscle development. A total of 254 DE miRNAs were identified, of which 126 miRNAs were up-regulated and 128 miRNAs were down-regulated. GO and KEGG enrichment analysis showed that the target genes of these DE miRNAs were significantly enriched in signaling pathways associated with muscle growth and development. By constructing a DE miRNA- DE mRNA interaction network, we screened 18 key miRNAs, and notably, four of the candidates (novel-m0143-3p, novel-m0024-3p, novel-m0128-5p, and novel-m0026-3p) targeted six genes associated with muscle growth and development (DDIT4, ADAMTS1, CRY2, AKIRIN2, SIX1, and FOXO1). These findings may provide theoretical references for further studies on the role of miRNAs in muscle growth and development in Gannan yaks.

Micro Target: MicroRNA Target Prediction and Validation with Experimentally Positive and Negative Examples

MicroRNAs (miRNAs) usually controls the gene by binding to complementary sites of 3’ untranslated region of its target genes. Numerous criteria-based and machine learning approaches are available in the literature to predict miRNA–mRNA interactions, but most of them struggle with either high false positive or false negative rates and also don’t show good validation with experimentally validated positive and negative examples. Here we present microTarget, a new computational approach for identifying miRNA target genes which are based on complementarity score, thermodynamic duplex stability and also independent of conservation of target sites in related genomes. In this article, we validated our algorithm using positive and negative data from the literature in various human tissues, and our method outperformed existing computational methods such as miRanda, RNA22, and PITA. Receiver operating characteristic curves (ROC) and Matthew's correlation coefficient (MCC) were calculated using experimentally validated data, and they reveal that microTarget greatly improves miRNA target prediction compared to the three algorithms employed individually. Additionally, an F-score analysis demonstrated that microTarget greatly enhances the relevance of the other techniques. Thus, microTarget is a useful tool for biologists looking for miRNA targets and integrating them into biological contexts.

Large Language Models and Genomics for Summarizing the Role of microRNA in Regulating mRNA Expression.

microRNA (miRNA)-messenger RNA (mRNA or gene) interactions are pivotal in various biological processes, including the regulation of gene expression, cellular differentiation, proliferation, apoptosis, and development, as well as the maintenance of cellular homeostasis and pathogenesis of numerous diseases, such as cancer, cardiovascular diseases, neurological disorders, and metabolic conditions. Understanding the mechanisms of miRNA-mRNA interactions can provide insights into disease mechanisms and potential therapeutic targets. However, extracting these interactions efficiently from a huge collection of published articles in PubMed is challenging. In the current study, we annotated a miRNA-mRNA Interaction Corpus (MMIC) and used it for evaluating the performance of a variety of machine learning (ML) models, deep learning-based transformer (DLT) models, and large language models (LLMs) in extracting the miRNA-mRNA interactions mentioned in PubMed. We used the genomics approaches for validating the extracted miRNA-mRNA interactions. Among the ML, DLT, and LLM models, PubMedBERT showed the highest precision, recall, and F-score, with all equal to 0.783. Among the LLM models, the performance of Llama-2 is better when compared to others. Llama 2 achieved 0.56 precision, 0.86 recall, and 0.68 F-score in a zero-shot experiment and 0.56 precision, 0.87 recall, and 0.68 F-score in a three-shot experiment. Our study shows that Llama 2 achieves better recall than ML and DLT models and leaves space for further improvement in terms of precision and F-score.

Exploring the targetome of IsrR, an iron-regulated sRNA controlling the synthesis of iron-containing proteins in Staphylococcus aureus.

Staphylococcus aureus is a common colonizer of the skin and nares of healthy individuals, but also a major cause of severe human infections. During interaction with the host, pathogenic bacteria must adapt to a variety of adverse conditions including nutrient deprivation. In particular, they encounter severe iron limitation in the mammalian host through iron sequestration by haptoglobin and iron-binding proteins, a phenomenon called "nutritional immunity." In most bacteria, including S. aureus, the ferric uptake regulator (Fur) is the key regulator of iron homeostasis, which primarily acts as a transcriptional repressor of genes encoding iron acquisition systems. Moreover, Fur can control the expression of trans-acting small regulatory RNAs that play an important role in the cellular iron-sparing response involving major changes in cellular metabolism under iron-limiting conditions. In S. aureus, the sRNA IsrR is controlled by Fur, and most of its predicted targets are iron-containing proteins and other proteins related to iron metabolism and iron-dependent pathways. To characterize the IsrR targetome on a genome-wide scale, we combined proteomics-based identification of potential IsrR targets using S. aureus strains either lacking or constitutively expressing IsrR with an in silico target prediction approach, thereby suggesting 21 IsrR targets, of which 19 were negatively affected by IsrR based on the observed protein patterns. These included several Fe-S cluster- and heme-containing proteins, such as TCA cycle enzymes and catalase encoded by katA. IsrR affects multiple metabolic pathways connected to the TCA cycle as well as the oxidative stress response of S. aureus and links the iron limitation response to metabolic remodeling. In contrast to the majority of target mRNAs, the IsrR-katA mRNA interaction is predicted upstream of the ribosome binding site, and further experiments including mRNA half-life measurements demonstrated that IsrR, in addition to inhibiting translation initiation, can downregulate target protein levels by affecting mRNA stability.

Relationship between the serum level, polymorphism and gene expression of IL-33 in samples of recurrent miscarriage Iraqi women infected with toxoplasmosis

One of the many warm-blooded hosts that toxoplasmosis-causing intracellular protozoan parasite Toxoplasma gondii can infect is humans. Cytokines are crucial to stimulate an effective immune response against T. gondii. Interleukin-33 (IL-33) is a unique anti-inflammatory cytokine that suppresses the immune response. The levels of cytokine gene expression are regulated by genetics, and the genetic polymorphisms of these cytokines play a functional role in this process. Single nucleotide polymorphisms (SNPs) are prognostic indicators of illnesses. This study aimed to determine whether toxoplasmosis interacts with serum levels of IL-33 and its SNP in miscarriage women as well as whether serum levels and IL-33 gene expression are related in toxoplasmosis-positive miscarriage women. Two hundred blood samples from patients and controls were collected from AL-Alawiya Maternity Teaching Hospital and AL-Yarmouk Teaching Hospital in Baghdad, Iraq from 2021 to 2022 in order to evaluate the serum level of IL-33 using ELISA test. For the SNP of IL-33, the allelic high-resolution approach was utilized, and real time-PCR was performed to assess gene expression. The results showed that compared to healthy and pregnant women, recurrent miscarriage with toxoplasmosis and recurrent miscarriage women had lower IL-33 concentrations. Additionally, there were significant differences among healthy women, pregnant women, and women with repeated miscarriage who experienced toxoplasmosis. Furthermore, no differences between patients and controls were revealed by gene expression data. The results revealed that recurrent miscarriage, pregnancy, and healthy women all had a slightly higher amount of the IL-33 gene fold. Additionally, the SNP of IL-33 data demonstrated that there was no significant genetic relationship between patients and controls. Recurrent miscarriage women with toxoplasmosis have showed significant differences from pregnant women in the genotypes GG and AA as well as the alleles A and G. There were notable variations between recurrent miscarriage with and without toxoplasmosis in terms of the genotypes AA and AC. The genotypes GG, AA, and allele A in recurrent miscarriage women with toxoplasmosis and recurrent miscarriage women is a protective factor. Taking together, there was a statistically significant negative correlation between toxoplasmosis and IL-33 gene expression, which calls for more quantitative investigation in order to fully comprehend the interaction of mRNA and protein.

PRIMITI: A computational approach for accurate prediction of miRNA-target mRNA interaction

Current medical research has been demonstrating the roles of miRNAs in a variety of cellular mechanisms, lending credence to the association between miRNA dysregulation and multiple diseases. Understanding the mechanisms of miRNA is critical for developing effective diagnostic and therapeutic strategies. miRNA-mRNA interactions emerge as the most important mechanism to be understood despite their experimental validation constraints. Accordingly, several computational models have been developed to predict miRNA-mRNA interactions, albeit presenting limited predictive capabilities, poor characterisation of miRNA-mRNA interactions, and low usability. To address these drawbacks, we developed PRIMITI, a PRedictive model for the Identification of novel miRNA-Target mRNA Interactions. PRIMITI is a novel machine learning model that utilises CLIP-seq and expression data to characterise functional target sites in 3’-untranslated regions (3’-UTRs) and predict miRNA-target mRNA repression activity. The model was trained using a reliable negative sample selection approach and the robust extreme gradient boosting (XGBoost) model, which was coupled with newly introduced features, including sequence and genetic variation information. PRIMITI achieved an area under the receiver operating characteristic (ROC) curve (AUC) up to 0.96 for a prediction of functional miRNA-target site binding and 0.96 for a prediction of miRNA-target mRNA repression activity on cross-validation and an independent blind test. Additionally, the model outperformed state-of-the-art methods in recovering miRNA-target repressions in an unseen microarray dataset and in a collection of validated miRNA-mRNA interactions, highlighting its utility for preliminary screening. PRIMITI is available on a reliable, scalable, and user-friendly web server at https://biosig.lab.uq.edu.au/primiti.

YTHDC1 inhibits autophagy-dependent NF-κB signaling by stabilizing Beclin1 mRNA in macrophages

BackgroundYTHDC1, a key m(6)A nuclear reader, plays a crucial role in regulating mRNA splicing, export, and stability. However, the functional significance and regulatory mechanisms of YTHDC1 in inflammatory bowel disease (IBD) remain to be explored.MethodsWe established a dextran sulfate sodium (DSS)-induced murine colitis model in vivo and LPS/IFN-γ-stimulated macrophage inflammation in vitro. The expression of YTHDC1 was determined. Colocalization of YTHDC1 and macrophages was assayed by immunofluorescence staining. LV-YTHDC1 or shYTHDC1 lentiviruses were applied for YTHDC1 overexpression or inhibition. For NF-κB inhibition, JSH-23 was utilized. The interaction of YTHDC1 and Beclin1 mRNA was determined by RIP, and the m6A modification of Beclin1 was confirmed by MeRIP.ResultsIn DSS-induced colitis and LPS/IFN-γ-treated RAW264.7 macrophages, we observed a significant downregulation of YTHDC1. Overexpression of YTHDC1 resulted in decreased levels of iNOS, CD86, and IL-6 mRNA, along with inhibited NF-κB activation in LPS/IFN-γ-treated RAW264.7 cells. Conversely, downregulation of YTHDC1 promoted iNOS expression and inhibited autophagy. Additionally, the effect of YTHDC1 knockdown on CD86 and IL-6 mRNA induced by LPS/IFN-γ was abolished by the NF-κB inhibitor JSH-23. Mechanistically, YTHDC1 interacted with Beclin1 mRNA, thereby stabilizing Beclin1 mRNA and enhancing Beclin1 expression and autophagy. These effects ultimately led to the inhibition of NF-κB signaling in LPS/IFN-γ-challenged macrophages.ConclusionsYTHDC1 inhibited the macrophage-mediated inflammatory response by stabilizing Beclin1 mRNA, which may be a potential therapeutic target for the treatment of IBD.

The Musashi-1–type 2 deiodinase pathway regulates astrocyte proliferation

Thyroid hormone (TH) is a critical regulator of cellular function and cell fate. The circulating TH level is relatively stable, while tissue TH action fluctuates according to cell type-specific mechanisms. Here, we focused on identifying mechanisms that regulate TH action through the type 2 deiodinase (D2) in glial cells. Dio2 mRNA has an unusually long 3'UTR where we identified multiple putative MSI1 binding sites for Musashi-1 (MSI1), a highly conserved RNA-binding cell cycle regulator. Binding to these sites was confirmed through electrophoretic mobility shift assay. In H4 glioma cells, shRNA-mediated MSI1 knockdown increased endogenous D2 activity, whereas MSI1 overexpression in HEK293T cells decreased D2 expression. This latter effect could be prevented by the deletion of a 3.6 kb region of the 3'UTR of Dio2 mRNA containing MSI1 binding sites. MSI1 immunoreactivity was observed in 2mouse Dio2-expressing cell types, that is, cortical astrocytes and hypothalamic tanycytes, establishing the anatomical basis for a potential invivo interaction of Dio2 mRNA and MSl1. Indeed, increased D2 expression was observed in the cortex of mice lacking MSI1 protein. Furthermore, MSI1 knockdown-induced D2 expression slowed down cell proliferation by 56% in primary cultures of mouse cortical astrocytes, establishing the functionality of the MSI1-D2-T3 pathway. In summary, Dio2 mRNA is a target of MSI1 and the MSI1-D2-T3 pathway is a novel regulatory mechanism of astrocyte proliferation with the potential to regulate the pathogenesis of human glioblastoma.

Liver cancer initiation requires translational activation by an oncofetal regulon involving LIN28 proteins.

It is unknown which posttranscriptional regulatory mechanisms are required for oncogenic competence. Here, we show that the LIN28 family of RNA-binding proteins (RBPs), which facilitate posttranscriptional RNA metabolism within ribonucleoprotein networks, is essential for the initiation of diverse oncotypes of hepatocellular carcinoma (HCC). In HCC models driven by NRASG12V/Tp53, CTNNB1/YAP/Tp53, or AKT/Tp53, mice without Lin28a and Lin28b were markedly impaired in cancer initiation. We biochemically defined an oncofetal regulon of 15 factors connected to LIN28 through direct mRNA and protein interactions. Interestingly, all were RBPs and only 1 of 15 was a Let-7 target. Polysome profiling and reporter assays showed that LIN28B directly increased the translation of 8 of these 15 RBPs. As expected, overexpression of LIN28B and IGFBP1-3 was able to genetically rescue cancer initiation. Using this platform to probe components downstream of LIN28, we found that 8 target RBPs were able to restore NRASG12V/Tp53 cancer formation in Lin28a/Lin28b-deficient mice. Furthermore, these LIN28B targets promote cancer initiation through an increase in protein synthesis. LIN28B, central to an RNP regulon that increases translation of RBPs, is important for tumor initiation in the liver.

Effect of miR-98/IL-6/STAT3 on Autophagy and Apoptosis of Cardiac Stem Cells Under Hypoxic Conditions In vitro.

The heavy burden of cardiovascular diseases demands innovative therapeutic strategies dealing with cardiomyocyte loss. Cardiac Stem Cells (CSCs) are renewable cells in the myocardium with differentiation and endocrine functions. However, their functions are significantly inhibited in conditions of severe hypoxia or inflammation. The mechanism of hypoxia affecting CSCs is not clear. Interleukin-6 (IL-6) appears active in both hypoxic and inflammatory microenvironments. The aim of this study was to explore whether IL-6 is related to CSC apoptosis and autophagy under severe hypoxia. In this study, rat CSCs were extracted by alternate digestion. The interaction of miR-98 and IL-6 mRNA was detected by the dual luciferase method, and qPCR was applied to confirm the effect of miR-98 on IL-6 expression. The effect of IL-6 on CSC apoptosis was measured by flow cytometry and the effect of IL-6 on CSC autophagy by transmission electron microscopy. The western blot method was applied to detect the effect of IL-6 on the expressions of proteins related to apoptosis and autophagy. ANOVA and Dunnett T3's test were employed in the statistical analysis. When p < 0.05, the difference was significant. Under severe hypoxia conditions, IL-6 increased CSC apoptosis and decreased p-STAT3 expression significantly. CSC apoptosis increased significantly after inhibition of the STAT3 signaling pathway under severe hypoxia. IL-6 could also significantly inhibit CSCs' autophagy and block their autophagy flow under severe hypoxic conditions. Meanwhile, it was confirmed that miR-98 had a binding site on IL-6 mRNA and miR-98 significantly inhibited IL-6 mRNA expression in CSCs under severe hypoxic conditions. miR-98/IL-6/STAT3 has been found to be involved in the regulation of CSCs' apoptosis and autophagy under severe hypoxic conditions and there might be a mutual linkage between CSCs' apoptosis and their autophagy.

Uncovering miRNA-mRNA Regulatory Networks Related to Olaparib Resistance and Resensitization of BRCA2MUT Ovarian Cancer PEO1-OR Cells with the ATR/CHK1 Pathway Inhibitors.

Resistance to olaparib is the major obstacle in targeted therapy for ovarian cancer (OC) with poly(ADP-ribose) polymerase inhibitors (PARPis), prompting studies on novel combination therapies to enhance olaparib efficacy. Despite identifying various mechanisms, understanding how OC cells acquire PARPi resistance remains incomplete. This study investigated microRNA (miRNA) expression in olaparib-sensitive (PEO1, PEO4) and previously established olaparib-resistant OC cell lines (PEO1-OR) using high-throughput RT-qPCR and bioinformatic analyses. The role of miRNAs was explored regarding acquired resistance and resensitization with the ATR/CHK1 pathway inhibitors. Differentially expressed miRNAs were used to construct miRNA-mRNA regulatory networks and perform functional enrichment analyses for target genes with miRNet 2.0. TCGA-OV dataset was analyzed to explore the prognostic value of selected miRNAs and target genes in clinical samples. We identified potential processes associated with olaparib resistance, including cell proliferation, migration, cell cycle, and growth factor signaling. Resensitized PEO1-OR cells were enriched in growth factor signaling via PDGF, EGFR, FGFR1, VEGFR2, and TGFβR, regulation of the cell cycle via the G2/M checkpoint, and caspase-mediated apoptosis. Antibody microarray analysis confirmed dysregulated growth factor expression. The addition of the ATR/CHK1 pathway inhibitors to olaparib downregulated FGF4, FGF6, NT-4, PLGF, and TGFβ1 exclusively in PEO1-OR cells. Survival and differential expression analyses for serous OC patients revealed prognostic miRNAs likely associated with olaparib resistance (miR-99b-5p, miR-424-3p, and miR-505-5p) and resensitization to olaparib (miR-324-5p and miR-424-3p). Essential miRNA-mRNA interactions were reconstructed based on prognostic miRNAs and target genes. In conclusion, our data highlight distinct miRNA profiles in olaparib-sensitive and olaparib-resistant cells, offering molecular insights into overcoming resistance with the ATR/CHK1 inhibitors in OC. Moreover, some miRNAs might serve as potential predictive signature molecules of resistance and therapeutic response.

Comprehensive analysis of changes in expression of lncRNA, microRNA and mRNA in liver tissues of chickens with high or low abdominal fat deposition

ABSTRACT 1. The liver of chickens is a dominant lipid biosynthetic tissue and plays a vital role in fat deposition, particularly in the abdomen. To determine the molecular mechanisms involved in its lipid metabolism, the livers of chickens with high (H) or low (L) abdominal fat content were sampled and sequencing on long non-coding RNA (lncRNA), messenger RNA (mRNA) and small RNA (microRNA) was performed. 2. In total, 351 expressed protein-coding genes for long non-coding RNA (DEL; 201 upregulated and 150 downregulated), 400 differentially expressed genes (DEG; 223 upregulated and 177 downregulated) and 10 differentially expressed miRNA (DEM; four upregulated and six downregulated) were identified between the two groups. Multiple potential signalling pathways related to lipogenesis and lipid metabolism were identified via pathway enrichment analysis. In addition, 173 lncRNA – miRNA – mRNA interaction regulatory networks were identified, including 30 lncRNA, 27 mRNA and seven miRNA. 3. These networks may help regulate lipid metabolism and fat deposition. Five promising candidate genes and two lncRNA may play important roles in the regulation of adipogenesis and lipid metabolism in chickens.