Untranslated Region Of mRNA Research Articles

MicroRNA-33b inhibits cell proliferation and glycolysis by targeting hypoxia-inducible factor-1α in malignant melanoma.

[This retracts the article DOI: 10.3892/etm.2017.4702.].

Biological Functions of Let-7e-5p in Promoting the Differentiation of MC3T3-E1 Cells.

MicroRNAs let-7c and let-7f, two members of the let-7 family, were involved in regulating osteoblast differentiation and have an important role in bone formation. Let-7e-5p, which also belonged to the let-7 family, presented in the differentiation of adipose-derived stem cells and mouse embryonic stem cells. However, the role of let-7e-5p in osteoblast differentiation was unclear. Thus, this study aimed to elucidate the function of let-7e-5p in osteoblast differentiation and its mechanism. Firstly, we found that the let-7e-5p mimic promoted osteoblast differentiation but not the proliferation of MC3T3-E1 cells by positively regulating the expression levels of osteogenic-associated genes (RUNX2, OCN, OPN, and OSX), the activity of ALP, and formation of mineralized nodules. Moreover, we ascertained that the let-7e-5p mimic downregulated the post-transcriptional expression of SOCS1 by specifically binding to the 3′ untranslated region of SOCS1 mRNA. Also, let-7e-5p-induced SOCS1 downregulation increased the protein levels of p-STAT5 and IGF-1, which were both modulated by SOCS1 molecules. Furthermore, let-7e-5p abrogated the inhibition of osteogenic differentiation mediated by SOCS1 overexpression. Therefore, these results suggested that let-7e-5p regulated the differentiation of MC3T3-E1 cells through the JAK2/STAT5 pathway to upregulate IGF-1 gene expression by inhibiting SOCS1. These findings may provide a new insight into the regulatory role of let-7e-5p in osteogenic differentiation and imply the existence of a novel mechanism underlying let-7e-5p-mediated osteogenic differentiation.

Structural recognition of the mRNA 3' UTR by PUF-8 restricts the lifespan of C. elegans.

The molecular mechanisms of aging are unsolved fundamental biological questions. Caenorhabditis elegans is an ideal model organism for investigating aging. PUF-8, a PUF (Pumilio and FBF) protein in C. elegans, is crucial for germline development through binding with the 3′ untranslated regions (3′ UTR) in the target mRNAs. Recently, PUF-8 was reported to alter mitochondrial dynamics and mitophagy by regulating MFF-1, a mitochondrial fission factor, and subsequently regulated longevity. Here, we determined the crystal structure of the PUF domain of PUF-8 with an RNA substrate. Mutagenesis experiments were performed to alter PUF-8 recognition of its target mRNAs. Those mutations reduced the fertility and extended the lifespan of C. elegans. Deep sequencing of total mRNAs from wild-type and puf-8 mutant worms as well as in vivo RNA Crosslinking and Immunoprecipitation (CLIP) experiments identified six PUF-8 regulated genes, which contain at least one PUF-binding element (PBE) at the 3′ UTR. One of the six genes, pqm-1, is crucial for lipid storage and aging process. Knockdown of pqm-1 could revert the lifespan extension of puf-8 mutant animals. We conclude that PUF-8 regulate the lifespan of C. elegans may not only via MFF but also via modulating pqm-1-related pathways.

A microRNA‑17‑5p/homeobox B13 axis participates in the phenotypic modulation of vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) serve a decisive role in intimal hyperplasia, a common pathophysiological process that leads to numerous vascular disorders. The present study aimed to investigate the unknown mechanisms underlying VSMC phenotypic modulation and identified a novel microRNA (miRNA/miR)-17-5p/homeobox B13 (HOXB13) axis involved in the phenotypic switching, proliferation and migration of VSMCs. VSMCs were isolated from the thoracic aorta of Sprague-Dawley rats, cell proliferation was determined by Cell Counting Kit-8 (CCK-8) assay, cell migration was examined by Transwell migration assay and gene expression was detected using reverse transcription-quantitative PCR and western blot analyses. It was firstly found that incubation with platelet-derived growth factor-BB (PDGF-BB) recombinant protein resulted in a significant increase in HOXB13 expression in VSMCs. Using multiple miRNA prediction tools, miR-17-5p was identified as a potential regulator for HOXB13, since it had a 7-base perfect binding site and a 5-base imperfect binding site with the 3′-untranslated region of HOXB13 mRNA, and these sequences were highly conserved across species. The regulatory effect of miR-17-5p on HOXB13 was validated using luciferase reporter assays. The expression level of miR-17-5p was increased in VSMCs under PDGF-BB stimulation, and was negatively correlated with HOXB13 mRNA and protein expression. Moreover, the miR-17-5p mimics significantly inhibited the proliferation and migration of VSMCs, while antagomiR-17-5p showed the opposite effects, which could be abolished by HOXB13 knockdown. The miR-17-5p/HOXB13 axis also regulated the expression levels of the markers of differentiated VSMCs (α-smooth muscle actin, transgelin and smoothelin), proliferating cell nuclear antigen and cell migration proteins, including MMP-2 and −9. In conclusion, the present study demonstrated that miR-17-5p inhibited the phenotypic modulation VSMCs stimulated by PDGF-BB by downregulating HOXB13, indicating that these factors may be potential therapeutic targets for intimal hyperplasia.

TNF-α-Induced miR-21-3p Promotes Intestinal Barrier Dysfunction by Inhibiting MTDH Expression.

Intestinal barrier dysfunction is characterized by increased intestinal permeability to lumen endotoxin, showing remarkable predisposition to immune enteropathy, and colorectal cancer tumor necrosis factor (TNF)-α is associated with this pathological process, while the mechanism remains unknown. In this study, different doses of TNF-α were used for Caco-2 cell treatment. We discovered that miR-21-3p expression was obviously increased by TNF-α in a dose-dependent manner. Further study demonstrated that TNF-α could upregulate miR-21-3p expression through the NF-κB signaling pathway. Then, TargetScan and miRWalk miRNA–mRNA interaction prediction online tools were introduced, and metadherin (MTDH) was screened out as a potential target of miR-21-3p. We subsequently found that miR-21-3p could directly target the 3′-untranslated region (UTR) of MTDH mRNA and inhibit its expression. Furthermore, it was demonstrated that miR-21-3p could regulate the Wnt signaling pathway by targeting MTDH mRNA, suggesting the effect of miR-21-3p/MTDH/Wnt axis on intestinal barrier dysfunction. Our findings provide a novel potential biomarker and therapeutic target for intestinal barrier dysfunction and related diseases.

ZFP36 promotes VDR mRNA degradation to facilitate cell death in oral and colonic epithelial cells

BackgroundVitamin D receptor (VDR) plays a vital protective role in oral and colonic epithelial cells. Albeit we know that VDR expression is reduced in the mucosal epithelial layers of autoimmune diseases, the mechanism by which VDR is decreased remains elusive.MethodsVDR and zinc finger protein 36 (ZFP36) levels in human samples and cell lines were detected by real-time PCR, western blot and immunostaining. Luciferase report assay was used to test cis-elements in VDR gene promoter, real-time PCR was applied to measure mRNA decay and western blot was performed to evaluate protein degradation. RNA affinity chromatography assay was used to test protein-mRNA interaction. Co-immunoprecipitation was used to detect protein–protein interaction. The role of ZFP36 in AU-rich elements (AREs) in the 3′ untranslated region (UTR) of VDR mRNA was also measured by luciferase report assay.ResultsWe identify ZFP36 can bind with the AREs in the 3’UTR of VDR mRNA, leading to mRNA degradation in oral and colonic epithelial cells under inflammatory circumstance. Either ZFP36 protein or AREs of VDR mRNA mutation abolishes this protein-mRNA binding process. After the key amino acid’s mutation, ZFP36 fails to decrease VDR mRNA expression. We also find that VDR physically binds with Y box-binding protein 1 (YBX-1) to block YBX-1’s nuclear translocation and ameliorate cell death in the presence of inflammation.ConclusionThese findings provide insights into the cause of VDR decrease in oral and colonic epithelial cells under inflammatory condition and explain how VDR maintains cell viability in these cells.6BzbZFy5QCY-oJ2EBzKUxNVideo abstract

3' untranslated region of Ckip-1 inhibits cardiac hypertrophy independently of its cognate protein.

3' untranslated region (3' UTR) of mRNA is more conserved than other non-coding sequences in vertebrate genomes, and its sequence space has substantially expanded during the evolution of higher organisms, which substantiates their significance in biological regulation. However, the independent role of 3' UTR in cardiovascular disease was largely unknown. Using bioinformatics, RNA fluorescent in situ hybridization and quantitative real-time polymerase chain reaction, we found that 3' UTR and coding sequence regions of Ckip-1 mRNA exhibited diverse expression and localization in cardiomyocytes. We generated cardiac-specific Ckip-1 3' UTR overexpression mice under wild type and casein kinase 2 interacting protein-1 (CKIP-1) knockout background. Cardiac remodelling was assessed by histological, echocardiography, and molecular analyses at 4 weeks after transverse aortic constriction (TAC) surgery. The results showed that cardiac Ckip-1 3' UTR significantly inhibited TAC-induced cardiac hypertrophy independent of CKIP-1 protein. To determine the mechanism of Ckip-1 3' UTR in cardiac hypertrophy, we performed transcriptome and metabolomics analyses, RNA immunoprecipitation, biotin-based RNA pull-down, and reporter gene assays. We found that Ckip-1 3' UTR promoted fatty acid metabolism through AMPK-PPARα-CPT1b axis, leading to its protection against pathological cardiac hypertrophy. Moreover, Ckip-1 3' UTR RNA therapy using adeno-associated virus obviously alleviates cardiac hypertrophy and improves heart function. These findings disclose that Ckip-1 3' UTR inhibits cardiac hypertrophy independently of its cognate protein. Ckip-1 3' UTR is an effective RNA-based therapy tool for treating cardiac hypertrophy and heart failure.

Marburg and Ebola Virus mRNA 3' Untranslated Regions Contain Negative Regulators of Translation That Are Modulated by ADAR1 Editing.

ABSTRACTThe filovirus family includes deadly pathogens such as Ebola virus (EBOV) and Marburg virus (MARV). A substantial portion of filovirus genomes encode 5′ and 3′ untranslated regions (UTRs) of viral mRNAs. Select viral genomic RNA sequences corresponding to 3′ UTRs are prone to editing by adenosine deaminase acting on RNA 1 (ADAR1). A reporter mRNA approach, in which different 5′ or 3′ UTRs were inserted into luciferase-encoding mRNAs, demonstrates that MARV 3′ UTRs yield different levels of reporter gene expression, suggesting modulation of translation. The modulation occurs in cells unable to produce microRNAs (miRNAs) and can be recapitulated in a MARV minigenome assay. Deletion mutants identified negative regulatory regions at the ends of the MARV nucleoprotein (NP) and large protein (L) 3′ UTRs. Apparent ADAR1 editing mutants were previously identified within the MARV NP 3′ UTR. Introduction of these changes into the MARV nucleoprotein (NP) 3′ UTR or deletion of the region targeted for editing enhances translation, as indicated by reporter assays and polysome analysis. In addition, the parental NP 3′ UTR, but not the edited or deletion mutant NP 3′ UTRs, induces a type I interferon (IFN) response upon transfection into cells. Because some EBOV isolates from the West Africa outbreak exhibited ADAR1 editing of the viral protein of 40 kDa (VP40) 3′ UTR, VP40 3′ UTRs with parental and edited sequences were similarly assayed. The EBOV VP40 3′ UTR edits also enhanced translation, but neither the wild-type nor the edited 3′ UTRs induced IFN. These findings implicate filoviral mRNA 3′ UTRs as negative regulators of translation that can be inactivated by innate immune responses that induce ADAR1.IMPORTANCE UTRs comprise a large percentage of filovirus genomes and are apparent targets of editing by ADAR1, an enzyme with pro- and antiviral activities. However, the functional significance of the UTRs and ADAR1 editing has been uncertain. This study demonstrates that MARV and EBOV 3′ UTRs can modulate translation, in some cases negatively. ADAR1 editing or deletion of select regions within the translation suppressing 3′ UTRs relieves the negative effects of the UTRs. These data indicate that filovirus 3′ UTRs contain translation regulatory elements that are modulated by activation of ADAR1, suggesting a complex interplay between filovirus gene expression and innate immunity.

Midazolam increases cisplatin-sensitivity in non-small cell lung cancer (NSCLC) via the miR-194-5p/HOOK3 axis

BackgroundsAs previously reported, midazolam anesthesia exerts tumor-suppressing effects in non-small cell lung cancer (NSCLC), but the regulating effects of this drug on cisplatin-resistance in NSCLC have not been studied. Thus, we designed this study to investigate this issue and preliminarily delineate the potential molecular mechanisms.MethodsWe performed MTT assay and trypan blue staining assay to measure cell proliferation and viability. Cell apoptosis was examined by FCM. qRT-PCR and immunoblotting were performed to determine the expression levels of genes. The targeting sites between genes were predicted by bioinformatics analysis and were validated by dual-luciferase reporter gene system assay. Mice tumor-bearing models were established and the tumorigenesis was evaluated by measuring tumor weight and volume. Immunohistochemistry (IHC) was used to examine the pro-proliferative Ki67 protein expressions in mice tumor tissues.ResultsThe cisplatin-resistant NSCLC (CR-NSCLC) cells were treated with high-dose cisplatin (50 μg/ml) and low-dose midazolam (10 μg/ml), and the results showed that midazolam suppressed cell proliferation and viability, and promoted cell apoptosis in cisplatin-treated CR-NSCLC cells. In addition, midazolam enhanced cisplatin-sensitivity in CR-NSCLC cell via modulating the miR-194-5p/hook microtubule-tethering protein 3 (HOOK3) axis. Specifically, midazolam upregulated miR-194-5p, but downregulated HOOK3 in the CR-NSCLC cells, and further results validated that miR-194-5p bound to the 3’ untranslated region (3’UTR) of HOOK3 mRNA for its inhibition. Also, midazolam downregulated HOOK3 in CR-NSCLC cells by upregulating miR-194-5p. Functional experiments validated that both miR-194-5p downregulation and HOOK3 upregulation abrogated the promoting effects of midazolam on cisplatin-sensitivity in CR-NSCLC cells.ConclusionsTaken together, this study found that midazolam anesthesia reduced cisplatin-resistance in CR-NSCLC cells by regulating the miR-194-5p/HOOK3 axis, implying that midazolam could be used as adjuvant drug for NSCLC treatment in clinical practices.

The U-to-C RNA editing affects the mRNA stability of nuclear genes in Arabidopsis thaliana

Cytidine-to-uridine (C-to-U) RNA editing has been generally observed in land plants; however, reverse (U-to-C) RNA editing is a rare phenomenon. In this study, we investigated the U-to-C RNA editing-related genes in Arabidopsis tissues and the effects on mRNA stability, with a special focus on PPR proteins. A previous study showed the extensive occurrence of U-to-C RNA editing in 12-day and 20-dayold Arabidopsis seedlings. Here, we have demonstrated the effects of this “reverse” RNA editing on the mRNA stability for all seven edited genes. We also identified U-to-C RNA editing in the nuclear PPR gene (AT2G19280) in 12-day-old seedlings of Arabidopsis thaliana. The U-to-C RNA editing sites were found in the untranslated region (3’ UTR) of the mature mRNA and may affect its secondary structure. We also examined the correlation between U-to-C RNA editing-related genes and their mRNA abundance. Furthermore, we investigated the effects of U-to-C RNA editing in Arabidopsis using the transcription inhibitor actinomycin D (Act D). The addition of Act D to the seedlings of transgenic Arabidopsis generated by Agrobacterium-mediated transformation showed that single nucleotide base conversion adversely affected the mRNA secondary structure and stability.

The Tumorigenic Properties of EZH2 are Mediated by MiR-26a in Uveal Melanoma.

Background: The polycomb group protein enhancer of zeste homolog 2 (EZH2) has been found to be highly expressed in various tumors, and microRNA-26a (miR-26a) is often unmodulated in cancers. However, the functions of these two molecules in uveal melanoma (UM) and their relationships have not been reported. Methods: We explored the effects of the miR-26a–EZH2 axis in UM by examining the levels of miR-26a and EZH2. The EZH2 levels in various tumor types and the correlations between EZH2 levels and overall survival and disease-free survival were reanalyzed. The binding of miR-26a to the 3′-untranslated region of EZH2 mRNA was measured using the luciferase reporter assay. The regulation of EZH2 gene expression by miR-26a was also identified, and the effect of elevated EZH2 expression on UM cell function was further examined. Results: miR-26a was downregulated and EZH2 was upregulated in UM cells. Overexpression of miR-26a inhibited cell proliferation, and knockdown of EZH2 suppressed cell growth. EZH2 was a direct target of miR-26a in UM cells. The knockout of EZH2 mimicked the tumor inhibition of miR-26a in UM cells, whereas the reintroduction of EZH2 abolished this effect. In addition, a network of EZH2 and its interacting proteins (UBC, CDK1, HDAC1, SUZ12, EED) was found to participate in miR-26a-mediated tumor progression. Conclusion: The newly identified miR-26a–EZH2 axis may be a potential target for the development of treatment strategies for UM.

Annotation of Novel Dynamic UTRs as Functional Targets of miRNAs During Cardiac Development

Small noncoding microRNAs (miRNAs) play essential roles in post-transcriptional gene regulation in development and disease, predominantly through interactions with mRNA untranslated regions (UTRs). However, the dynamic and tissue specific expression of miRNAs and target UTRs throughout development is poorly characterized. In order to understand the RNA regulatory events that drive cardiac morphogenesis, we captured a simultaneous small RNA and total RNA-seq time course in zebrafish hearts. We extracted de novo transcriptome annotation of dynamically changing UTRs, revealing over ten thousand significantly altered transcripts, with novel 5’ and 3’UTRs for approximately half of all protein coding genes, and predicted regulatory miRNA/UTR interactions during development. In addition to miRNA and mRNA transcriptome resources for studies of RNA regulatory events in cardiac development and disease, this study provides a robust bioinformatic pipeline that should facilitate discovery of posttranscriptional regulatory networks in other developmental systems.

The Role of DND1 in Cancers.

Simple SummaryThe Dead-End (DND1) protein can interact with different messenger RNAs (mRNAs) in the cell. It uses multiple mechanisms to regulate expression of proteins from their cognate mRNAs. High levels of DND1 are found in the progenitor cells that develop into the egg and sperm. Here we review how and why defects in DND1 cause tumors in the testes and ovaries of vertebrates. Unexpectedly, some recent reports indicate that DND1 may also participate in human cancer development in cells other than those of the testes and ovaries. The goal of this review is to summarize the literature on the role of DND1 in cancers to obtain perspective regarding future scientific endeavors on DND1 function.The Ter mutation in Dead-End 1 (Dnd1), Dnd1Ter, which leads to a premature stop codon, has been determined to be the cause for primordial germ cell deficiency, accompanied with a high incidence of congenital testicular germ cell tumors (TGCTs) or teratomas in the 129/Sv-Ter mice. As an RNA-binding protein, DND1 can bind the 3′-untranslated region (3′-UTR) of mRNAs and function in translational regulation. DND1 can block microRNA (miRNA) access to the 3′-UTR of target mRNAs, thus inhibiting miRNA-mediated mRNA degradation and up-regulating translation or can also function to degrade or repress mRNAs. Other mechanisms of DND1 activity include promoting translation initiation and modifying target protein activity. Although Dnd1Ter mutation causes spontaneous TGCT only in male 129 mice, it can also cause ovarian teratomas in mice when combined with other genetic defects or cause germ cell teratomas in both genders in the WKY/Ztm rat strain. Furthermore, studies on human cell lines, patient cancer tissues, and the use of human cancer genome analysis indicate that DND1 may possess either tumor-suppressive or -promoting functions in a variety of somatic cancers. Here we review the involvement of DND1 in cancers, including what appears to be its emerging role in somatic cancers.

E2F3 promotes liver cancer progression under the regulation of circ-PRKAR1B

Liver cancer is one of the most lethal malignant tumors in the world. The high recurrence and mortality rate make it urgent for scientists and clinicians to find new targets for better treatment of liver cancer. Here, we found that circ-PRKAR1B expression was increased in the paired intrahepatic metastasis sample through high-throughput sequencing. Further experiments also confirmed its high expression both in carcinoma and metastasis when compared to the paired para-carcinoma and the paired carcinoma, respectively. Mechanism study showed that circ-PRKAR1B could promote liver cancer progression through the miR-432-5p/E2F3 pathway, and microRNA-432-5p could directly target the 3′ untranslated region (UTR) of E2F3 mRNA to suppress its translation, thereby influencing liver cancer cell invasion and migration capacities. Clinical data obtained by using online databases based on The Cancer Genome Atlas (TCGA) samples and the clinicopathological data of liver cancer patients who underwent surgery in our hospital in the past 2 years also confirmed the significance of circ-PRKAR1B/miR-432-5p/E2F3 signaling in liver cancer progression. Animal experiments also indicated that targeting this newly identified signaling by overexpressing microRNA-432-5p could suppress the progression of liver cancer. Together, our study suggests that circ-PRKAR1B plays an important role in the regulation of liver cancer progression, and targeting this new circ-PRKAR1B/miR-432-5p/E2F3 signaling may help us find new treatment strategies to better suppress liver cancer progression.

CircRNA FUT10 regulates the regenerative potential of aged skeletal muscle stem cells by targeting HOXA9.

Skeletal muscle is capable of repairing itself after injury to maintain the stability of its own tissue, but this ability declines with aging. Circular RNAs (circRNAs) are involved in cell aging. However, there is little research into their role and underlying mechanisms, especially in skeletal muscle stem cells (SkMSCs). In this study, we assessed circRNA FUT10 expression in aged and adult SkMSCs. We observed that circRNA FUT10 was upregulated in aged SkMSCs compared with that in adult SkMSCs. Furthermore, we identified putative miR-365-3p binding sites on circRNA FUT10, suggesting that this circRNA sponges miR-365a-3p. We also found that HOXA9 is a downstream target of miR-365a-3p and confirmed that miR-365a-3p can bind to circRNA FUT10 and the 3′-untranslated region of HOXA9 mRNA. This finding indicated that miR-365a-3p might serve as a “bridge” between circRNA FUT10 and HOXA9. Finally, we found that the circRNA FUT10/miR365a-3p/HOXA9 axis is involved in SkMSC aging. Collectively, our results show that the circRNA FUT10/miR365a-3p/HOXA9 axis is a promising therapeutic target and are expected to facilitate the development of therapeutic strategies to improve the prognosis of degenerative muscle disease.

Dissection of a rice OsMac1 mRNA 5' UTR to uncover regulatory elements that are responsible for its efficient translation.

The untranslated regions (UTRs) of mRNAs are involved in many posttranscriptional regulatory pathways. The rice OsMac1 mRNA has three splicing variants of the 5' UTR (UTRa, UTRb, and UTRc), which include a CU-rich region and three upstream open reading frames (uORFs). UTRc contains an additional 38-nt sequence, termed sp38, which acts as a strong translational enhancer of the downstream ORF; reporter analysis revealed translational efficiencies >15-fold higher with UTRc than with the other splice variants. Mutation analysis of UTRc demonstrated that an optimal sequence length of sp38, rather than its nucleotide sequence is essential for UTRc to promote efficient translation. In addition, the 5' 100 nucleotides of CU-rich region contribute to UTRc translational enhancement. Strikingly, three uORFs did not reveal their inhibitory potential within the full-length leader, whereas deletion of the 5' leader fragment preceding the leader region with uORFs nearly abolished translation. Computational prediction of UTRc structural motifs revealed stem-loop structures, termed SL1-SL4, and two regions, A and B, involved in putative intramolecular interactions. Our data suggest that SL4 binding to Region-A and base pairing between Region-B and the UTRc 3'end are critically required for translational enhancement. Since UTRc is not capable of internal initiation, we presume that the three-dimensional leader structures can allow translation of the leader downstream ORF, likely allowing the bypass of uORFs.

Mutation of the 5'-untranslated region stem-loop mRNA structure reduces type I collagen deposition and arterial stiffness in male obese mice.

Arterial stiffening, a characteristic feature of obesity and type 2 diabetes, contributes to the development and progression of cardiovascular diseases (CVD). Currently, no effective prophylaxis or therapeutics is available to prevent or treat arterial stiffening. A better understanding of the molecular mechanisms underlying arterial stiffening is vital to identify newer targets and strategies to reduce CVD burden. A major contributor to arterial stiffening is increased collagen deposition. In the 5'-untranslated regions of mRNAs encoding for type I collagen, an evolutionally conserved stem-loop (SL) structure plays an essential role in its stability and post-transcriptional regulation. Here, we show that feeding a high-fat/high-sucrose (HFHS) diet for 28 wk increases adiposity, insulin resistance, and blood pressure in male wild-type littermates. Moreover, arterial stiffness, assessed in vivo via aortic pulse wave velocity, and ex vivo using atomic force microscopy in aortic explants or pressure myography in isolated femoral and mesenteric arteries, was also increased in those mice. Notably, all these indices of arterial stiffness, along with collagen type I levels in the vasculature, were reduced in HFHS-fed mice harboring a mutation in the 5'SL structure, relative to wild-type littermates. This protective vascular phenotype in 5'SL-mutant mice did not associate with a reduction in insulin resistance or blood pressure. These findings implicate the 5'SL structure as a putative therapeutic target to prevent or reverse arterial stiffening and CVD associated with obesity and type 2 diabetes.NEW & NOTEWORTHY In the 5'-untranslated (UTR) regions of mRNAs encoding for type I collagen, an evolutionally conserved SL structure plays an essential role in its stability and posttranscriptional regulation. We demonstrate that a mutation of the SL mRNA structure in the 5'-UTR decreases collagen type I deposition and arterial stiffness in obese mice. Targeting this evolutionarily conserved SL structure may hold promise in the management of arterial stiffening and CVD associated with obesity and type 2 diabetes.

Platelet-rich plasma attenuates intervertebral disc degeneration via delivering miR-141-3p-containing exosomes

ABSTRACT Oxidative stress mediated apoptotic and pyroptotic cell death contributes to intervertebral disc (IVD) degeneration, and platelet-rich plasma (PRP) exerts protective effects to attenuate IVD degeneration. Hence, the present study aimed to validate this issue and uncover the potential underlying mechanisms. The mice and cellular models for IVD degeneration were established by using puncture method and H2O2 exposure, respectively, and we evidenced that NLRP3-mediated cell pyroptosis, apoptosis and inflammatory responses occurred during IVD degeneration progression in vitro and in vivo. Then, the PRP-derived exosomes (PRP-exo) were isolated and purified, and we noticed that both PRP-exo and ROS scavenger (NAC) reversed the detrimental effects of H2O2 treatment on the nucleus pulposus (NP) cells. Further results supported that PRP-exo exerted its protective effects on H2O2 treated NP cells by modulating the Keap1-Nrf2 pathway. Mechanistically, PRP-exo downregulated Keap1, resulting in the release of Nrf2 from the Keap1-Nrf2 complex, which further translocated from cytoplasm to nucleus to achieve its anti-oxidant biological functions, and H2O2 treated NP cells with Nrf2-deficiency did not respond to PRP-exo treatment. In addition, miR-141-3p was enriched in PRP-exo, and miR-141-3p targeted the 3' untranslated region (3'UTR) of Keap1 mRNA for its degradation, leading to Nrf2 translocation. Furthermore, overexpression of miR-141-3p ameliorated the cytotoxic effects of H2O2 on NP cells, which were abrogated by upregulating Keap1 and silencing Nrf2. Taken together, we concluded that PRP secreted exosomal miR-141-3p to activate the Keap1-Nrf2 pathway, which helped to slow down IVD degeneration.

α-Lipoic Acid Alleviates Hepatic Lipid Deposition by Inhibiting FASN Expression via miR-3548 in Rats.

Excessive liver lipid deposition is a vital risk factor for the development of many diseases. Here, we fed Sprague-Dawley rats with a control or α-lipoic acid-supplemented diet (0.2%) for 5 weeks to elucidate the effects of α-lipoic acid on preventive ability, hepatic lipid metabolism-related gene expression, and the involved regulatory mechanisms. In the current study, α-lipoic acid supplementation lowered plasma triglyceride level and hepatic triglyceride content. Reduced hepatic lipid deposition was closely associated with inhibiting fatty acid-binding protein 1 and fatty acid synthase expression, as well as increasing phosphorylated hormone-sensitive lipase expression at the protein level in α-lipoic acid-exposed rats. Hepatic miRNA sequencing revealed increased expression of miR-3548 targeting the 3′untranslated region of Fasn mRNA, and the direct regulatory link between miRNA-3548 and FASN was verified by dual-luciferase reporter assay. Taken together, α-lipoic acid lowered hepatic lipid accumulation, which involved changes in miRNA-mediated lipogenic genes.

A nitrogen stress-inducible small RNA regulates CO2 fixation in Nostoc.

In the absence of fixed nitrogen, some filamentous cyanobacteria differentiate heterocysts, specialized cells devoted to fixing atmospheric nitrogen (N2). This differentiation process is controlled by the global nitrogen regulator NtcA and involves extensive metabolic reprogramming, including shutdown of photosynthetic CO2 fixation in heterocysts, to provide a microaerobic environment suitable for N2 fixation. Small regulatory RNAs (sRNAs) are major post-transcriptional regulators of gene expression in bacteria. In cyanobacteria, responding to nitrogen deficiency involves transcribing several nitrogen-regulated sRNAs. Here, we describe the participation of nitrogen stress-inducible RNA 4 (NsiR4) in post-transcriptionally regulating the expression of two genes involved in CO2 fixation via the Calvin cycle: glpX, which encodes bifunctional sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (SBPase), and pgk, which encodes phosphoglycerate kinase (PGK). Using a heterologous reporter assay in Escherichia coli, we show that NsiR4 interacts with the 5′-untranslated region (5′-UTR) of glpX and pgk mRNAs. Overexpressing NsiR4 in Nostoc sp. PCC 7120 resulted in a reduced amount of SBPase protein and reduced PGK activity, as well as reduced levels of both glpX and pgk mRNAs, further supporting that NsiR4 negatively regulates these two enzymes. In addition, using a gfp fusion to the nsiR4 promoter, we show stronger expression of NsiR4 in heterocysts than in vegetative cells, which could contribute to the heterocyst-specific shutdown of Calvin cycle flux. Post-transcriptional regulation of two Calvin cycle enzymes by NsiR4, a nitrogen-regulated sRNA, represents an additional link between nitrogen control and CO2 assimilation.