CXCL1: Gene, Promoter, Regulation of Expression, mRNA Stability, Regulation of Activity in the Intercellular Space

CXCL1 is one of the most important chemokines, part of a group of chemotactic cytokines involved in the development of many inflammatory diseases. It activates CXCR2 and, at high levels, CXCR1. The expression of CXCL1 is elevated in inflammatory reactions and also has important functions in physiology, including the induction of angiogenesis and recruitment of neutrophils. Due to a lack of reviews that precisely describe the regulation of CXCL1 expression and function, in this paper, we present the mechanisms of CXCL1 expression regulation with a special focus on cancer. We concentrate on the regulation of CXCL1 expression through the regulation of CXCL1 transcription and mRNA stability, including the involvement of NF-κB, p53, the effect of miRNAs and cytokines such as IFN-γ, IL-1β, IL-17, TGF-β and TNF-α. We also describe the mechanisms regulating CXCL1 activity in the extracellular space, including proteolytic processing, CXCL1 dimerization and the influence of the ACKR1/DARC receptor on CXCL1 localization. Finally, we explain the role of CXCL1 in cancer and possible therapeutic approaches directed against this chemokine.

Small uORFs favor translation re-initiation but do not protect mRNAs from nonsense-mediated decay

It is estimated that nearly 50% of mammalian transcripts contain at least one upstream open reading frame (uORF), which are typically one to two orders of magnitude smaller than the downstream main ORF. Most uORFs are thought to be inhibitory as they sequester the scanning ribosome, but in some cases allow for translation re-initiation. However, termination in the 5ʹ UTR at the end of uORFs resembles pre-mature termination that is normally sensed by the nonsense-mediated mRNA decay (NMD) pathway. Translation re-initiation has been proposed as a method for mRNAs to prevent NMD. Here we test how uORF length influences translation re-initiation and mRNA stability. Using custom 5ʹ UTRs and uORF sequences, we show that re-initiation can occur on heterologous mRNA sequences, favors small uORFs, and is supported when initiation occurs with more initiation factors. After determining reporter mRNA half-lives and mining available mRNA half-life datasets for cumulative uORF length, we conclude that translation re-initiation after uORFs is not a robust method for mRNAs to evade NMD. Together, these data support a model where uORFs have evolved to balance coding capacity, translational control, and mRNA stability.

RNA N6-methyladenosine reader IGF2BP2 promotes lymphatic metastasis and epithelial-mesenchymal transition of head and neck squamous carcinoma cells via stabilizing slug mRNA in an m6A-dependent manner

Background Lymph node metastasis is the main cause of poor prognosis of head and neck squamous carcinoma (HNSCC) patients. N6-methyladenosine (m6A) RNA modification is an emerging epigenetic regulatory mechanism for gene expression, and as a novel m6A reader protein, IGF2BP2 has been implicated in tumor progression and metastasis. However, not much is currently known about the functional roles of IGF2BP2 in HNSCC, and whether IGF2BP2 regulates lymphatic metastasis through m6A modification in HNSCC remains to be determined. Methods The expression and overall survival (OS) probability of m6A-related regulators in HNSCC were analyzed with The Cancer Genome Atlas (TCGA) dataset and GEPIA website tool, respectively. The expression levels of IGF2BP2 were measured in HNSCC tissues and normal adjacent tissues. To study the effects of IGF2BP2 on HNSCC cell metastasis in vitro and in vivo, gain- and loss- of function methods were employed. RIP, MeRIP, luciferase reporter and mRNA stability assays were performed to explore the epigenetic mechanism of IGF2BP2 in HNSCC. Results We investigated 20 m6A-related regulators in HNSCC and discovered that only the overexpression of IGF2BP2 was associated with a poor OS probability and an independent prognostic factor for HNSCC patients. Additionally, we demonstrated that IGF2BP2 was overexpressed in HNSCC tissues, and significantly correlated to lymphatic metastasis and poor prognosis. Functional studies have shown that IGF2BP2 promotes both HNSCC cell migration as well as invasion via the epithelial-mesenchymal transition (EMT) process in vitro, and IGF2BP2 knockdown significantly inhibited lymphatic metastasis and lymphangiogenesis in vivo. Mechanistic investigations revealed that Slug, a key EMT-related transcriptional factor, is the direct target of IGF2BP2, and essential for IGF2BP2-regulated EMT and metastasis in HNSCC. Furthermore, we demonstrated that IGF2BP2 recognizes and binds the m6A site in the coding sequence (CDS) region of Slug and promotes its mRNA stability. Conclusions Collectively, our study uncovers the oncogenic role and potential mechanism of IGF2BP2, which serves as a m6A reader, in controlling lymphatic metastasis and EMT in HNSCC, suggesting that IGF2BP2 may act as a therapeutic target and prognostic biomarker for HNSCC patients with metastasis.

METTL14 suppresses pyroptosis and diabetic cardiomyopathy by downregulating TINCR lncRNA

N6-methyladenosine (m6A) is one of the most important epigenetic regulation of RNAs, such as lncRNAs. However, the underlying regulatory mechanism of m6A in diabetic cardiomyopathy (DCM) is very limited. In this study, we sought to define the role of METTL14-mediated m6A modification in pyroptosis and DCM progression. DCM rat model was established and qRT-PCR, western blot, and immunohistochemistry (IHC) were used to detect the expression of METTL14 and TINCR. Gain-and-loss functional experiments were performed to define the role of METTL14-TINCR-NLRP3 axis in pyroptosis and DCM. RNA pulldown and RNA immunoprecipitation (RIP) assays were carried out to verify the underlying interaction. Our results showed that pyroptosis was tightly involved in DCM progression. METTL14 was downregulated in cardiomyocytes and hear tissues of DCM rat tissues. Functionally, METTL14 suppressed pyroptosis and DCM via downregulating lncRNA TINCR, which further decreased the expression of key pyroptosis-related protein, NLRP3. Mechanistically, METTL14 increased m6A methylation level of TINCR gene, resulting in its downregulation. Moreover, the m6A reader protein YTHDF2 was essential for m6A methylation and mediated the degradation of TINCR. Finally, TINCR positively regulated NLRP3 by increasing its mRNA stability. To conclude, our work revealed the novel role of METTL14-mediated m6A methylation and lncRNA regulation in pyroptosis and DCM, which could help extend our understanding the epigenetic regulation of pyroptosis in DCM progression.

ZC3H13 Inhibits the Progression of Hepatocellular Carcinoma through m6A-PKM2-Mediated Glycolysis and Enhances Chemosensitivity

Objective. N6-Methyladenosine (m6A) is the most prevalent RNA epigenetic modulation in eukaryotic cells, which serves a critical role in diverse physiological processes. Emerging evidences indicate the prognostic significance of m6A regulator ZC3H13 in hepatocellular carcinoma (HCC). Herein, this study was conducted for revealing biological functions and mechanisms of ZC3H13 in HCC. Methods. Expression of ZC3H13 was examined in collected HCC and normal tissues, and its prognostic significance was investigated in a public database. Gain/loss of functional assays were presented for defining the roles of ZC3H13 in HCC progression. The specific interactions of ZC3H13 with PKM2 were validated in HCC cells via mRNA stability, RNA immunoprecipitation, and luciferase reporter and MeRIP‐qPCR assays. Moreover, rescue experiments were carried out for uncovering the mechanisms. Results. ZC3H13 expression was downregulated in HCC, and its loss was in relation to dismal survival outcomes. Functionally, overexpressed ZC3H13 suppressed proliferation, migration, and invasion and elevated apoptotic levels of HCC cells. Moreover, ZC3H13 overexpression sensitized to cisplatin and weakened metabolism reprogramming of HCC cells. Mechanically, ZC3H13-induced m6A modified patterns substantially abolished PKM2 mRNA stability. ZC3H13 facilitated malignant behaviors of HCC cells through PKM2-dependent glycolytic signaling. Conclusion. Collectively, ZC3H13 suppressed the progression of HCC through m6A-PKM2-mediated glycolysis and sensitized HCC cells to cisplatin, which offered a fresh insight into HCC therapy.

Mature Neurons sensitivity to oxidative stress is epigenetically programmed by alternative splicing and mRNA stability

Neuronal differentiation is a complex process that entails extensive morphological, transcriptional, metabolic, and functional changes that dictate neuronal lineage commitment. Much less understood is the role that epigenetic and epi-transcriptional reprogramming plays in the process of neuronal differentiation and maturation. To depict the whole landscape of transcriptomics and epigenetic changes during neuronal differentiation and maturation, we differentiated SH-SY5Y cells and performed RNA sequencing on differentiated and undifferentiated cells. 728 differentially expressed genes (DEGs) enriched in synaptic signaling and cell morphogenesis pathways were observed. Moreover, transcriptome-wide mRNA stability profiling revealed that genes with altered stability were exceptionally enriched for redox homeostasis pathways. Mature neurons are known to be highly sensitive to oxidative stress, which is crucial in the pathophysiology of neurodegenerative disease. Our results suggest that this heightened sensitivity is regulated at the mRNA stability level (i.e., epigenetic) rather than at the transcriptional level. Alternative splicing analysis revealed the exon skipping and alternative mRNA isoforms enriched for morphogenesis related pathway. Alternatively, alternative 5 and 3 prime splicing site, intron retention and mutually exclusive exon events exclusively clustered in the translation and translation initiation pathways, suggesting the potential effect of alternative splicing on translation following neuronal maturation. Splice motif analysis revealed enriched motifs for RBPs that regulate various splice types and can be further correlated to distinct phenotypical changes during neuronal differentiation and maturation. Here we present an extensive exploration of the transcriptional and epigenetic changes and their potential association with the process of neuronal differentiation, providing a new insight into understanding the molecular mechanism of neuronal function and behavior.

The m6A reader IGF2BP3 Facilitates Gastric Cancer Progression Through Activating EMT via Upregulating MYC mRNA Stability

Background: N6-methyladenosine (m6A) RNA methylation plays an important biological role in cancer progression. Even so, the role of m6A modification in gastric cancer (GC) still needs further research. Methods: Firstly, based on the bioinformatics databases and human GC tissues analysis. Secondly, the IGF2BP3 expression in GC cells was measured by the quantificational real-time polymerase chain reaction and Western Blot. Then, the IGF2BP3 knockdown stable cells model was successfully constructed with the specific lentivirus-mediated short-hairpin RNA to explore the functions and mechanism of IGF2BP3 in GC. Next, the functions of IGF2BP3 on the cell phenotypes, including proliferation, invasion, migration, and Epithelial-mesenchymal transition process were clarified by the Cell Counting Kit-8, transwell, and WB experiments. Subsequently, RNA Immunoprecipitation analysis and mRNA stability experiments were used to verify the relationship between IGF2BP3 and MYC. Finally, in the rescue experiment, MYC was overexpressed and transfected into IGF2BP3 knockdown cells to further detect the influences on the cell phenotypes and the EMT process.Results: IGF2BP3 was up-regulated in GC. Meanwhile, IGF2BP3 had diagnostic and prognosis values for GC. Functionally, knockdown IGF2BP3 repressed gastric cancer cells proliferation, migration invasion and EMT process. Mechanically, IGF2BP3 activated the EMT process by improving the expression of MYC via combining with MYC mRNA and promoting its stability. Conclusions: Taken together, IGF2BP3 could activate the EMT process via increasing the MYC mRNA stability and expression to promote GC development, which provided insight into promising early diagnose and treatment for gastric cancer.

TCF4 and HuR mediated-METTL14 suppresses dissemination of colorectal cancer via N6-methyladenosine-dependent silencing of ARRDC4

Metastasis remains the major obstacle to improved survival for colorectal cancer (CRC) patients. Dysregulation of N6-methyladenosine (m6A) is causally associated with the development of metastasis through poorly understood mechanisms. Here, we report that METTL14, a key component of m6A methylation, is functionally related to the inhibition of ARRDC4/ZEB1 signaling and to the consequent suppression of CRC metastasis. We unveil METTL14-mediated m6A modification profile and identify ARRDC4 as a direct downstream target of METTL14. Knockdown of METTL14 significantly enhanced ARRDC4 mRNA stability relying on the “reader” protein YHTDF2 dependent manner. Moreover, we demonstrate that TCF4 can induce METTL14 protein expression, and HuR suppress METTL14 expression by directly binding to its promoter. Clinically, our results show that decreased METTL14 is correlated with poor prognosis and acts as an independent predictor of CRC survival. Collectively, our findings propose that METTL14 functions as a metastasis suppressor, and define a novel signaling axis of TCF4/HuR-METTL14-YHTDF2-ARRDC4-ZEB1 in CRC, which might be potential therapeutic targets for CRC.

CPEB4 Inhibit Cell Proliferation via Upregulating p21 mRNA Stability in Renal Cell Carcinoma

Cytoplasmic polyadenylation element-binding protein 4 (CPEB4) has been reported to be dysregulated in a variety of cancers and seems to play paradoxical roles in different cancers. However, the functional roles of CPEB4 in Renal cell carcinoma (RCC) are still unclear. This study aims to explore the role and underlying mechanism of CPEB4 in RCC. We found that the relative expression level of CPEB4 is down-regulated in RCC tissues and cell lines, and the low CPEB4 expression is correlated with short overall and disease-free survival of RCC patients. CPEB4 significantly inhibits RCC tumor growth both in vivo and in vitro. CPEB4 exerts an anti-tumor effect by increasing p21 mRNA stability and inducing G1 cell cycle arrest in RCC. Our data revealed that CPEB4 is a tumor suppressor gene that restrains cell cycle progression upstream of p21 in RCC. These findings revealed that CPEB4 may become a promising predictive biomarker for prognosis in patients with RCC.

Chronic kidney disease alters Pin1 phosphorylation and parathyroid hormone mRNA binding proteins leading to secondary hyperparathyroidism

Parathyroid hormone (PTH) regulates calcium metabolism and bone strength. Chronic kidney disease (CKD) leads to secondary hyperparathyroidism (SHP) which increases morbidity and mortality. High PTH expression in SHP is due to increased PTH mRNA stability mediated by changes in PTH mRNA interaction with stabilizing AUF1 and destabilizing KSRP. Pin1 isomerizes target proteins, including mRNA binding proteins. In SHP, Pin1 isomerase activity is decreased and phosphorylated KSRP fails to bind PTH mRNA, resulting in high PTH mRNA stability and levels. The molecular mechanisms underlying Pin1 regulation and their effect to increase PTH expression are unknown. We show by mass-spectrometry (MS) the CKD induced changes in rat parathyroid proteome and phosphoproteome profiles. Parathyroid Pin1 Ser16 and Ser71 phosphorylation, that disrupts Pin1 activity, is enhanced in acute and chronic kidney failure rats. Accordingly, pharmacologic Pin1 inhibition increases PTH expression in parathyroid organ cultures and transfected cells, through the PTH mRNA protein binding cis element and KSRP phosphorylation. Therefore, CKD leads to parathyroid loss of Pin1 activity by inducing Pin1 phosphorylation. This predisposes parathyroids to increase PTH production through modified PTH mRNA-KSRP interaction that is dependent on KSRP phosphorylation. CKD induced Pin1 and KSRP phosphorylation and the Pin1-KSRP-PTH mRNA axis thus drive secondary hyperparathyroidism.