m6a methylation orchestrates IMP1 regulation of microtubules during human neuronal differentiation.

Neuronal differentiation requires building a complex intracellular architecture, and therefore the coordinated regulation of defined sets of genes. RNA-binding proteins (RBPs) play a key role in this regulation. However, while their action on individual mRNAs has been explored in depth, the mechanisms used to coordinate gene expression programs shaping neuronal morphology are poorly understood. To address this, we studied how the paradigmatic RBP IMP1 (IGF2BP1), an essential developmental factor, selects and regulates its RNA targets during the human neuronal differentiation. We perform a combination of system-wide and molecular analyses, revealing that IMP1 developmentally transitions to and directly regulates the expression of mRNAs encoding essential regulators of the microtubule network, a key component of neuronal morphology. Furthermore, we show that m6A methylation drives the selection of specific IMP1 mRNA targets and their protein expression during the developmental transition from neural precursors to neurons, providing a molecular principle for the onset of target selectivity.

RBM15-Mediated N6-Methyl Adenosine (m6A) Modification of EZH2 Drives the Epithelial-Mesenchymal Transition of Cervical Cancer.

RBM15 functions as an oncogene in multi-type cancers. However, the reports on the roles of RBM15 in cervical cancer are limited. The purpose of this study was to investigate the potentials of RBM15 in cervical cancer. RT-qPCR was conducted to determine mRNA levels. Western was carried out to detect protein expression. CCK-8, colony formation and EdU assays were conducted to determine cell proliferation. Scratch and transwell assays were conducted to determine cell migration and invasion. MeRIP assay was conducted to determine N6-methyl adenosine (m6A) levels. Luciferase assay was conducted to verify the m6A sites of EZH2 and binding sites between EZH2 and promoter of FN1. ChIP assay was conducted to verify the interaction between EZH2 and FN1. The results showed that RBM15 was upregulated in cervical cancer patients and cells. Moreover, high levels of RBM15 predicted poor clinical outcomes. RBM15 knockdown inhibited the proliferation and epithelial-mesenchymal transition (EMT) of cervical cancer cells. RBM15 promoted the m6A modification of EZH2 as well as its protein translation. Additionally, EZH2 bound to the promoter of fibronectin 1 (FN1) and EZH2-FN1 axis is the cascade downstream of RBM15. Overexpressed EZH2 antagonized the effects of RBM15 knockdown and promoted the aggressiveness of cervical cancer cells. In summary, RBM15/EZH2/FN1 signaling cascade induces the proliferation and EMT of cervical cancer. Therefore, RBM15/EZH2/FN1 signaling may be a promising strategy for cervical cancer.

PURPL Promotes M2 Macrophage Polarization in Lung Cancer by Regulating RBM4/xCT Signaling.

Lung cancer is the most common malignancy worldwide. Long non-coding RNA (lncRNA) p53 upregulated regulator of P53 levels (PURPL) is abnormally in various cancers. However, the reports on its roles in lung cancer are limited. The purpose of present study is to investigate the potentials of lncRNA PURPL in lung cancer. PURPL and mRNA expression was determined using real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). The location of PURPL was detected using RNA fluorescence in situ hybridization (FISH) assay. Protein expression was detected using western blot. Cellular functions were determined using flow cytometry. The interaction between PURPL and RNA-binding motif 4 (RBM4) was confirmed using RNA immunoprecipitation (RIP) assay. PURPL was overexpressed in lung cancer cells and patients. Overexpressed PURPL promoted M2 macrophage polarization and suppressed ferroptosis. Additionally, PURPL maintained the mRNA stability of cystine glutamate reverse transporter (xCT) via regulating RBM4. xCT knockdown antagonized the effects of overexpressed PURPL and inhibited M2 macrophage polarization via inducing macrophage ferroptosis. PURPL/RBM4/xCT axis promoted M2 macrophage polarization in lung cancer. Therefore, PURPL may be a potential target of lung cancer.

An axonemal intron splicing program sustains Plasmodium male development.

Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito transmission of the malaria parasite. RNA-binding protein (RBP)-mediated post-transcriptional regulation of mRNA plays important roles in eukaryotic sexual development, including the development of female Plasmodium. However, the role of RBP in defining the Plasmodium male transcriptome and its function in male gametogenesis remains incompletely understood. Here, we performed genome-wide screening for gender-specific RBPs and identified an undescribed male-specific RBP gene Rbpm1 in the Plasmodium. RBPm1 is localized in the nucleus of male gametocytes. RBPm1-deficient parasites fail to assemble the axoneme for male gametogenesis and thus mosquito transmission. RBPm1 interacts with the spliceosome E complex and regulates the splicing initiation of certain introns in a group of 26 axonemal genes. RBPm1 deficiency results in intron retention and protein loss of these axonemal genes. Intron deletion restores axonemal protein expression and partially rectifies axonemal defects in RBPm1-null gametocytes. Further splicing assays in both reporter and endogenous genes exhibit stringent recognition of the axonemal introns by RBPm1. The splicing activator RBPm1 and its target introns constitute an axonemal intron splicing program in the post-transcriptional regulation essential for Plasmodium male development.

Oridonin represses lung cancer cell proliferation and migration by modulating AFAP1-AS1/IGF2BP1.

Oridonin belongs to a small molecule from the Chinese herb Rabdosia rubescens with potent anticancer activity. In spite of the lncRNA AFAP1-AS1 has been proven to exert promoting function in lung cancer, its relationship with oridonin in lung cancer is obscure. Therefore, our study planned to explore the potential of oridonin in lung cancer as well as unveil the regulatory mechanism of oridonin on AFAP1-AS1 in lung cancer cells. In the present study, oridonin inhibited lung cancer cell proliferation, migration, as well as invasion, as evidenced by MTT, wound healing, as well as transwell assays. Besides, we observed that oridonin could downregulate AFAP1-AS1 expression, and overexpressed AFAP1-AS1 could reverse the repressive effects of oridonin on lung cancer cell proliferation, migration, as well as invasion. More importantly, we found that AFAP1-AS1 could bind to IGF2BP1 through starBase prediction and RIP assay. The expression level of IGF2BP1 was also reduced by oridonin treatment but reversed after AFAP1-AS1 overexpression. Additionally, we proved that overexpressed IGF2BP1 could reverse the repressive impacts of oridonin on lung cancer cell proliferation, migration, as well as invasion. Further, in vivo experiments validated the repressive role of oridonin on tumor growth of lung cancer. Together, oridonin inhibits lung cancer cell proliferation as well as migration by modulating AFAP1-AS1/IGF2BP1, and AFAP1-AS1/IGF2BP1 possesses the potential to be a promising therapy targeting for lung cancer, especially in oridonin treatment.

Deoxynivalenol induces m6A-mediated upregulation of p21 and growth arrest of mouse hippocampal neuron cells in vitro.

Hippocampal neurons maintain the ability of proliferation throughout life to support neurogenesis. Deoxynivalenol (DON) is a mycotoxin that exhibits brain toxicity, yet whether and how DON affects hippocampal neurogenesis remains unknown. Here, we use mouse hippocampal neuron cells (HT-22) as a model to illustrate the effects of DON on neuron proliferation and to explore underlying mechanisms. DON exposure significantly inhibits the proliferation of HT-22 cells, which is associated with an up-regulation of cell cycle inhibitor p21 at both mRNA and protein levels. Global and site-specific m6A methylation levels on the 3'UTR of p21 mRNA are significantly increased in response to DON treatment, whereas inhibition of m6A hypermethylation significantly alleviates DON-induced cell cycle arrest. Further mechanistic studies indicate that the m6A readers YTHDF1 and IGF2BP1 are responsible for m6A-mediated increase in p21 mRNA stability. Meanwhile, 3'UTR of E3 ubiquitin ligase TRIM21 mRNA is also m6A hypermethylated, and another m6A reader YTHDF2 binds to the m6A sites, leading to decreased TRIM21 mRNA stability. Consequently, TRIM21 suppression impairs ubiquitin-mediated p21 protein degradation. Taken together, m6A-mediated upregulation of p21, at both post-transcriptional and post-translational levels, contributes to DON-induced inhibition of hippocampal neuron proliferation. These results may provide new insights for epigenetic therapy of neurodegenerative diseases.

ARID1A loss promotes RNA editing of CDK13 in an ADAR1-dependent manner.

BACKGROUND: ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, is thought to play a significant role both in tumor suppression and tumor initiation, which is highly dependent upon context. Previous studies have suggested that ARID1A deficiency may contribute to cancer development. The specific mechanisms of whether ARID1A loss affects tumorigenesis by RNA editing remain unclear. RESULTS: Our findings indicate that the deficiency of ARID1A leads to an increase in RNA editing levels and alterations in RNA editing categories mediated by adenosine deaminases acting on RNA 1 (ADAR1). ADAR1 edits the CDK13 gene at two previously unidentified sites, namely Q113R and K117R. Given the crucial role of CDK13 as a cyclin-dependent kinase, we further observed that ADAR1 deficiency results in changes in the cell cycle. Importantly, the sensitivity of ARID1A-deficient tumor cells to SR-4835, a CDK12/CDK13 inhibitor, suggests a promising therapeutic approach for individuals with ARID1A-mutant tumors. Knockdown of ADAR1 restored the sensitivity of ARID1A deficient cells to SR-4835 treatment. CONCLUSIONS: ARID1A deficiency promotes RNA editing of CDK13 by regulating ADAR1.

Loss of endogenous estrogen alters mitochondrial metabolism and muscle clock-related protein Rbm20 in female mdx mice.

Female carriers of a Duchenne muscular dystrophy (DMD) gene mutation manifest exercise intolerance and metabolic anomalies that may be exacerbated following menopause due to the loss of estrogen, a known regulator of skeletal muscle function and metabolism. Here, we studied the impact of estrogen depletion (via ovariectomy) on exercise tolerance and muscle mitochondrial metabolism in female mdx mice and the potential of estrogen replacement therapy (using estradiol) to protect against functional and metabolic perturbations. We also investigated the effect of estrogen depletion, and replacement, on the skeletal muscle proteome through an untargeted proteomic approach with TMT-labelling. Our study confirms that loss of estrogen in female mdx mice reduces exercise capacity, tricarboxylic acid cycle intermediates, and citrate synthase activity but that these deficits are offset through estrogen replacement therapy. Furthermore, ovariectomy downregulated protein expression of RNA-binding motif factor 20 (Rbm20), a critical regulator of sarcomeric and muscle homeostasis gene splicing, which impacted pathways involving ribosomal and mitochondrial translation. Estrogen replacement modulated Rbm20 protein expression and promoted metabolic processes and the upregulation of proteins involved in mitochondrial dynamics and metabolism. Our data suggest that estrogen mitigates dystrophinopathic features in female mdx mice and that estrogen replacement may be a potential therapy for post-menopausal DMD carriers.

Reduced protein-coding transcript diversity in severe dengue emphasises the role of alternative splicing.

Dengue fever, a neglected tropical arboviral disease, has emerged as a global health concern in the past decade. Necessitating a nuanced comprehension of the intricate dynamics of host-virus interactions influencing disease severity, we analysed transcriptomic patterns using bulk RNA-seq from 112 age- and gender-matched NS1 antigen-confirmed hospital-admitted dengue patients with varying severity. Severe cases exhibited reduced platelet count, increased lymphocytosis, and neutropenia, indicating a dysregulated immune response. Using bulk RNA-seq, our analysis revealed a minimal overlap between the differentially expressed gene and transcript isoform, with a distinct expression pattern across the disease severity. Severe patients showed enrichment in retained intron and nonsense-mediated decay transcript biotypes, suggesting altered splicing efficiency. Furthermore, an up-regulated programmed cell death, a haemolytic response, and an impaired interferon and antiviral response at the transcript level were observed. We also identified the potential involvement of the RBM39 gene among others in the innate immune response during dengue viral pathogenesis, warranting further investigation. These findings provide valuable insights into potential therapeutic targets, underscoring the importance of exploring transcriptomic landscapes between different disease sub-phenotypes in infectious diseases.

Crosstalk between Regnase-1 and -3 shapes mast cell survival and cytokine expression.

Post-transcriptional regulation of immune-related transcripts by RNA-binding proteins (RBPs) impacts immune cell responses, including mast cell functionality. Despite their importance in immune regulation, the functional role of most RBPs remains to be understood. By manipulating the expression of specific RBPs in murine mast cells, coupled with mass spectrometry and transcriptomic analyses, we found that the Regnase family of proteins acts as a potent regulator of mast cell physiology. Specifically, Regnase-1 is required to maintain basic cell proliferation and survival, whereas both Regnase-1 and -3 cooperatively regulate the expression of inflammatory transcripts upon activation, with Tnf being a primary target in both human and mouse cells. Furthermore, Regnase-3 directly interacts with Regnase-1 in mast cells and is necessary to restrain Regnase-1 expression through the destabilization of its transcript. Overall, our study identifies protein interactors of endogenously expressed Regnase factors, characterizes the regulatory interplay between Regnase family members in mast cells, and establishes their role in the control of mast cell homeostasis and inflammatory responses.

N6-methyladenosine-modified SENP1, identified by IGF2BP3, is a novel molecular marker in acute myeloid leukemia and aggravates progression by activating AKT signal via de-SUMOylating HDAC2.

BACKGROUND: Elevated evidence suggests that the SENPs family plays an important role in tumor progression. However, the role of SENPs in AML remains unclear. METHODS: We evaluated the expression pattern of SENP1 based on RNA sequencing data obtained from OHSU, TCGA, TARGET, and MILE datasets. Clinical samples were used to verify the expression of SENP1 in the AML cells. Lentiviral vectors shRNA and sgRNA were used to intervene in SENP1 expression in AML cells, and the effects of SENP1 on AML proliferation and anti-apoptosis were detected using in vitro and in vivo models. Chip-qPCR, MERIP-qPCR, CO-IP, RNA pulldown, and dual-luciferase reporter gene assays were used to explore the regulatory mechanisms of SNEP1 in AML. RESULTS: SENP1 was significantly upregulated in high-risk AML patients and closely related to poor prognosis. The AKT/mTOR signaling pathway is a key downstream pathway that mediates SENP1's regulation of AML proliferation and anti-apoptosis. Mechanistically, the CO-IP assay revealed binding between SENP1 and HDAC2. SUMO and Chip-qPCR assays suggested that SENP1 can desumoylate HDAC2, which enhances EGFR transcription and activates the AKT pathway. In addition, we found that IGF2BP3 expression was upregulated in high-risk AML patients and was positively correlated with SENP1 expression. MERIP-qPCR and RIP-qPCR showed that IGF2BP3 binds SENP1 3-UTR in an m6A manner, enhances SENP1 expression, and promotes AKT pathway conduction. CONCLUSIONS: Our findings reveal a distinct mechanism of SENP1-mediated HDAC2-AKT activation and establish the critical role of the IGF2BP3/SENP1signaling axis in AML development.

Transcription factor FOXF2 promotes the development and progression of pancreatic cancer by targeting MSI2.

Pancreatic cancer (PC) is a malignant tumor possessing high mortality. The role of transcription factor Forkhead Box F2 (FOXF2) in PC remains unverified. The current study investigated the roles of FOXF2 in developing PC in vitro and in vivo. A xenograft tumor model was constructed with nude mice injected using FOXF2­overexpressing PC cells or FOXF2­silenced PC cells. High FOXF2 expression significantly enhanced the proliferation ability of PC cells in vitro and pancreatic tumor growth in vivo. The cell cycle analysis indicated that transition of G1­S phase was promoted by FOXF2. The cell cycle­associated proteins cyclin D1, CDK2, phosphorylated (p)­CDK2 and p­RB were upregulated in the FOXF2­overexpressing cells and downregulated in the cells with FOXF2 knockdown. Flow cytometric analysis and Hoechst staining showed that the percentage of apoptotic cells was significantly increased after FOXF2 was silenced. FOXF2 knockdown promoted expression of pro­apoptotic proteins (Bad, Bax and cleaved caspase­3) while suppressing the anti­apoptotic proteins (Bcl­2 and Bcl­xl) at the protein level. FOXF2 improved the migration and invasion of PC cells in vitro. Moreover, luciferase and chromatin immunoprecipitation assays revealed that FOXF2 binds to the MSI2 promoter, promoting its transcriptional expression. FOXF2 knockdown inhibited the MSI2 protein translation while enhancing the translation of NUMB protein, suppressing PC development in vivo. MSI2 silencing reversed the promotive effect mediated by FOXF2 on cell proliferation. These results demonstrated that FOXF2 is essential in PC progression, and the potential mechanism includes regulating MSI2 transcription.

Integrated analysis of the role of PR/SET domain 14 in gastric cancer.

BACKGROUND: Gastric cancer is one of the most common tumors worldwide, and most patients are deprived of treatment options when diagnosed at advanced stages. PRDM14 has carcinogenic potential in breast and non-small cell lung cancer. however, its role in gastric cancer has not been elucidated. METHODS: We aimed to elucidate the expression of PRDM14 using pan-cancer analysis. We monitored the expression of PRDM14 in cells and patients using quantitative polymerase chain reaction, western blotting, and immunohistochemistry. We observed that cell phenotypes and regulatory genes were influenced by PRDM14 by silencing PRDM14. We evaluated and validated the value of the PRDM14-derived prognostic model. Finally, we predicted the relationship between PRDM14 and small-molecule drug responses using the Connectivity Map and The Genomics of Drug Sensitivity in Cancer databases. RESULTS: PRDM14 was significantly overexpressed in gastric cancer, which identified in cell lines and patients' tissues. Silencing the expression of PRDM14 resulted in apoptosis promotion, cell cycle arrest, and inhibition of the growth and migration of GC cells. Functional analysis revealed that PRDM14 acts in epigenetic regulation and modulates multiple DNA methyltransferases or transcription factors. The PRDM14-derived differentially expressed gene prognostic model was validated to reliably predict the patient prognosis. Nomograms (age, sex, and PRDM14-risk score) were used to quantify the probability of survival. PRDM14 was positively correlated with sensitivity to small-molecule drugs such as TPCA-1, PF-56,227, mirin, and linsitinib. CONCLUSIONS: Collectively, our findings suggest that PRDM14 is a positive regulator of gastric cancer progression. Therefore, it may be a potential therapeutic target for gastric cancer.

METTL3 promotes the progression of osteosarcoma through the N6-methyladenosine modification of MCAM via IGF2BP1.

BACKGROUND: The molecular mechanisms of osteosarcoma (OS) are complex. In this study, we focused on the functions of melanoma cell adhesion molecule (MCAM), methyltransferase 3 (METTL3) and insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) in OS development. METHODS: qRT-PCR assay and western blot assay were performed to determine mRNA and protein expression of MCAM, METTL3, IGF2BP1 and YY1. MTT assay and colony formation assay were conducted to assess cell proliferation. Cell apoptosis, invasion and migration were evaluated by flow cytometry analysis, transwell assay and wound-healing assay, respectively. Methylated RNA Immunoprecipitation (MeRIP), dual-luciferase reporter, Co-IP, RIP and ChIP assays were performed to analyze the relationships of MCAM, METTL3, IGF2BP1 and YY1. The functions of METTL3 and MCAM in tumor growth were explored through in vivo experiments. RESULTS: MCAM was upregulated in OS, and MCAM overexpression promoted OS cell growth, invasion and migration and inhibited apoptosis. METTL3 and IGF2BP1 were demonstrated to mediate the m6A methylation of MCAM. Functionally, METTL3 or IGF2BP1 silencing inhibited OS cell progression, while MCAM overexpression ameliorated the effects. Transcription factor YY1 promoted the transcription level of METTL3 and regulated METTL3 expression in OS cells. Additionally, METTL3 deficiency suppressed tumor growth in vivo, while MCAM overexpression abated the effect. CONCLUSION: YY1/METTL3/IGF2BP1/MCAM axis aggravated OS development, which might provide novel therapy targets for OS.

Metadherin-driven promotion of cancer stem cell phenotypes and its effect on immunity in hepatocellular carcinoma.

In this editorial we provide commentary on the article published by Wang et al, featured in the recent issue of the World Journal of Gastroenterology in 2024. We focus on the metadherin (MTDH), also known as astrocyte elevated gene-1 or lysine rich CEACAM1, and its effects on cancer stem cells (CSCs) and immunity in hepatocellular carcinoma (HCC). HCC is the most common primary liver cancer and one of the leading causes of cancer-related deaths worldwide. Most HCC cases develop in the context of liver cirrhosis. Among the pivotal mechanisms of carcinogenesis are gene mutations, dysregulation of diverse signaling pathways, epigenetic alterations, hepatitis B virus-induced hepatocarcinogenesis, chronic inflammation, impact of tumor microenvironment, oxidative stress. Over the years, extensive research has been conducted on the MTDH role in various tumor pathologies, such as lung, breast, ovarian, gastric, hepatocellular, colorectal, renal carcinoma, neuroblastoma, melanoma, and leukemias. Specifically, its involvement in tumor development processes including transformation, apoptosis evasion, angiogenesis, invasion, and metastasis via multiple signaling pathways. It has been demonstrated that knockdown or knockout of MTDH disrupt tumor development and metastasis. In addition, numerous reports have been carried out regarding the MTDH influence on HCC, demonstrating its role as a predictor of poor prognosis, aggressive tumor phenotypes prone to metastasis and recurrence, and exhibiting significant potential for therapy resistance. Finally, more studies finely investigated the influence of MTDH on CSCs. The CSCs are a small subpopulation of tumor cells that sharing traits with normal stem cells like self-renewal and differentiation abilities, alongside a high plasticity that alters their phenotype. Beyond their presumed role in tumor initiation, they can drive also disease relapse, metastasis, and resistance to chemo and radiotherapy.

Nono induces Gadd45b to mediate DNA repair.

RNA-binding proteins are frequently deregulated in cancer and emerge as effectors of the DNA damage response (DDR). The non-POU domain-containing octamer-binding protein NONO/p54nrb is a multifunctional RNA-binding protein that not only modulates the production and processing of mRNA, but also promotes the repair of DNA double-strand breaks (DSBs). Here, we investigate the impact of Nono deletion in the murine KP (KRas G12D , Trp53 -/- ) cell-based lung cancer model. We show that the deletion of Nono impairs the response to DNA damage induced by the topoisomerase II inhibitor etoposide or the radiomimetic drug bleomycin. Nono-deficient KP (KPN) cells display hyperactivation of DSB signalling and high levels of DSBs. The defects in the DDR are accompanied by reduced RNA polymerase II promoter occupancy, impaired nascent RNA synthesis, and attenuated induction of the DDR factor growth arrest and DNA damage-inducible beta (Gadd45b). Our data characterise Gadd45b as a putative Nono-dependent effector of the DDR and suggest that Nono mediates a genome-protective crosstalk of the DDR with the RNA metabolism via induction of Gadd45b.

Alternative 3'UTR expression induced by T cell activation is regulated in a temporal and signal dependent manner.

The length of 3' untranslated regions (3'UTR) is highly regulated during many transitions in cell state, including T cell activation, through the process of alternative polyadenylation (APA). However, the regulatory mechanisms and functional consequences of APA remain largely unexplored. Here we present a detailed analysis of the temporal and condition-specific regulation of APA following activation of primary human CD4+ T cells. We find that global APA changes are regulated temporally and CD28 costimulatory signals enhance a subset of these changes. Most APA changes upon T cell activation involve 3'UTR shortening, although a set of genes enriched for function in the mTOR pathway exhibit 3'UTR lengthening. While upregulation of the core polyadenylation machinery likely induces 3'UTR shortening following prolonged T cell stimulation; a significant program of APA changes occur prior to cellular proliferation or upregulation of the APA machinery. Motif analysis suggests that at least a subset of these early changes in APA are driven by upregulation of RBM3, an RNA-binding protein which competes with the APA machinery for binding. Together this work expands our understanding of the impact and mechanisms of APA in response to T cell activation and suggests new mechanisms by which APA may be regulated.

Clinical significance of PNO1 as a novel biomarker and therapeutic target of hepatocellular carcinoma.

The RNA-binding protein PNO1 plays an essential role in ribosome biogenesis. Recent studies have shown that it is involved in tumorigenesis; however, its role in hepatocellular carcinoma (HCC) is not well understood. The purpose of this study was to examine whether PNO1 can be used as a biomarker of HCC and also examine the therapeutic potential of PNO1 knockout for the treatment of HCC. PNO1 expression was upregulated in HCC and associated with poor prognosis. PNO1 expression was positively associated with tumour stage, lymph node metastasis and poor survival. PNO1 expression was significantly higher in HCC compared to that in fibrolamellar carcinoma or normal tissues. Furthermore, HCC tissues with mutant Tp53 expressed higher PNO1 than those with wild-type Tp53. PNO1 knockout suppressed cell viability, colony formation and EMT of HCC cells. Since activation of Notch signalling pathway promotes HCC, we measured the effects of PNO1 knockout on the components of Notch pathway and its targets. PNO1 knockout suppressed Notch signalling by modulating the expression of Notch ligands and their receptors, and downstream targets. PNO1 knockout also inhibited genes involved in surface adhesion, cell cycle, inflammation and chemotaxis. PNO1 knockout also inhibited colony and spheroid formation, cell migration and invasion, and markers of stem cells, pluripotency and EMT in CSCs. Overall, our data suggest that PNO1 can be used as a diagnostic and prognostic biomarker of HCC, and knockout of PNO1 by CRISPR/Cas9 can be beneficial for the management of HCC by targeting CSCs.

Deficiency of ADAR2 ameliorates metabolic-associated fatty liver disease via AMPK signaling pathways in obese mice.

Non-alcoholic fatty liver disease (NAFLD) is a chronic disease caused by hepatic steatosis. Adenosine deaminases acting on RNA (ADARs) catalyze adenosine to inosine RNA editing. However, the functional role of ADAR2 in NAFLD is unclear. ADAR2+/+/GluR-BR/R mice (wild type, WT) and ADAR2-/-/GluR-BR/R mice (ADAR2 KO) mice are fed with standard chow or high-fat diet (HFD) for 12 weeks. ADAR2 KO mice exhibit protection against HFD-induced glucose intolerance, insulin resistance, and dyslipidemia. Moreover, ADAR2 KO mice display reduced liver lipid droplets in concert with decreased hepatic TG content, improved hepatic insulin signaling, better pyruvate tolerance, and increased glycogen synthesis. Mechanistically, ADAR2 KO effectively mitigates excessive lipid production via AMPK/Sirt1 pathway. ADAR2 KO inhibits hepatic gluconeogenesis via the AMPK/CREB pathway and promotes glycogen synthesis by activating the AMPK/GSK3ß pathway. These results provide evidence that ADAR2 KO protects against NAFLD progression through the activation of AMPK signaling pathways.

Calreticulin regulates the expression of MMP14 and ADAR1 through EIF2AK2 signaling to promote the proliferation and progression of malignant melanoma cells.

It has been demonstrated that calreticulin (CALR) is expressed abnormally in various tumors and is involved in the occurrence and development of tumors. In this study, CALR and EIF2AK2 expression was measured in the clinical specimens of 39 patients with melanoma. Then, we constructed knockdown and overexpression cell models of CALR and EIF2AK2 and used wound healing and Transwell assays to observe cell migration and invasion. Apoptosis, EDU, and ROS assays were used to measure cell apoptosis and proliferation, as well as ROS levels. The effect of CALR on endoplasmic reticulum stress was detected using endoplasmic reticulum fluorescent probes. Western blotting was used to detect protein levels of CALR, EIF2AK2, ADAR1, and MMP14. The results indicated that CALR and EIF2AK2 expression levels were significantly higher in human melanoma tissues than in adjacent non-tumor tissue. In addition, we found a correlation between CALR and the expression of EIF2AK2 and MMP14, and the experimental results indicated that overexpression of CALR significantly upregulated the expression of EIF2AK2, MMP14, and ADAR1, while knockdown of CALR inhibited their expression. Notably, the knockdown of EIF2AK2 in the CALR overexpression group blocked the upregulation of MMP14 and ADAR1 expression by CALR, and the knockdown of both CALR and EIF2AK2 significantly inhibited MMP14 and ADAR1 expression. In conclusion, CALR and EIF2AK2 play a promoting role in melanoma progression, and knockdown of CALR and EIF2AK2 may be an effective anti-tumor target, and its mechanism may be through MMP14, ADAR1 signaling.