The role of GADD45G methylation in endometrial cancer: Insights into CDK1/CCNB1 activation and therapeutic opportunities.

INTRODUCTION: Accumulating evidence suggests the significant involvement of GADD45G in the development of various cancers. This study investigates GADD45G's involvement and methylation status in endometrial cancer (EC), along with molecular mechanisms and potential therapies. METHODS: The expression of GADD45G in EC tissues and controls was evaluated using RNA-seq, quantitative real-time polymerase chain reaction (qRT-PCR), and western blotting (WB). Methylation-specific PCR (MSP) evaluated GADD45G's methylation status. Protein-protein interaction (PPI) prediction identified potential interactors of GADD45G, and co-immunoprecipitation (co-IP) confirmed GADD45G interact with Cyclin-dependent kinase 1 (CDK1) and cyclin B1 (CCNB1). Several cell behavior assays were conducted in both in vitro and in vivo settings to comprehensively understand the impact of GADD45G dysregulation in EC. RESULTS: Our findings revealed a significant decrease in the expression of GADD45G in endometrial cancer tissues and cells, which was attributed to its methylation status. Reduced GADD45G expression correlated with increased invasive behaviors in EC cells. Furthermore, GADD45G negatively regulated CDK1 and CCNB1, promoting invasive behaviors at transcript and protein levels. CONCLUSION: This study demonstrated that the downregulation of GADD45G, mediated by methylation, facilitates the invasive behaviors of EC cells through interaction with the CDK1/CCNB1. These findings enhance understanding of the molecular mechanisms underlying endometrial cancer and suggest potential therapeutic strategies targeting GADD45G for treatment.

Perinatal protein malnutrition alters maternal behavior and leads to maladaptive stress response, neurodevelopmental delay and disruption on DNA methylation machinery in female mice offspring.

Deficiencies in maternal nutrition have long-term consequences affecting brain development of the progeny and its behavior. In the present work, female mice were exposed to a normal-protein or a low-protein diet during gestation and lactation. We analyzed behavioral and molecular consequences of malnutrition in dams and how it affects female offspring at weaning. We have observed that a low-protein diet during pregnancy and lactation leads to anxiety-like behavior and anhedonia in dams. Protein malnutrition during the perinatal period delays physical and neurological development of female pups. Glucocorticoid levels increased in the plasma of malnourished female offspring but not in dams when compared to the control group. Interestingly, the expression of glucocorticoid receptor (GR) was reduced in hippocampus and amygdala on both malnourished dams and female pups. In addition, malnourished pups exhibited a significant increase in the expression of Dnmt3b, Gadd45b, and Fkbp5 and a reduction in Bdnf VI variant mRNA in hippocampus. In contrast, a reduction on Dnmt3b has been observed on the amygdala of weaned mice. No changes have been observed on global methylation levels (5-methylcytosine) in hippocampal genomic DNA neither in dams nor female offspring. In conclusion, deregulated behaviors observed in malnourished dams might be mediated by a low expression of GR in brain regions associated with emotive behaviors. Additionally, low-protein diet differentially deregulates the expression of genes involved in DNA methylation/demethylation machinery in female offspring but not in dams, providing an insight into regional- and age-specific mechanisms due to protein malnutrition.

Functional evaluation of germline TP53 variants identified in Brazilian families at-risk for Li-Fraumeni syndrome.

Germline TP53 pathogenic variants can lead to a cancer susceptibility syndrome known as Li-Fraumeni (LFS). Variants affecting its activity can drive tumorigenesis altering p53 pathways and their identification is crucial for assessing individual risk. This study explored the functional impact of TP53 missense variants on its transcription factor activity. We selected seven TP53 missense variants (c.129G > C, c.320A > G, c.417G > T, c.460G > A, c,522G > T, c.589G > A and c.997C > T) identified in Brazilian families at-risk for LFS. Variants were created through site-directed mutagenesis and transfected into SK-OV-3 cells to assess their transcription activation capabilities. Variants K139N and V197M displayed significantly reduced transactivation activity in a TP53-dependent luciferase reporter assay. Additionally, K139N negatively impacted CDKN1A and MDM2 expression and had a limited effect on GADD45A and PMAIP1 upon irradiation-induced DNA damage. Variant V197M demonstrated functional impact in all target genes evaluated and loss of Ser15 phosphorylation. K139N and V197M variants presented a reduction of p21 levels after irradiation. Our data show that K139N and V197M negatively impact p53 functions, supporting their classification as pathogenic variants. This underscores the significance of conducting functional studies on germline TP53 missense variants classified as variants of uncertain significance to ensure proper management of LFS-related cancer risks.

Enriched environment treatment promotes neurofunctional recovery by regulating the ALK5/Smad2/3/Gadd45ß signaling pathway in rats with cerebral ischemia /reperfusion injury.

It has been demonstrated that an enriched environment (EE) treatment can alter neuroplasticity in neurodegenerative diseases. However, the role of EE treatment in ischemic stroke remains unclear. Previous findings have revealed that EE treatment can promote cerebral activin-receptor-like-kinase-5 (ALK5) expression after cerebral ischemia/reperfusion (I/R) injury. ALK5 has been identified as a potential mediator of neuroplasticity through its modulation of Smad2/3 and Gadd45ß. Therefore, the aim of this study was to investigate whether EE treatment could promote neurofunctional recovery by regulating the ALK5/Smad2/3/Gadd45ß pathway. The study utilized the rat model of middle cerebral artery occlusion/reperfusion (MCAO/R). The ALK5/Smad2/3/Gadd45ß signaling pathway changes were evaluated using western blotting (WB). Brain injury was assessed by infarct volume and neurobehavioral scores. The effect of EE treatment on neurogenesis was evaluated using Doublecortin (DCX) and Nestin, axonal plasticity with biotinylated dextran amine (BDA) nerve tracing, and dendritic plasticity was assessed using Golgi-Cox staining. EE treatment has been demonstrated to modulate the Smad2/3/Gadd45ß pathway by regulating the expression of ALK5. The protective effects of EE treatment on brain infarct volume, neurological function, newborn neurons, dendritic and axonal plasticity following cerebral I/R injury were counteracted by ALK5 silencing. EE treatment can enhance neurofunctional recovery after cerebral I/R injury, which is achieved by regulating the ALK5/Smad2/3/Gadd45ß signaling pathway to promote neuroplasticity.

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.

AGEs induce MMP-9 promoter demethylation through the GADD45α-mediated BER pathway to promote breast cancer metastasis in patients with diabetes.

Scientific evidence has linked diabetes to a higher incidence and increased aggressiveness of breast cancer; however, mechanistic studies of the numerous regulators involved in this process are insufficiently thorough. Advanced glycation end products (AGEs) play an important role in the chronic complications of diabetes, but the mechanisms of AGEs in breast cancer are largely unexplored. In this study, we first demonstrate that high AGE levels in breast cancer tissues are associated with the diabetic state and poor patient outcomes. Furthermore, AGEs interact with the receptor for AGEs (RAGE) to promote breast cancer cell migration and invasion. Mechanistically, based on RNA sequencing (RNA-seq) analysis, we reveal that growth arrest and DNA damage gene 45α (GADD45α) is a vital protein upregulated by AGEs through a P53-dependent pathway. Next, GADD45α recruits thymine DNA glycosylase for base excision repair to form the demethylation complex at the promoter region of MMP-9 and enhance MMP-9 transactivation through DNA demethylation. Overall, our results indicate a critical regulatory role of AGEs in patients with breast cancer and diabetes and reveal a novel mechanism of epigenetic modification in promoting breast cancer metastasis.

Harmine inhibits pulmonary fibrosis through regulating DNA damage repair-related genes and activation of TP53-Gadd45α pathway.

BACKGROUND: Harmine has many pharmacological activities and has been found to significantly inhibit the fibrosis of keloid fibroblasts. DNA damage repair (DDR) is essential to prevent fibrosis. This study aimed to investigate the effects of harmine on pulmonary fibrosis and its underlying mechanisms. METHODS: Bleomycin and TGF-ß1 were used to construct pulmonary fibrosis models in vivo and in vitro, then treated with harmine to explore harmine's effects in treating experimental pulmonary fibrosis and its related mechanisms. Then, RNA sequencing was applied to investigate further the crucial DDR-related genes and drug targets of harmine against pulmonary fibrosis. Finally, the expression levels of DDR-related genes were verified by real-time quantitative PCR (RT-qPCR) and western blot. RESULTS: Our in vivo experiments showed that harmine treatment could improve weight loss and lung function and reduce tissue fibrosis in mice with pulmonary fibrosis. The results confirmed that harmine could inhibit the viability and migration of TGF-ß1-induced MRC-5 cells, induce their apoptosis, and suppress the F-actin expression, suggesting that harmine could suppress the phenotypic transition from lung fibroblasts to lung myoblasts. In addition, RNA sequencing identified 1692 differential expressed genes (DEGs), and 10 DDR-related genes were screened as critical DDR-related genes. RT-qPCR and western blotting showed that harmine could down-regulate the expression of CHEK1, ERCC1, ERCC4, POLD1, RAD51, RPA1, TOP1, and TP53, while up-regulate FEN1, H2AX and GADD45α expression. CONCLUSIONS: Harmine may inhibit pulmonary fibrosis by regulating DDR-related genes and activating the TP53-Gadd45α pathway.

Evolution of a novel adrenal cell type that promotes parental care.

Cell types with specialized functions fundamentally regulate animal behaviour, and yet the genetic mechanisms that underlie the emergence of novel cell types and their consequences for behaviour are not well understood1. Here we show that the monogamous oldfield mouse (Peromyscus polionotus) has recently evolved a novel cell type in the adrenal gland that expresses the enzyme AKR1C18, which converts progesterone into 20α-hydroxyprogesterone. We then demonstrate that 20α-hydroxyprogesterone is more abundant in oldfield mice, where it induces monogamous-typical parental behaviours, than in the closely related promiscuous deer mice (Peromyscus maniculatus). Using quantitative trait locus mapping in a cross between these species, we ultimately find interspecific genetic variation that drives expression of the nuclear protein GADD45A and the glycoprotein tenascin N, which contribute to the emergence and function of this cell type in oldfield mice. Our results provide an example by which the recent evolution of a new cell type in a gland outside the brain contributes to the evolution of social behaviour.

Exploring CDKN1A Upregulation Mechanisms: Insights into Cell Cycle Arrest Induced by NC2603 Curcumin Analog in MCF-7 Breast Cancer Cells.

Breast cancer stands out as one of the most prevalent malignancies worldwide, necessitating a nuanced understanding of its molecular underpinnings for effective treatment. Hormone receptors in breast cancer cells substantially influence treatment strategies, dictating therapeutic approaches in clinical settings, serving as a guide for drug development, and aiming to enhance treatment specificity and efficacy. Natural compounds, such as curcumin, offer a diverse array of chemical structures with promising therapeutic potential. Despite curcumin's benefits, challenges like poor solubility and rapid metabolism have spurred the exploration of analogs. Here, we evaluated the efficacy of the curcumin analog NC2603 to induce cell cycle arrest in MCF-7 breast cancer cells and explored its molecular mechanisms. Our findings reveal potent inhibition of cell viability (IC50 = 5.6 µM) and greater specificity than doxorubicin toward MCF-7 vs. non-cancer HaCaT cells. Transcriptome analysis identified 12,055 modulated genes, most notably upregulation of GADD45A and downregulation of ESR1, implicating CDKN1A-mediated regulation of proliferation and cell cycle genes. We hypothesize that the curcumin analog by inducing GADD45A expression and repressing ESR1, triggers the expression of CDKN1A, which in turn downregulates the expression of many important genes of proliferation and the cell cycle. These insights advance our understanding of curcumin analogs' therapeutic potential, highlighting not just their role in treatment, but also the molecular pathways involved in their activity toward breast cancer cells.

Activation of the MEK/ERK Pathway Mediates the Inhibitory Effects of Silvestrol on Nasopharyngeal Carcinoma Cells via RAP1A, HK2, and GADD45A.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant tumor associated with Epstein-Barr virus (EBV) infection. Chemoradiotherapy is the mainstream treatment for locally advanced NPC, and chemotherapeutic drugs are an indispensable part of NPC treatment. However, the toxic side-effects of chemotherapy drugs limit their therapeutic value, and new chemotherapy drugs are urgently needed for NPC. Silvestrol, an emerging natural plant anticancer molecule, has shown promising antitumor activity in breast cancer, melanoma, liver cancer, and other tumor types by promoting apoptosis in cancer cells to a greater extent than in normal cells. However, the effects of silvestrol on NPC and its possible molecular mechanisms have yet to be fully explored. METHODS: Cell counting kit-8 (CCK-8), cell scratch, flow cytometry, 5-ethynyl-2'-deoxyuridine (EdU), and Western blot (WB) assays were used to evaluate the effects of silvestrol on the cell viability, cell cycle, apoptosis, and migration of NPC cells. RNA sequencing (RNA-Seq) was used to study the effect of extracellular signal-regulated kinase (ERK) inhibitors on the cell transcriptome, and immunohistochemistry (IHC) to assess protein expression levels in patient specimens. RESULTS: Silvestrol inhibited cell migration and DNA replication of NPC cells, while promoting the expression of cleaved caspase-3, apoptosis, and cell cycle arrest. Furthermore, silvestrol altered the level of ERK phosphorylation. The ERK-targeted inhibitor LY3214996 attenuated silvestrol-mediated inhibition of NPC cell proliferation but not migration. Analysis of RNA-Seq data and WB were used to identify and validate the downstream regulatory targets of silvestrol. Expression of GADD45A, RAP1A, and hexokinase-II (HK2) proteins was inhibited by silvestrol and LY3214996. Finally, IHC revealed that GADD45A, RAP1A, and HK2 protein expression was more abundant in cancer tissues than in non-tumor tissues. CONCLUSIONS: Silvestrol inhibits the proliferation of NPC cells by targeting ERK phosphorylation. However, the inhibition of NPC cell migration by silvestrol was independent of the Raf-MEK-ERK pathway. RAP1A, HK2, and GADD45A may be potential targets for the action of silvestrol.

Loss of the stress sensor GADD45A promotes stem cell activity and ferroptosis resistance in LGR4/HOXA9-dependent AML.

ABSTRACT: The overall prognosis of acute myeloid leukemia (AML) remains dismal, largely because of the inability of current therapies to kill leukemia stem cells (LSCs) with intrinsic resistance. Loss of the stress sensor growth arrest and DNA damage-inducible 45 alpha (GADD45A) is implicated in poor clinical outcomes, but its role in LSCs and AML pathogenesis is unknown. Here, we define GADD45A as a key downstream target of G protein-coupled receptor (LGR)4 pathway and discover a regulatory role for GADD45A loss in promoting leukemia-initiating activity and oxidative resistance in LGR4/HOXA9-dependent AML, a poor prognosis subset of leukemia. Knockout of GADD45A enhances AML progression in murine and patient-derived xenograft (PDX) mouse models. Deletion of GADD45A induces substantial mutations, increases LSC self-renewal and stemness in vivo, and reduces levels of reactive oxygen species (ROS), accompanied by a decreased response to ROS-associated genotoxic agents (eg, ferroptosis inducer RSL3) and acquisition of an increasingly aggressive phenotype on serial transplantation in mice. Our single-cell cellular indexing of transcriptomes and epitopes by sequencing analysis on patient-derived LSCs in PDX mice and subsequent functional studies in murine LSCs and primary AML patient cells show that loss of GADD45A is associated with resistance to ferroptosis (an iron-dependent oxidative cell death caused by ROS accumulation) through aberrant activation of antioxidant pathways related to iron and ROS detoxification, such as FTH1 and PRDX1, upregulation of which correlates with unfavorable outcomes in patients with AML. These results reveal a therapy resistance mechanism contributing to poor prognosis and support a role for GADD45A loss as a critical step for leukemia-initiating activity and as a target to overcome resistance in aggressive leukemia.

GADD45B in the ventral hippocampal CA1 modulates aversive memory acquisition and spatial cognition.

AIMS: This study was designed to investigate the role of growth arrest and DNA damage-inducible ß (GADD45B) in modulating fear memory acquisition and elucidate its underlying mechanisms. MAIN METHODS: Adeno-associated virus (AAV) that knockdown or overexpression GADD45B were injected into ventral hippocampal CA1 (vCA1) by stereotactic, and verified by fluorescence and Western blot. The contextual fear conditioning paradigm was employed to examine the involvement of GADD45B in modulating aversive memory acquisition. The Y-maze and novel location recognition (NLR) tests were used to examine non-aversive cognition. The synaptic plasticity and electrophysiological properties of neurons were measured by slice patch clamp. KEY FINDINGS: Knockdown of GADD45B in the vCA1 significantly enhanced fear memory acquisition, accompanied by an upregulation of long-term potentiation (LTP) expression and intrinsic excitability of vCA1 pyramidal neurons (PNs). Conversely, overexpression of GADD45B produced the opposite effects. Notably, silencing the activity of vCA1 neurons abolished the impact of GADD45B knockdown on fear memory development. Moreover, mice with vCA1 GADD45B overexpression exhibited impaired spatial cognition, whereas mice with GADD45B knockdown did not display such impairment. SIGNIFICANCE: These results provided compelling evidence for the crucial involvement of GADD45B in the formation of aversive memory and spatial cognition.

OCA-B/Pou2af1 is sufficient to promote CD4+ T cell memory and prospectively identifies memory precursors.

The molecular mechanisms leading to the establishment of immunological memory are inadequately understood, limiting the development of effective vaccines and durable antitumor immune therapies. Here, we show that ectopic OCA-B expression is sufficient to improve antiviral memory recall responses, while having minimal effects on primary effector responses. At peak viral response, short-lived effector T cell populations are expanded but show increased Gadd45b and Socs2 expression, while memory precursor effector cells show increased expression of Bcl2, Il7r, and Tcf7 on a per-cell basis. Using an OCA-B mCherry reporter mouse line, we observe high OCA-B expression in CD4+ central memory T cells. We show that early in viral infection, endogenously elevated OCA-B expression prospectively identifies memory precursor cells with increased survival capability and memory recall potential. Cumulatively, the results demonstrate that OCA-B is both necessary and sufficient to promote CD4 T cell memory in vivo and can be used to prospectively identify memory precursor cells.

GADD45A: With or without you.

The growth arrest and DNA damage inducible (GADD)45 family includes three small and ubiquitously distributed proteins (GADD45A, GADD45B, and GADD45G) that regulate numerous cellular processes associated with stress signaling and injury response. Here, we provide a comprehensive review of the current literature investigating GADD45A, the first discovered member of the family. We first depict how its levels are regulated by a myriad of genotoxic and non-genotoxic stressors, and through the combined action of intricate transcriptional, posttranscriptional, and even, posttranslational mechanisms. GADD45A is a recognized tumor suppressor and, for this reason, we next summarize its role in cancer, as well as the different mechanisms by which it regulates cell cycle, DNA repair, and apoptosis. Beyond these most well-known actions, GADD45A may also influence catabolic and anabolic pathways in the liver, adipose tissue and skeletal muscle, among others. Not surprisingly, GADD45A may trigger AMP-activated protein kinase activity, a master regulator of metabolism, and is known to act as a transcriptional coregulator of numerous nuclear receptors. GADD45A has also been reported to display a cytoprotective role by regulating inflammation, fibrosis and oxidative stress in several organs and tissues, and is regarded an important contributor for the development of heart failure. Overall data point to that GADD45A may play an important role in metabolic, neurodegenerative and cardiovascular diseases, and also autoimmune-related disorders. Thus, the potential mechanisms by which dysregulation of GADD45A activity may contribute to the progression of these diseases are also reviewed below.

Malignant peritoneal mesotheliomas of rats induced by multiwalled carbon nanotubes and amosite asbestos: transcriptome and epigenetic profiles.

BACKGROUND: Malignant mesothelioma is an aggressive cancer that often originates in the pleural and peritoneal mesothelium. Exposure to asbestos is a frequent cause. However, studies in rodents have shown that certain multiwalled carbon nanotubes (MWCNTs) can also induce malignant mesothelioma. The exact mechanisms are still unclear. To gain further insights into molecular pathways leading to carcinogenesis, we analyzed tumors in Wistar rats induced by intraperitoneal application of MWCNTs and amosite asbestos. Using transcriptomic and epigenetic approaches, we compared the tumors by inducer (MWCNTs or amosite asbestos) or by tumor type (sarcomatoid, epithelioid, or biphasic). RESULTS: Genome-wide transcriptome datasets, whether grouped by inducer or tumor type, showed a high number of significant differentially expressed genes (DEGs) relative to control peritoneal tissues. Bioinformatic evaluations using Ingenuity Pathway Analysis (IPA) revealed that while the transcriptome datasets shared commonalities, they also showed differences in DEGs, regulated canonical pathways, and affected molecular functions. In all datasets, among highly- scoring predicted canonical pathways were Phagosome Formation, IL8 Signaling, Integrin Signaling, RAC Signaling, and TREM1 Signaling. Top-scoring activated molecular functions included cell movement, invasion of cells, migration of cells, cell transformation, and metastasis. Notably, we found many genes associated with malignant mesothelioma in humans, which showed similar expression changes in the rat tumor transcriptome datasets. Furthermore, RT-qPCR revealed downregulation of Hrasls, Nr4a1, Fgfr4, and Ret or upregulation of Rnd3 and Gadd45b in all or most of the 36 tumors analyzed. Bisulfite sequencing of Hrasls, Nr4a1, Fgfr4, and Ret revealed heterogeneity in DNA methylation of promoter regions. However, higher methylation percentages were observed in some tumors compared to control tissues. Lastly, global 5mC DNA, m6A RNA and 5mC RNA methylation levels were also higher in tumors than in control tissues. CONCLUSIONS: Our findings may help better understand how exposure to MWCNTs can lead to carcinogenesis. This information is valuable for risk assessment and in the development of safe-by-design strategies.

Shikonin reactivates TSGs GADD45B and PPP3CC to block NSCLC cell proliferation and migration through JNK/P38/MAPK signaling pathways.

BACKGROUND: Shikonin, a natural naphthoquinone compound extracted from the Chinese traditional herbal medicine "Lithospermum erythrorhizon", possesses antitumor activity against various cancer types. Tumor-suppressor genes (TSGs) negatively regulate cell growth, proliferation, and differentiation, thereby inhibiting tumor formation. However, the molecular mechanism of action of shikonin on TSGs in non-small-cell lung cancer (NSCLC) remains unclear. METHODS: The inhibitory effect of shikonin on the proliferation and migration abilities of lung cancer cells were measured by Cell Counting Kit 8 (CCK8) and wound healing assays. The alteration of genes by shikonin treatment was detected by mRNA high-throughput sequencing and further confirmed by qPCR and western blotting experiments. The dominant functions of the upregulated genes were analyzed by GO and KEGG profiling. RESULTS: Shikonin inhibited the proliferation and migration of A549 and H1299 NSCLC cells in a dose-dependent manner. mRNA high-throughput sequencing revealed a total of 1794 upregulated genes in shikonin-treated NSCLC cells. Moreover, bioinformatic analysis of GO and KEGG profiling revealed that the up-regulated genes were mostly involved in the JNK/P38/MAPK signaling pathway, among which the expression of GADD45B and PPP3CC was significantly enhanced. Finally, we confirmed that GADD45B and PPP3CC were indeed upregulated in JNK/P38/MAPK pathway. CONCLUSIONS: Taken together, these results suggested that shikonin might affect the expression of GADD45B and PPP3CC through the JNK/P38/MAPK pathway, therefore exerting an inhibitory effect on the proliferation and migration of cancer cells. To our knowledge, this is the first study reporting the role of shikonin in upregulating TSGs to activate the JNK/P38/MAPK signaling pathways in NSCLC.

Has-miR-300-GADD45B promotes melanoma growth via cell cycle.

Response to oncogenic factors like UV, GADD45 family in skin participates in scavenging ROS, DNA repair and cell cycle control. Because of this, the previous study of the chronic UVB injury model has found that hsa-miR-300 can conduct intercellular transport by exosomes and target regulation of GADD45B. Whether the hsa-miR-300-GADD45B still regulates tumor development by cell cycle pathway is unclear. Through transcriptomic analysis of primary (n=39) and metastatic (n=102) melanoma, it was confirmed that in metastatic samples, some of the 97 down-regulated genes participate in maintaining skin homeostasis while 42 up-regulated genes were enriched in cancer-related functions. Furthermore, CDKN1A, CDKN2A, CXCR4 and RAD51 in the melanoma pathway, were also differentially expressed between normal skin and melanoma. CDKN1A and CDKN2A were also found to be involved in TP53-dependent cell cycle regulation. In conclusion, it was speculated that CDKN1A, CDKN2A, TP53, GADD45B and hsa-miR-300 may have regulatory relationships. It was demonstrated that there is a bidirectional regulation between hsa-miR-300 and TP53. In addition, miR-300 can regulate CDKN1A by GADD45B/TP53 and promote melanoma growth by accelerating the cell cycle transition from G1/S to G2 phase.

Circular RNA circCHFR downregulation protects against oxidized low-density lipoprotein-induced endothelial injury via regulation of microRNA-15b-5p/growth arrest and DNA damage inducible gamma.

Atherosclerosis is the leading cause of coronary heart disease. In recent years, circ_0029589 (circCHFR) has been found to be associated with atherosclerosis development. However, the molecular mechanism of circCHFR action in atherosclerosis development is unknown. This study was aimed to investigate the function and action mechanism of circCHFR in atherosclerosis development. An atherosclerosis cell model was created by exposing human vascular endothelial cells (HUVECs) to oxidized low-density lipoprotein. The expression of circCHFR, microRNA(miR)-15b-5p, growth arrest and DNA damage inducible gamma (GADD45G), and their associated proteins was evaluated using quantitative reverse transcription-polymerase chain reaction and Western blotting. Additionally, cell viability, apoptosis, and cytokine levels were determined using Cell Counting Kit-8 (CCK8) assay, flow cytometry, and enzyme-linked immunosorbent assay, respectively. circCHFR expression was upregulated in patients with atherosclerosis and oxidized low-density lipoprotein (ox-LDL)-exposed HUVECs, whereas miR-15b-5p expression was downregulated. circCHFR silencing significantly improved viability and reduced apoptosis of HUVECs. In addition, the pro-apoptotic protein Bax and atherosclerosis-associated cytokines (interleukin-1ß, interleukin-6, and tumor necrosis factor-α) were significantly downregulated, whereas the anti-apoptotic protein Bcl-2 was upregulated. Further, we discovered that circCHFR serves as a molecular sponge of miR-15b-5p. GADD45G was found to be an important target of miR-15b-5p; miR-15b-5p mimic inhibited GADD45G expression, reduced apoptosis and proinflammatory cytokine secretion, and improved cell survival. However, these effects of miR-15b-5p on (ox-LDL) induced HUVECs were reversed with GADD45G plasmid co-transfection. In conclusion, circCHFR promotes atherosclerosis progression via the miR-15b-5p/GADD45G axis and may be an important target for atherosclerosis treatment.

METTL3-mediated RNA m6A Hypermethylation Promotes Tumorigenesis and GH Secretion of Pituitary Somatotroph Adenomas.

INTRODUCTION: Pituitary growth hormone-secreting (GH) pituitary adenomas (PAs) cause mass effects and dysregulated hypersecretion of GH. However, somatic mutation burden is low in PAs. While progress has been made in identifying the epigenetic changes involved in GH-PA initiation, the precise details of its tumorigenesis in GH-PA patients remains to be elucidated. As N6-methyladenosine (m6A) has been shown to often play a critical role in various tumors, it represents a possible initiation point for the tumorigenesis of pituitary adenomas. However, the role of RNA methylation in GH adenomas remains unclear. METHODS: Protein expression of m6A regulators was measured by immunohistochemistry. Global levels and distribution of m6A methylation were separately analyzed by m6A enzyme-linked immunosorbent assay and m6A sequencing (m6A-seq). RNA interference and lentivirus knockdown system were used to investigate the role of methyltransferase-like 3 (METTL3) and its m6A- dependent regulatory mechanism in tumor progression and GH secretion. RESULTS: We show that both METTL3 messenger RNA and protein expression are elevated in GH-PA samples when compared with both normal pituitary tissue specimens and nonsecreting pituitary adenomas. Levels of m6A modification increased in GH-PAs, and hypermethylated RNAs are involved in hormone secretion and cell development. Knockdown of METTL3 in GH3 cell line resulted in decreased cell growth and GH secretion. Importantly, we found that GNAS and GADD45γ act as the downstream targets in this process. CONCLUSION: Our findings strongly suggest that m6A methyltransferase METTL3 promotes tumor growth and hormone secretion by increasing expression of GNAS and GADD45γ in a m6A-dependent manner. Thus, METTL3 and the methylated RNAs constitute suitable targets for clinical treatment of GH-PAs.

Expression of Human Mutant Preproinsulins Induced Unfolded Protein Response, Gadd45 Expression, JAK-STAT Activation, and Growth Inhibition in Drosophila.

Mutations in the insulin gene (INS) are frequently associated with human permanent neonatal diabetes mellitus. However, the mechanisms underlying the onset of this genetic disease is not sufficiently decoded. We induced expression of two types of human mutant INSs in Drosophila using its ectopic expression system and investigated the resultant responses in development. Expression of the wild-type preproinsulin in the insulin-producing cells (IPCs) throughout the larval stage led to a stimulation of the overall and wing growth. However, ectopic expression of human mutant preproinsulins, hINSC96Y and hINSLB15YB16delinsH, neither of which secreted from the ß-cells, could not stimulate the Drosophila growth. Furthermore, neither of the mutant polypeptides induced caspase activation leading to apoptosis. Instead, they induced expression of several markers indicating the activation of unfolded protein response, such as ER stress-dependent Xbp1 mRNA splicing and ER chaperone induction. We newly found that the mutant polypeptides induced the expression of Growth arrest and DNA-damage-inducible 45 (Gadd45) in imaginal disc cells. ER stress induced by hINSC96Y also activated the JAK-STAT signaling, involved in inflammatory responses. Collectively, we speculate that the diabetes-like growth defects appeared as a consequence of the human mutant preproinsulin expression was involved in dysfunction of the IPCs, rather than apoptosis.