The transcription of the main gene associated with Treacher-Collins syndrome (TCOF1) is regulated by G-quadruplexes and cellular nucleic acid binding protein (CNBP).

Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with Kd values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter (nolc1). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD.

Peeling the onion: additional layers of regulation in the acid stress response.

Bacteria are capable of withstanding large changes in osmolality and cytoplasmic pH, unlike eukaryotes that tightly regulate their pH and cellular composition. Previous studies on the bacterial acid stress response described a rapid, brief acidification, followed by immediate recovery. More recent experiments with better pH probes have imaged single living cells, and we now appreciate that following acid stress, bacteria maintain an acidic cytoplasm for as long as the stress remains. This acidification enables pathogens to sense a host environment and turn on their virulence programs, for example, enabling survival and replication within acidic vacuoles. Single-cell analysis identified an intracellular pH threshold of ~6.5. Acid stress reduces the internal pH below this threshold, triggering the assembly of a type III secretion system in Salmonella and the secretion of virulence factors in the host. These pathways are significant because preventing intracellular acidification of Salmonella renders it avirulent, suggesting that acid stress pathways represent a potential therapeutic target. Although we refer to the acid stress response as singular, it is actually a complex response that involves numerous two-component signaling systems, several amino acid decarboxylation systems, as well as cellular buffering systems and electron transport chain components, among others. In a recent paper in the Journal of Bacteriology, M. G. Gorelik, H. Yakhnin, A. Pannuri, A. C. Walker, C. Pourciau, D. Czyz, T. Romeo, and P. Babitzke (J Bacteriol 206:e00354-23, 2024, https://doi.org/10.1128/jb.00354-23) describe a new connection linking the carbon storage regulator CsrA to the acid stress response, highlighting new additional layers of complexity.

LncRNA GAS6-AS1 contributes to 5-fluorouracil resistance in colorectal cancer by facilitating the binding of PCBP1 with MCM3.

5-Fluorouracil (5-FU) resistance has always been a formidable obstacle in the adjuvant treatment of advanced colorectal cancer (CRC). In recent years, long non-coding RNAs have emerged as key regulators in various pathophysiological processes including 5-FU resistance. TRG is a postoperative pathological score of the chemotherapy effectiveness for CRC, of which TRG 0-1 is classified as chemotherapy sensitivity and TRG 3 as chemotherapy resistance. Here, RNA-seq combined with weighted gene correlation network analysis confirmed the close association of GAS6-AS1 with TRG. GAS6-AS1 expression was positively correlated with advanced clinicopathological features and poor prognosis in CRC. GAS6-AS1 increased the 50% inhibiting concentration of 5-FU, enhanced cell proliferation and accelerated G1/S transition, both with and without 5-FU, both in vitro and in vivo. Mechanistically, GAS6-AS1 enhanced the stability of MCM3 mRNA by recruiting PCBP1, consequently increasing MCM3 expression. Furthermore, PCBP1 and MCM3 counteracted the effects of GAS6-AS1 on 5-FU resistance. Notably, the PDX model indicated that combining chemotherapeutic drugs with GAS6-AS1 knockdown yielded superior outcomes in vivo. Together, our findings elucidate that GAS6-AS1 directly binds to PCBP1, enhancing MCM3 expression and thereby promoting 5-FU resistance. GAS6-AS1 may serve as a robust biomarker and potential therapeutic target for combination therapy in CRC.

Dioscin decreases M2 polarization via inhibiting a positive feedback loop between RBM47 and NF-κB in glioma.

BACKGROUND: The role of the glioblastoma (GBM) microenvironment is pivotal in the development of gliomas. Discovering drugs that can traverse the blood-brain barrier and modulate the tumor microenvironment is crucial for the treatment of GBM. Dioscin, a steroidal saponin derived from various kinds of plants and herbs known to penetrate the blood-brain barrier, has shown its powerful anti-tumor activity. However, little is known about its effects on GBM microenvironment. METHODS: Bioinformatics analysis was conducted to assess the link between GBM patients and their prognosis. Multiple techniques, including RNA sequencing, immunofluorescence staining, Western blot analysis, RNA-immunoprecipitation (RIP) assays, and Chromatin immunoprecipitation (CHIP) analysis were employed to elucidate the mechanism through which Dioscin modulates the immune microenvironment. RESULTS: Dioscin significantly impaired the polarization of macrophages into the M2 phenotype and enhanced the phagocytic ability of macrophages in vitro and in vivo. A strong correlation between high expression of RBM47 in GBM and a detrimental prognosis for patients was demonstrated. RNA-sequencing analysis revealed an association between RBM47 and the immune response. The inhibition of RBM47 significantly impaired the recruitment and polarization of macrophages into the M2 phenotype and enhanced the phagocytic ability of macrophages. Moreover, RBM47 could stabilize the mRNA of inflammatory genes and enhance the expression of these genes by activating the NF-κB pathway. In addition, NF-κB acts as a transcription factor that enhances the transcriptional activity of RBM47. Notably, we found that Dioscin could significantly inhibit the activation of NF-κB and then downregulate the expression of RBM47 and inflammatory genes protein. CONCLUSION: Our study reveals that the positive feedback loop between RBM47 and NF-κB could promote immunosuppressive microenvironment in GBM. Dioscin effectively inhibits M2 polarization in GBM by disrupting the positive feedback loop between RBM47 and NF-κB, indicating its potential therapeutic effects in GBM treatment.

Pien Tze Huang Inhibits Proliferation of Colorectal Cancer Cells through Suppressing PNO1 Expression and Activating p53/p21 Signaling Pathway.

OBJECTIVE: To explore the regulatory effect of Pien Tze Huang (PZH) on targeting partner of NOB1 (PNO1) and it's down-stream mediators in colorectal cancer (CRC) cells. METHODS: Quantitative polymerase chain reaction was performed to determine mRNA levels of PNO1, TP53, and CDKN1A. Western blotting was performed to determine protein levels of PNO1, p53, and p21. HCT-8 cells were transduced with a lentivirus over-expressing PNO1. Colony formation assay was used to detect cell survival in PNO1 overexpression of HCT-8 cells after PZH treatment. Cell-cycle distribution, cell viability and cell apoptosis were performed to identify the effect of PNO1 overexpression on cell proliferation and apoptosis of HCT-8 cells after PZH treatment. Xenograft BALB/c nude mice bearing HCT116 cells transduced with sh-PNO1 or sh-Ctrl lentivirus were evaluated. Western blot assay was performed to detect PNO1, p53, p21 and PCNA expression in tumor sections. Terminal deoxynucleotidyl transferase dUTP nick end labling (TUNEL) assay was used to determine the apoptotic cells in tissues. RESULTS: PZH treatment decreased cell viability, down-regulated PNO1 expression, and up-regulated p53 and p21 expressions in HCT-8 cells (P<0.05). PNO1 overexpression attenuated the effects of PZH treatment, including the expression of p53 and p21, cell growth, cell viability, cell cycle arrest and cell apoptosis in vitro (P<0.05). PNO1 knockdown eliminated the effects of PZH treatment on tumor growth, inhibiting cell proliferation inhibition and apoptosis induction in vivo (P<0.05). Similarly, PNO1 knockdown attenuated the effects of PZH treatment on the down-regulation of PNO1 and up-regulation of p53 and p21 in vivo (P<0.05). CONCLUSION: The mechanism by which PZH induces its CRC anti-proliferative effect is at least in part by regulating the expression of PNO1 and its downstream targets p53 and p21.

Tandem Expression of a Mobile RNA and Its RNA-Binding Protein(s) Enhances Tuber Productivity in Potato.

A significant number of discoveries in past two decades have established the importance of long-distance signaling in controlling plant growth, development, and biotic and abiotic stress responses. Numerous mobile signals, such as mRNAs, proteins, including RNA-binding proteins, small RNAs, sugars, and phytohormones, are shown to regulate various agronomic traits such as flowering, fruit, seed development, and tuberization. Potato is a classic model tuber crop, and several mobile signals are known to govern tuber development. However, it is unknown if these mobile signals have any synergistic effects on potato crop improvement. Here, we employed a simple innovative strategy to test the cumulative effects of a key mobile RNA, StBEL5, and its RNA-binding proteins, StPTB1, and -6 on tuber productivity of two potato cultivars, Solanum tuberosum cv. Désirée and subspecies andigena, using a multi-gene stacking approach. In this approach, the coding sequences of StBEL5 and StPTB1/6 are driven by their respective native promoters to efficiently achieve targeted expression in phloem for monitoring tuber productivity. We demonstrate that this strategy resulted in earliness for tuberization and enhanced tuber productivity by 2-4 folds under growth chamber, greenhouse, and field conditions. This multi-gene stacking approach could be adopted to other crops, whose agronomic traits are governed by mobile macromolecules, expanding the possibilities to develop crops with improved traits and enhanced yields.

PUM1 Promotes Tumor Progression by Activating DEPTOR-Meditated Glycolysis in Gastric Cancer.

RNA-binding proteins (RBPs) play essential roles in tumorigenesis and progression, but their functions in gastric cancer (GC) remain largely elusive. Here, it is reported that Pumilio 1 (PUM1), an RBP, induces metabolic reprogramming through post-transcriptional regulation of DEP domain-containing mammalian target of rapamycin (mTOR)-interacting protein (DEPTOR) in GC. In clinical samples, elevated expression of PUM1 is associated with recurrence, metastasis, and poor survival. In vitro and in vivo experiments demonstrate that knockdown of PUM1 inhibits the proliferation and metastasis of GC cells. In addition, RNA-sequencing and bioinformatics analyses show that PUM1 is enriched in the glycolysis gene signature. Metabolomics studies confirm that PUM1 deficiency suppresses glycolytic metabolism. Mechanistically, PUM1 binds directly to DEPTOR mRNA pumilio response element to maintain the stability of the transcript and prevent DEPTOR degradation through post-transcriptional pathway. PUM1-mediated DEPTOR upregulation inhibits mTORC1 and alleviates the inhibitory feedback signal transmitted from mTORC1 to PI3K under normal conditions, thus activating the PI3K-Akt signal and glycolysis continuously. Collectively, these results reveal the critical epigenetic role of PUM1 in modulating DEPTOR-dependent GC progression. These conclusions support further clinical investigation of PUM1 inhibitors as a metabolic-targeting treatment strategy for GC.

ROS-responsive nanomodulators downregulate IFITM3 expression and eliminate ROS for Alzheimer's disease combination treatment.

Neuronal damage caused by ß-amyloid (Aß) aggregates and excess reactive oxygen species (ROS) is a crucial pathogenic event in Alzheimer's disease (AD). However, current Aß-targeting RNA interference (RNAi) treatments have shown limited therapeutic efficacy due to ineffective intracerebral siRNA delivery and overlooked crosstalk between excess ROS and Aß aggregates in the brain. Herein, a ROS-responsive nanomodulator (NM/CM) was developed for the combinational treatment of RNAi and ROS elimination for AD. NM/CM was coated with 4T1 cell membranes, which endowed NM/CM with the capability to cross blood-brain barrier (BBB). After being internalized by neural cells, NM/CM releases curcumin (Cur) and siIFITM3 spontaneously into the cytoplasm. The released Cur can eliminate ROS, protecting neurons from oxidative damage and reducing the production of Aß induced by ROS-related neuroinflammation. The released siIFITM3 can downregulate the expression of interferon-induced transmembrane protein 3 (IFITM3), thereby reducing the abnormal Aß production mediated by IFITM3. As a result, NM/CM remarkably alleviated ROS- and Aß aggregate-induced neurotoxicity in vitro, showing significant neuroprotective effects. This work demonstrates the potential of NM/CM in the development of novel and effective AD combination therapies.

RNA-binding domain 2 of nucleolin is important for the autophagy induction of curcumol in nasopharyngeal carcinoma cells.

BACKGROUND & AIMS: Excessive autophagy induces cell death and is regarded as the treatment of cancer therapy. We have confirmed that the anti-cancer mechanism of curcumol is related to autophagy induction. As the main target protein of curcumol, RNA binding protein nucleolin (NCL) interacted with many tumor promoters accelerating tumor progression. However, the role of NCL in cancer autophagy and in curcumol's anti-tumor effects haven't elucidated. The purpose of the study is to identify the role of NCL in nasopharyngeal carcinoma autophagy and reveal the immanent mechanisms of NCL played in cell autophagy. METHODS & RESULTS: In the current study, we have found that NCL was markedly upregulated in nasopharyngeal carcinoma (NPC) cells. NCL overexpression effectively attenuated the level of autophagy in NPC cells, and NCL silence or curcumol treatment obviously aggravated the autophagy of NPC cells. Moreover, the attenuation of NCL by curcumol lead a significant suppression on PI3K/AKT/mTOR signaling pathway in NPC cells. Mechanistically, NCL was found to be directly interact with AKT and accelerate AKT phosphorylation, which caused the activation of the PI3K/AKT/mTOR pathway. Meanwhile, the RNA Binding Domain (RBD) 2 of NCL interacts with Akt, which was also influenced by curcumol. Notably, the RBDs of NCL delivered AKT expression was related with cell autophagy in the NPC. CONCLUSION: The results demonstrated that NCL regulated cell autophagy was related with interaction of NCL and Akt in NPC cells. The expression of NCL play an important role in autophagy induction and further found that was associated with its effect on NCL RNA-binding domain 2. This study may provide a new perspective on the target protein studies for natural medicines and confirm the effect of curcumol not only regulating the expression of its target protein, but also influencing the function domain of its target protein.

Deciphering the molecular mechanisms of Maxing Huoqiao Decoction in treating pulmonary fibrosis via transcriptional profiling and circRNA-miRNA-mRNA network analysis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung condition with unknown etiology and high mortality. Chinese herbal medicine has been used for more than a thousand years to treat various lung diseases. PURPOSE: The current study aimed to examine whether Chinese herbal Maxing Huoqiao Decoction (MXHQD) exerts therapeutic effects on IPF and to further uncover its underlying molecular mechanisms. METHODS: Mouse model of acute lung injury (ALI) or IPF was induced by intratracheal instillation of LPS or bleomycin, respectively. ALI mice were treated with MXHQD for 7 days, and lung tissues were taken for test after modeling 24 h. IPF mice were gavaged for 21 days after modeling. Lung tissues were subjected to whole transcriptome detection, and the differential RNAs were experimentally verified. RESULTS: The results showed that MXHQD alleviated the computed tomography (CT) and the pathological degree changes in mice with IPF, improved changes in the expression of fibrosis related genes and reduced the hydroxyproline expression in IPF mice. MXHQD also decreased the cell numbers in bronchoalveolar lavage fluids, and the expression levels of the inflammatory factors in the ALI mice lung tissues were significantly inhibited. By applying whole transcriptome analysis, results showed that MXHQD acted on 40 mRNAs, 15 miRNAs, 25 novel lncRNAs and 17 circRNAs to resist pulmonary fibrosis. The competing endogenous RNA (ceRNA) network diagram showed that the multiple components of MXHQD against fibrosis through a network of multiple targets. The differential mRNAs were mainly related to the innate immune response and the defense response to virus. Then the expression of mRNAs in the differential mRNA-miRNA-differential circRNA network in the lung tissue of IPF was verified. The expression of ZBP1 and ISG15 related to immune system and anti virus was verified at both gene and protein expressions. MXHQD could significantly inhibit the elevation of ZBP1 and ISG15 factors induced by the fibrosis model. CONCLUSION: Overall, our findings provide compelling evidence that MXHQD can alleviate IPF by modulating innate immunity. This is the first study to reveal the molecular mechanism underlying the multi-components, multi-channels and multi-targets anti-IPF immune injury of MXHQD, and supports its potential clinical application for IPF.

Chronic Kidney Disease Induces Proarrhythmic Remodeling.

BACKGROUND: Patients with chronic kidney disease (CKD) are at increased risk of developing cardiac arrhythmogenesis and sudden cardiac death; however, the basis for this association is incompletely known. METHODS: Here, using murine models of CKD, we examined interactions between kidney disease progression and structural, electrophysiological, and molecular cardiac remodeling. RESULTS: C57BL/6 mice with adenine supplemented in their diet developed progressive CKD. Electrocardiographically, CKD mice developed significant QT prolongation and episodes of bradycardia. Optical mapping of isolated-perfused hearts using voltage-sensitive dyes revealed significant prolongation of action potential duration with no change in epicardial conduction velocity. Patch-clamp studies of isolated ventricular cardiomyocytes revealed changes in sodium and potassium currents consistent with action potential duration prolongation. Global transcriptional profiling identified dysregulated expression of cellular stress response proteins RBM3 (RNA-binding motif protein 3) and CIRP (cold-inducible RNA-binding protein) that may underlay the ion channel remodeling. Unexpectedly, we found that female sex is a protective factor in the progression of CKD and its cardiac sequelae. CONCLUSIONS: Our data provide novel insights into the association between CKD and pathologic proarrhythmic cardiac remodeling. Cardiac cellular stress response pathways represent potential targets for pharmacologic intervention for CKD-induced heart rhythm disorders.

NOVA1 acts on Impact to regulate hypothalamic function and translation in inhibitory neurons.

We describe a patient haploinsufficient for the neuronal RNA binding protein NOVA1 who developed a behavioral motor hyperactivity disorder, suggesting a role of NOVA1 in postnatal motor inhibition. To investigate Nova1's action in adult Gad2+ inhibitory neurons, we generated a conditional Nova1-null mouse (Nova1-cKOGad2-cre). Strikingly, the phenotypes of these mice show many similarities to the NOVA1 haploinsufficient patient and identify a function of Nova1 in the hypothalamus. Molecularly, Nova1 loss in Gad2-positive neurons alters downstream expression of Impact mRNA, along with a subset of RNAs encoding electron transport chain-related factors and ribosomal proteins. NOVA1 stabilizes Impact mRNA by binding its 3' UTR, antagonizing the actions of miR-138 and miR-124. Together, these studies demonstrate actions of NOVA1 in adult hypothalamic neurons, mechanisms by which it functions in translation and metabolism, including through direct binding to Impact mRNA, and illuminate its role in human neurologic disease.

Carob extract induces spermatogenesis in an infertile mouse model via upregulation of Prm1, Plzf, Bcl-6b, Dazl, Ngn3, Stra8, and Smc1b.

ETHNOPHARMACOLOGICAL RELEVANCE: Ethnopharmacological studies for drug discovery from natural compounds play an important role for developing current therapeutical platforms. Plants are a group of natural sources which have been served as the basis in the treatment of many diseases for centuries. In this regard, Ceratonia siliqua (carob) is one of the herbal medicine which is traditionally used for male infertility treatments. But so far the main mechanisms for effects of carob are unknown. Here, we intend to investigate the ability of carob extract to induce spermatogenesis in an azoospermia mouse model and determine the mechanisms that underlie its function. AIM OF THE STUDY: This is a pre-clinical animal model study to evaluate the effect of carob extract in spermatogenesis recovery. METHODS: We established an infertile mouse model with the intent to examine the ability of carob extract as a potential herbal medicine for restoration of male fertility. Sperm parameters, as well as gene expression dynamics and levels of spermatogenesis hormones, were evaluated 35 days after carob administration. RESULTS: Significant enhanced sperm parameters (P < 0.05) showed that the carob extract could induce spermatogenesis in the infertile mouse model. Our data suggested an anti-apototic and inducer role in the expressions of cell cycle regulating genes. Carob extract improved the spermatogenesis niche by considerable affecting Sertoli and Leydig cells (P < 0.05). The carob-treated mice were fertile and contributed to healthy offspring that matured. Our data confirmed that this extract triggered the hormonal system, the spermatogenesis-related gene expression network, and signaling pathways to induce and promote sperm production with notable level (P < 0.05). We found that the aqueous extract consisted of a polar and mainly well water-soluble substance. Carob extract might upregulate spermatogenesis hormones via its amino acid components, which were detected in the extract by liquid chromatography-mass spectrometry (LC-MS). CONCLUSION: Our results strongly suggest that carob extract might be a promising future treatment option for male infertility. This finding could pave the way for clinical trials in infertile men. This is the first study that has provided reliable, strong pre-clinical evidence for carob extract as an effective candidate for fertility recovery in cancer-related azoospermia.

Qilan preparation inhibits proliferation and induces apoptosis by down-regulating microRNA-21 in human Tca8113 tongue squamous cell carcinoma cells.

OBJECTIVE: The aim of this study was to examine the antitumor effects of Qilan preparation on oral squamous cell carcinoma (OSCC) and to investigate its underlying mechanisms of action. METHODS: Cell proliferation, cell cycle distribution and apoptosis were examined using cell counting kit-8 (CCK8) and flow cytometry (FCM). The expression of PTEN and PDCD4 were determined by western blot. Changes in miR-21 levels were quantified using TaqMan stem-loop real-time PCR. After miR-21 was transiently transfected into Tca8113 cells using Lipofectamine®3000, cell proliferation, apoptosis and miR-21 and PDCD4 expression levels were measured. RESULTS: Qilan preparation inhibited Tca8113 cell growth in a dose- and time-dependent manner by inducing apoptosis and cell cycle arrest in S-phase, decreasing miR-21 levels and increasing PTEN and PDCD4 expression. MiR-21 overexpression reversed the Qilan preparation-induced suppression of cell proliferation and induction of apoptosis while also blocking the increase in PDCD4. CONCLUSIONS: Our study revealed, for the first time, the ability of Qilan preparation to suppress TSCC cell growth and elucidated that Qilan preparation elicits its anti-cancer actions either the miR-21/PDCD4 or PTEN pathway.

Optimizing component formula suppresses lung cancer by blocking DTL-mediated PDCD4 ubiquitination to regulate the MAPK/JNK pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza Bunge and Panax ginseng C. A. Meyer have special curative effect on cancer treatment. The optimizing component formula (OCF) extracted from those two herbs was in line with the anti-lung cancer treatment principle of activating blood and supplementing 'Qi'. However, the study on the mechanism of component formula has always been an insurmountable challenge. Nowadays, the application of network pharmacology and artificial intelligence (AI) in the field of TCM provides new ideas for the study of new targets and mechanisms of TCM, which promotes the modernization of TCM. AIM OF THE STUDY: This study aims to further explore the anti-lung cancer mechanism of OCF by using an integrated strategy of network pharmacology and AI technology. MATERIALS AND METHODS: Bioinformatic analysis was used to analyze the expression levels, prognosis and survival of DTL and PDCD4 in cancer patients. The binding strength of OCF and DTL was simulated by molecular docking, and the affinity between them was detected by Bio-layer interferometry. Network pharmacology was used to predict the active components, potential targets and pathways of OCF. The association between key targets and their corresponding components and DTL was analyzed by Ingenuity Pathway Analysis (IPA). MTT assay, colony formation assay, wound-healing assay and transwell assay were used to verify the inhibitory effects of OCF on lung cancer cells in vitro. qRT-PCR and Western blot assay were used to detect the effects of OCF on mRNA and protein expression of DTL, PDCD4 and key genes in MAPK/JNK pathways. RESULTS: Bioinformatics analysis showed that DTL was significantly up-regulated in lung cancer, which was associated with high malignancy rate, high metastasis rate and poor prognosis of primary tumor. PDCD4 was down-regulated in lung cancer, and associated with high metastasis rate and poor prognosis. The good affinity between OCF and DTL was predicted and verified by molecular docking and Bio-layer interferometry. Based on the network pharmacological databases, 40 active components and 220 corresponding targets of OCF were screened out. KEGG analysis showed that OCF component targets were mainly enriched in MAPK signaling pathway. IPA results showed the interrelationship between DTL, PDCD4, MAPK pathway genes and their corresponding OCF components. In addition, in vitro experiments demonstrated anti-lung cancer activity of OCF, as validated, via impairing cell viability and cell proliferation, as well as inhibiting migration and invasion abilities in lung cancer cells. qRT-PCR showed that OCF down-regulated the mRNA expression of DTL, MAP4K1, JNK, c-Jun and c-Myc, and up-regulated the mRNA expression of PDCD4 and P53 genes in A549 lung cancer cells. Western blot suggested that OCF suppressed the protein level of DTL and blocked the ubiquitination of PDCD4 in A549 lung cancer cells, and down-regulated the protein levels of MAP4K1, p-JNK and p-c-Jun while up-regulated the proteins expression level of P53. CONCLUSIONS: OCF might elicit an anti-lung cancer effect by blocking DTL-mediated PDCD4 ubiquitination and suppression of the MAPK/JNK pathway. Meanwhile, our work revealed that network pharmacology and AI technology strategy are cogent means of studying the active components and mechanism of TCM.

Isoliquiritigenin inhibits non-small cell lung cancer progression via m6A/IGF2BP3-dependent TWIST1 mRNA stabilization.

BACKGROUND: N6-methyladenosine (m6A) has been identified to regulate the tumorigenesis and development of various tumors, including non-small cell lung cancer (NSCLC). Isoliquiritigenin (ISL), derived from the Chinese herb licorice, shows a significant anti-tumor activity on multiple human cancers. However, the role of ISL on NSCLC through m6A is still unclear. PURPOSE: Here, we investigated the anti-tumor effect of ISL on NSCLC, and explored whether ISL affected the NSCLC phenotype by modulating its m6A modification. METHODS: Cell proliferation, migration and invasion assays were performed to evaluate the inhibitory effects of ISL on NSCLC cells. M6A enrichment was determined by m6A quantitative analysis. The mechanism regarding IGF2BP3 was explored using RIP-PCR, MeRIP-qPCR and RNA decay analysis. RESULTS: ISL significantly repressed the proliferation, migration and invasion of NSCLC cells in vitro. In addition, m6A reader IGF2BP3 expression significantly increased in NSCLC tissues compared to adjacent tissues, and was positively correlated with NSCLC patients' poor survival. Mechanistically, ISL reduced m6A modification and down-regulated IGF2BP3 expression in NSCLC. Furthermore, IGF2BP3 enhanced the mRNA stability of twist family bHLH transcription factor 1 (TWIST1) in m6A-dependent manner. Moreover, ISL treatment combined with TWSIT1 knockdown effectively reversed IGF2BP3 overexpression-induced NSCLC cells' proliferation, migration and invasion. CONCLUSION: Our findings uncover that ISL might function as an anticarcinogen through targeting IGF2BP3/m6A/TWIST1 axis for NSCLC.

The selenoprotein P 3' untranslated region is an RNA binding protein platform that fine tunes selenocysteine incorporation.

Selenoproteins contain the 21st amino acid, selenocysteine (Sec), which is incorporated at select UGA codons when a specialized hairpin sequence, the Sec insertion sequence (SECIS) element, is present in the 3' UTR. Aside from the SECIS, selenoprotein mRNA 3' UTRs are not conserved between different selenoproteins within a species. In contrast, the 3'-UTR of a given selenoprotein is often conserved across species, which supports the hypothesis that cis-acting elements in the 3'-UTR other than the SECIS exert post-transcriptional control on selenoprotein expression. In order to determine the function of one such SECIS context, we chose to focus on the plasma selenoprotein, SELENOP, which is required to maintain selenium homeostasis as a selenium transport protein that contains 10 Sec residues. It is unique in that its mRNA contains two SECIS elements in the context of a highly conserved 843-nucleotide 3' UTR. Here we have used RNA affinity chromatography and identified PTBP1 as the major RNA binding protein that specifically interacts with the sequence between the two SECIS elements. We then used CRISPR/Cas9 genome editing to delete two regions surrounding the first SECIS element. We found that these sequences are involved in regulating SELENOP mRNA and protein levels, which are inversely altered as a function of selenium concentrations.

Discovery of dearomatized isoprenylated acylphloroglucinols with colon tumor suppressive activities in mice via inhibiting NFκB-FAT1-PDCD4 signaling activation.

Dearomatized isoprenylated acylphloroglucinols (DIAPs) are specific natural products mainly distributed in the plants of genus Hypericum. In this study, guided by HPLC-UV screening, 46 DIAPs (approximately 70% of all DIAPs) including 20 new ones and an unprecedented architecture, were discovered from the roots of Hypericum henryi, which were elucidated by comprehensive spectroscopic, X-ray crystallography, and ECD methods. Compounds 1-7, 39, and 41-42 exhibited remarkable cytotoxicities (IC50 = 0.84-5.63 µM) in human colon cancer HCT116 cells, in which 2 and 6 possessed selective cytotoxicities towards colon cancer cells. The preliminary structure-activity relationships of these tested compounds were discussed. In addition, mechanistic investigations demonstrated that 2 and 6 could significantly suppress the expressions of NFκB, FAT1, and promoted novel tumor suppressor gene PDCD4 in HCT116 cells. Furthermore, in HCT116 colon xenograft-bearing mouse model, treatments with 2 and 6 reduced the growth of xenograft tumors in dose-dependent manner. Expressions of FAT1 in tumors were also decreased in mice treated with 2 and 6, suggesting their anti-tumor effects were via FAT1 signaling pathway. In conclusion, this is the first report on the mechanistic and in vivo studies of DIAP, indicating that these metabolites can be considered as a new type of anti-colon cancer lead agents for further drug development.

Carnosol, a diterpene present in rosemary, increases ELP1 levels in familial dysautonomia patient-derived cells and healthy adults: a possible therapy for FD.

Recent research on familial dysautonomia (FD) has focused on the development of therapeutics that facilitate the production of the correctly spliced, exon 20-containing, transcript in cells and individuals bearing the splice-altering, FD-causing mutation in the elongator acetyltransferase complex subunit I (ELP1) gene. We report here the ability of carnosol, a diterpene present in plant species of the Lamiaceae family, including rosemary, to enhance the cellular presence of the correctly spliced ELP1 transcript in FD patient-derived fibroblasts by upregulating transcription of the ELP1 gene and correcting the aberrant splicing of the ELP1 transcript. Carnosol treatment also elevates the level of the RNA binding motif protein 24 (RBM24) and RNA binding motif protein 38 (RBM38) proteins, two multifunctional RNA-binding proteins. Transfection-mediated expression of either of these RNA binding motif (RBMs) facilitates the inclusion of exon 20 sequence into the transcript generated from a minigene-bearing ELP1 genomic sequence containing the FD-causing mutation. Suppression of the carnosol-mediated induction of either of these RBMs, using targeting siRNAs, limited the carnosol-mediated inclusion of the ELP1 exon 20 sequence. Carnosol treatment of FD patient peripheral blood mononuclear cells facilitates the inclusion of exon 20 into the ELP1 transcript. The increased levels of the ELP1 and RBM38 transcripts and the alternative splicing of the sirtuin 2 (SIRT2) transcript, a sentinel for exon 20 inclusion in the FD-derived ELP1 transcript, are observed in RNA isolated from whole blood of healthy adults following the ingestion of carnosol-containing rosemary extract. These findings and the excellent safety profile of rosemary together justify an expedited clinical study of the impact of carnosol on the FD patient population.

Zinc supplementation prevents arsenic-induced dysregulation of ZRANB2 splice function.

Environmentally relevant (100 nM) inorganic arsenic (iAs) exposure displaces zinc from zinc fingers of upstream splice regulator ZRANB2 disrupting the splicing of its target TRA2B. Excess zinc displaced iAs from ZRANB2 zinc fingers in cell free system. Thus, the hypothesis that zinc supplementation could prevent iAs-mediated disruption of ZRANB2 splice function in human keratinocytes was tested. The data show that zinc supplementation prevented iAs-induced dysregulation of TRA2B splicing by ZRANB2 as well as the induction of ZRANB2 protein expression. These results provide additional support for the hypothesis that zinc supplementation could prevent iAs-mediated disease in iAs-exposed populations.