PEI, a new transfection method, augments the inhibitory effect of RBM5 on prostate cancer.

PEI is a cationic polymer, serving as a non-viral transfection carrier grounded in nanotechnology that enhances transfection efficiency via the proton sponge effect. RBM5 is an RNA-binding protein that can inhibit tumor development. This study involved the transfection of RBM5 in prostate cancer cells with PEI, Lipo2000, and their combination. Transwell and wound healing assays were used to observe invasion and migration of prostate cancer cells and flow cytometry was used to observe the apoptosis. Detect the expression of invasion and migration-related protein MMP9 through western blotting experiment. An activity detection kit was used to detect the activity of apoptotic protein caspase-3. We found that there was no significant difference in transfection efficiency when PEI and Lipo2000 were used alone but it significantly improved when they are combined. RBM5 reduced invasion, migration, and proliferation of prostate cancer and enhanced apoptosis. MMP9 expression was reduced, and the activity of caspase-3 was increased. PEI transfection could improve the inhibition of RBM5 on tumors more than Lipo2000. The inhibitory effect is more obvious when the two are used together. RBM5 transfected with PEI can amplify its inhibitory effect on prostate cancer, and this effect is more evident when combined with Lipo2000.

Dual Role of IGF2BP2 in Osteoimmunomodulation during Periodontitis.

Periodontitis is a complex disease characterized by distinct inflammatory stages, with a peak of inflammation in the early phase and less prominent inflammation in the advanced phase. The insulin-like growth factor 2-binding proteins 2 (IGF2BP2) has recently been identified as a new m6A reader that protects m6A-modified messenger RNAs (mRNAs) from decay, thus participating in multiple biological processes. However, its role in periodontitis remains unexplored. Here, we investigated the role of IGF2BP2 in inflammation and osteoclast differentiation using a ligature-induced periodontitis model. Our findings revealed that IGF2BP2 responded to bacterial-induced inflammatory stimuli and exhibited differential expression patterns in early and advanced periodontitis stages, suggesting its dual role in regulating this disease. Depletion of Igf2bp2 contributed to increased release of inflammatory cytokines, thereby exacerbating periodontitis after 3 d of ligature while suppressing osteoclast differentiation and ameliorating periodontitis after 14 d of ligature. Mechanistically, we demonstrated that IGF2BP2 directly interacted with Cd5l and Cd36 mRNA via RNA immunoprecipitation assay. Overexpression of CD36 or recombinant CD5L rescued the osteoclast differentiation ability of Igf2bp2-null cells upon lipopolysaccharide stimulus, and thus the downregulation of Cd36 and Cd5l effectively reversed periodontitis in the advanced stage. Altogether, this study deepens our understanding of the potential mechanistic link among the dysregulated m6A reader IGF2BP2, immunomodulation, and osteoclastogenesis during different stages of periodontitis.

Down-regulation of Musashi-2 exerts antileukemic effects on acute lymphoblastic leukemia cells and increases sensitivity to dexamethasone.

Musashi-2 (MSI2), implicated in the oncogenesis and propagation of a broad array of malignancies, inclusive of certain leukemia, remains a nascent field of study within the context of acute lymphoblastic leukemia (ALL). Using lentiviral transfection, ALL cells with stable MSI2 knockdown were engineered. A suite of analytic techniques - a CCK-8 assay, flow cytometry, qRT-PCR, and western blotting - were employed to evaluate cellular proliferation, cell cycle arrest, and apoptosis and to confirm differential gene expression. The suppression of MSI2 expression yielded significant results: inhibition of cell proliferation, G0/G1 cell cycle arrest, and induced apoptosis in ALL cell lines. Furthermore, it was noted that MSI2 inhibition heightened the responsiveness of ALL cells to dexamethasone. Significantly, the depletion of MSI2 prompted the translocation of GR from the cytoplasm to the nucleus upon dexamethasone treatment, consequently leading to enhanced sensitivity. Additionally, the FOXO1/4 signaling pathway contributed to the biological effects of ALL cells evoked by MSI2 silencing. Our study offers novel insight into the inhibitory effects of MSI2 suppression on ALL cells, positing MSI2 as a promising therapeutic target in the treatment of ALL.

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.

MEX3A promotes angiogenesis in colorectal cancer via glycolysis.

As a gastrointestinal malignancy, colorectal cancer (CRC) is a main cause of cancer-related deaths worldwide. Mex-3 RNA-binding family member A (MEX3A) is upregulated in multiple types of tumors and plays a critical role in tumor proliferation and metastasis. However, the function of MEX3A in CRC angiogenesis has not been fully understood. Hence, the aim of this study was to explore the role of MEX3A in CRC angiogenesis and investigate its underlying mechanisms. MEX3A expression in CRC was first investigated by bioinformatics means and then measured by qRT-PCR and Western blot. CCK-8 assay was employed to test cell viability. Angiogenesis assay was used to assess angiogenesis. The protein levels of VEGF, FGF and SDF-1 were evaluated using Western blot. The expression levels of MYC, HK2 and PGK1 were investigated by qRT-PCR. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were determined by Seahorse XP 96. The levels of pyruvate, lactate, citric acid and malate were measured by corresponding kits. Bioinformatics analysis demonstrated high MEX3A expression in CRC tissues and MEX3A enrichment in glycolysis and angiogenesis pathways. Cell assays showed high MEX3A expression in CRC cells and its promoting effects in CRC cell proliferation and glycolysis as well as angiogenesis. Rescue experiment confirmed that glycolysis inhibitor 2-DG could offset the promoting effects of MEX3A on the proliferation, angiogenesis and glycolysis of CRC cells. In conclusion, MEX3A could facilitate CRC angiogenesis by activating the glycolytic pathway, suggesting that MEX3A may be a novel therapeutic target for CRC.

BPDE exposure promotes trophoblast cell pyroptosis and induces miscarriage by up-regulating lnc-HZ14/ZBP1/NLRP3 axis.

Environmental Benzo(a)pyrene (BaP) and its ultimate metabolite BPDE (benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide) are typical persistent organic pollutants and endocrine disrupting chemicals. BaP/BPDE exposure might cause human trophoblast cell dysfunctions and induce miscarriage. However, the underlying mechanisms remain largely elusive. In this study, we found that BPDE exposure induced human trophoblast cell pyroptosis by up-regulating NLRP3/Caspase1/GSDMD pathway. We also identified that lnc-HZ14 was highly expressed in BPDE-exposed trophoblast cells and in recurrent miscarriage (RM) vs healthy control (HC) villous tissues. Lnc-HZ14 promoted trophoblast cell pyroptosis by promoting IRF1-mediated ZBP1 transcription, increasing METTL3-mediated m6A methylation on NLRP3 mRNA and its stability, and also enhancing ZBP1/NLRP3 protein interactions. Knockdown of lnc-HZ14/ZBP1/NLRP3 axis could efficiently alleviate BPDE-induced trophoblast cell pyroptosis. Higher level of pyroptosis, as indicated by the up-regulation of lnc-HZ14/ZBP1/NLRP3 axis, was found in RM vs HC villous tissues. In BaP-exposed mouse model, BaP exposure induced placental tissue pyroptosis and miscarriage by up-regulating murine Zbp1/Nlrp3 axis, and knockdown of Nlrp3 could efficiently reduce placenta pyroptosis and alleviate BaP-induced mouse miscarriage. Serum IL-1ß protein level might act as a promising indicator to predict the risk of miscarriage. These findings provided new insights into BaP/BPDE-induced trophoblast cell pyroptosis and miscarriage and might be helpful for further assessment of the toxicological effects of BaP/BPDE on the female reproduction.

The protective effect of H151, a novel STING inhibitor, in renal ischemia-reperfusion-induced acute kidney injury.

Renal ischemia-reperfusion (RIR)-induced acute kidney injury (AKI) is a common renal functional disorder with high morbidity and mortality. Stimulator of interferon (IFN) genes (STING) is the cytosolic DNA-activated signaling pathway that mediates inflammation and injury. Our recent study showed that extracellular cold-inducible RNA-binding protein (eCIRP), a newly identified damage-associated molecular pattern, activates STING and exacerbates hemorrhagic shock. H151 is a small molecule that selectively binds to STING and inhibits STING-mediated activity. We hypothesized that H151 attenuates eCIRP-induced STING activation in vitro and inhibits RIR-induced AKI in vivo. In vitro, renal tubular epithelial cells incubated with eCIRP showed increased levels of IFN-ß, STING pathway downstream cytokine, IL-6, tumor necrosis factor-α, and neutrophil gelatinase-associated lipocalin, whereas coincubation with eCIRP and H151 diminished those increases in a dose-dependent manner. In vivo, 24 h after bilateral renal ischemia-reperfusion, glomerular filtration rate was decreased in RIR-vehicle-treated mice, whereas glomerular filtration rate was unchanged in RIR-H151-treated mice. In contrast to sham, serum blood urea nitrogen, creatinine, and neutrophil gelatinase-associated lipocalin were increased in RIR-vehicle, but in RIR-H151, these levels were significantly decreased from RIR-vehicle. In contrast to sham, kidney IFN-ß mRNA, histological injury score, and TUNEL staining were also increased in RIR-vehicle, but in RIR-H151, these levels were significantly decreased from RIR-vehicle. Importantly, in contrast to sham, in a 10-day survival study, survival decreased to 25% in RIR-vehicle, but RIR-H151 had a survival of 63%. In conclusion, H151 inhibits eCIRP-induced STING activation in renal tubular epithelial cells. Therefore, STING inhibition by H151 can be a promising therapeutic intervention for RIR-induced AKI.NEW & NOTEWORTHY Renal ischemia-reperfusion (RIR)-induced acute kidney injury (AKI) is a common renal functional disorder with a high morbidity and mortality rate. Stimulator of interferon genes (STING) is the cytosolic DNA-activated signaling pathway responsible for mediating inflammation and injury. Extracellular cold-inducible RNA-binding protein (eCIRP) activates STING and exacerbates hemorrhagic shock. H151, a novel STING inhibitor, attenuated eCIRP-induced STING activation in vitro and inhibited RIR-induced AKI. H151 shows promise as a therapeutic intervention for RIR-induced AKI.

Variations in the motility and biofilm formation abilities of Escherichia coli O157:H7 during noodle processing.

Motility and biofilm formation help to protect bacteria from host immune responses and facilitate tolerance of environmental stimuli to improve their adaptability. However, few reports have investigated the adaptability of bacteria that live in food substrates undergoing food processing-induced stress. In this study, variations in the surface morphology, bacterial count, motility, and biofilm formation abilities of Escherichia coli O157:H7 NCTC12900 were investigated during noodle processing, including the kneading, squeezing, resting, and sheeting phases. The results showed that bacterial surface morphology, count, and motility were impaired in the squeezing phase, whereas biofilm biomass continuously increased across all processing phases. Twenty-one genes and sRNAs were measured using RT-qPCR to reveal the mechanisms underlying these changes. Of these, the genes adrA, csrA, flgM, flhD, fliM, ydaM, and the sRNA McaS were significantly upregulated, whereas the genes fliA, fliG, and the sRNAs CsrC, DsrA, GcvB, and OxyS were evidently repressed. According to the correlation matrix results based on the reference gene adrA, we found that csrA, GcvB, McaS, and OxyS were the most relevant genes and sRNAs for biofilm formation and motility. For each of them, their overexpressions was found to inhibit bacterial motility and biofilm formation to varying degrees during noodle processing. Among these, 12900/pcsrA had the highest inhibitory potential against motility, yielding a minimum of 11.2 mm motility diameter in the resting phase. Furthermore, 12900/pOxyS showed the most significant inhibitory effect against biofilm formation, yielding a minimum biofilm formation value of 5% of that exhibited the wild strain in the sheeting phase. Therefore, we prospect to find an effective and feasible novel approach to weaken bacterial survival during food processing by regulating the genes or sRNAs related to motility and biofilm formation.

FTO-Nrf2 axis regulates bisphenol F-induced leydig cell toxicity in an m6A-YTHDF2-dependent manner.

Studies have shown that Bisphenol F (BPF) as an emerging bisphenol pollutant also has caused many hazards to the reproductive systems of humans and animals. However, its specific mechanism is still unclear. The mouse TM3 Leydig cell was used to explore the mechanism of BPF-induced reproductive toxicity in this study. The results showed BPF (0, 20, 40 and 80 µM) exposure for 72 h significantly increased cell apoptosis and decreased cell viability. Correspondingly, BPF increased the expression of P53 and BAX, and decreased the expression of BCL2. Moreover, BPF significantly increased the intracellular ROS level in TM3 cells, and significantly decreased oxidative stress-related molecule Nrf2. BPF decreased the expression of FTO and YTHDF2, and increased the total cellular m6A level. ChIP results showed that AhR transcriptionally regulated FTO. Differential expression of FTO revealed that FTO reduced the apoptosis rate of BPF-exposed TM3 cells and increased the expression of Nrf2, MeRIP confirmed that overexpression of FTO reduced the m6A of Nrf2 mRNA. After differential expression of YTHDF2, it was found that YTHDF2 enhanced the stability of Nrf2, and RIP assay showed that YTHDF2 was bound to Nrf2 mRNA. Nrf2 agonist enhanced the protective effect of FTO on TM3 cells exposure to BPF. Our study is the first to demonstrate that AhR transcriptionally regulated FTO, and then FTO regulated Nrf2 in a m6A-modified manner through YTHDF2, thereby affecting apoptosis in BPF-exposed TM3 cells to induce reproductive damage. It provides new insights into the importance of FTO-YTHDF2-Nrf2 signaling axis in BPF-induced reproductive toxicity and provided a new idea for the prevention of male reproductive injury.

LncRNA NIPA1-SO confers atherosclerotic protection by suppressing the transmembrane protein NIPA1.

Long non-coding RNAs (lncRNAs) are emerging as important players in gene regulation and cardiovascular diseases. However, the roles of lncRNAs in atherosclerosis are poorly understood. In the present study, we found that the levels of NIPA1-SO were decreased while those of NIPA1 were increased in human atherosclerotic plaques. Furthermore, NIPA1-SO negatively regulated NIPA1 expression in human umbilical vein endothelial cells (HUVECs). Mechanistically, NIPA1-SO interacted with the transcription factor FUBP1 and the NIPA1 gene. The effect of NIPA1-SO on NIPA1 protein levels was reversed by the knockdown of FUBP1. NIPA1-SO overexpression increased, whilst NIPA1-SO knockdown decreased BMPR2 levels; these effects were enhanced by the knockdown of NIPA1. The overexpression of NIPA1-SO reduced while NIPA1-SO knockdown increased monocyte adhesion to HUVECs; these effects were diminished by the knockdown of BMPR2. The lentivirus-mediated-overexpression of NIPA1-SO or gene-targeted knockout of NIPA1 in low-density lipoprotein receptor-deficient mice reduced monocyte-endothelium adhesion and atherosclerotic lesion formation. Collectively, these findings revealed a novel anti-atherosclerotic role for the lncRNA NIPA1-SO and highlighted its inhibitory effects on vascular inflammation and intracellular cholesterol accumulation by binding to FUBP1 and consequently repressing NIPA1 expression.

RNA binding protein YTHDF1 mediates bisphenol S-induced Leydig cell damage by regulating the mitochondrial pathway of BCL2 and the expression of CDK2-CyclinE1.

Bisphenol S (BPS) causes reproductive adverse effects on humans and animals. However, the detailed mechanism is still unclear. This research aimed to clarify the role of RNA binding protein YTHDF1 in Leydig cell damage induced by BPS. The mouse TM3 Leydig cells were exposed to BPS of 0, 20, 40, and 80 µmol/L for 72 h. Results showed that TM3 Leydig cells apoptosis rate markedly increased in BPS exposure group. Meanwhile, the apoptosis-related molecule BCL2 protein level decreased significantly, and Caspase9, Caspase3, and BAX increased significantly. Moreover, the cell cycle was blocked in the G1/S phase, CDK2 and CyclinE1 were considerably down-regulated in BPS exposure groups, and the protein level of RNA binding protein YTHDF1 decreased sharply. Furthermore, after overexpression of YTHDF1, the cell viability significantly increased, and the apoptosis rate significantly decreased in TM3 Leydig cells. In the meantime, BCL2, CDK2, and CyclinE1 were significantly up-regulated, and BAX, Caspase9, and Caspase3 were significantly down-regulated. Conversely, interference with YTHDF1 decreased cell proliferation and promoted apoptosis. Importantly, overexpression of YTHDF1 alleviated the cell viability decrease induced by BPS, and interference with YTHDF1 exacerbated the situation. RIP assays showed that the binding of YTHDF1 to CDK2, CyclinE1, and BCL2 significantly increased after overexpressing YTHDF1. Collectively, our study suggested that YTHDF1 plays an essential role in BPS-induced TM3 Leydig cell damage by regulating CDK2-CyclinE1 and BCL2 mitochondrial pathway at the translational level.

Mechanism of METTL3-Mediated m6A Modification in Cardiomyocyte Pyroptosis and Myocardial Ischemia-Reperfusion Injury.

OBJECTIVE: Myocardial ischemia/reperfusion (MI/R) injury is a complicated pathophysiological process associated with cardiomyocyte pyroptosis. Methyltransferase-like protein 3 (METTL3) catalyzes the formation of N6-methyl-adenosine (m6A) and participates in various biological processes. This study probed into the mechanism of METTL3 in cardiomyocyte pyroptosis in MI/R injury. METHODS: A rat model of MI/R was established. Rat cardiomyocytes were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) treatment for the establishment of a cell model in vitro. METTL3 expression in myocardial tissues of MI/R rats and OGD/R-treated cardiomyocytes was determined using RT-qPCR and Western blot. The pathological changes of rat myocardial tissues were observed using hematoxylin and eosin staining. The positive expression of NLRP3 in myocardial tissues or cardiomyocytes was observed through immunohistochemistry or immunofluorescence. The activity of caspase-1 was measured using the colorimetric method. The expressions of GSDMD and cleaved caspase-1, as well as the levels of IL-1ß and IL-18 in rat myocardial tissues or cardiomyocytes were determined. m6A modification level was quantified. The binding relationship between pri-miR-143-3p and DGCR8 and the enrichment of m6A on pri-miR-143-3p were detected. The binding relationship between miR-143-3p and protein kinase C epsilon (PRKCE) was verified. RESULTS: METTL3 expression was elevated in MI/R rats and OGD/R cardiomyocytes. METTL3 silencing alleviated myocardial injury, reduced the number of NLRP3-positive cardiomyocytes, suppressed caspase-1 activity, decreased the protein levels of GSDMD-N and cleaved caspase-1, and decreased IL-1ß and IL-18 levels. METTL3 increased the total m6A level in MI/R rats and injured cardiomyocytes, promoted DGCR8 binding to pri-miR-143-3p, and enhanced miR-143-3p expression. miR-143-3p suppressed PRKCE transcription, and miR-143-3p overexpression reversed the inhibitory effect of METTL3 silencing on cardiomyocyte pyroptosis. CONCLUSION: METTL3 promoted DGCR8 binding to pri-miR-143-3p through m6A modification, thus enhancing miR-143-3p expression to inhibit PRKCE transcription and further aggravating cardiomyocyte pyroptosis and MI/R injury.

Hypoxia and Wnt signaling inversely regulate expression of chondroprotective molecule ANP32A in articular cartilage.

OBJECTIVES: ANP32A is a key protector of cartilage health, via preventing oxidative stress and Wnt hyper-activation. We aimed to unravel how ANP32A is regulated in cartilage. METHODS: A bioinformatics pipeline was applied to identify regulators of ANP32A. Pathways of interest were targeted to study their impact on ANP32A in in vitro cultures of the human chondrocyte C28/I2 cell-line and primary human articular chondrocytes (hACs) from up to five different donors, using Wnt-activator CHIR99021, hypoxia-mimetic IOX2 and a hypoxia chamber. ANP32A was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. In vivo, the effect of hypoxia was examined by immunohistochemistry in mice injected intra-articularly with IOX2 after destabilization of the medial meniscus. Effects of Wnt hyper-activation were investigated using Frzb-knockout mice and wild-type mice treated intra-articularly with CHIR99021. Wnt inhibition effects were assessed upon intra-articular injection of XAV939. RESULTS: The hypoxia and Wnt signaling pathways were identified as networks controlling ANP32A expression. In vitro and in vivo experiments demonstrated increases in ANP32A upon hypoxic conditions (1.3-fold in hypoxia in C28/I2 cells with 95% confidence interval (CI) [1.11-1.54] and 1.90-fold in hACs [95% CI: 1.56-2] and 1.67-fold in ANP32A protein levels after DMM surgery with IOX2 injections [95% CI: 1.33-2.08]). Wnt hyper-activation decreased ANP32A in chondrocytes in vitro (1.23-fold decrease [95% CI: 1.02-1.49]) and in mice (1.45-fold decrease after CHIR99021 injection [95% CI: 1.22-1.72] and 1.41-fold decrease in Frzb-knockout mice [95% CI: 1.00-1.96]). Hypoxia and Wnt modulated ataxia-telangiectasia mutated serine/threonine kinase (ATM), an ANP32A target gene, in hACs (1.89-fold increase [95% CI: 1.38-2.60] and 1.41-fold decrease [95% CI: 1.02-1.96]). CONCLUSIONS: Maintaining hypoxia and limiting Wnt activation sustain ANP32A and protect against osteoarthritis.

A Totipotent "All-In-One" Peptide Sequentially Blocks Immune Checkpoint and Reverses the Immunosuppressive Tumor Microenvironment.

Immune checkpoint blockade combined with reversal of the immunosuppressive tumor microenvironment (TME) can dramatically enhance anti-tumor immunity, which can be achieved by using multiple-agent therapy. However, the optimal dose and order of administration of different agents remain elusive. To address this dilemma, multiple agents are often grafted together to construct "all-in-one" totipotent drugs, but this usually comes at the cost of a lack of synergy between the agents. Herein, by comprehensively analyzing the conserved sites of the immune checkpoint and TME drug targets, peptide secondary structures, assembly properties, and other physicochemical properties, a high-content peptide library is designed. By using the "3D-molecular-evolution" screening strategy, an efficient and totipotent "all-in-one" peptide (TAP) is obtained, which possesses the abilities of self-assembling, blocking the PD-1/PD-L1 axis, inhibiting Rbm38-eIF4E complex formation, and activating p53. It is shown that in mice treated with TAP, with either subcutaneous tumors or patient-derived xenografts, PD-L1 is blocked, with increased activation of both T and NK cells whilst reversing the immunosuppressive TME. Moreover, TAP can mitigate tumor activity and suppress tumor growth, showing superior therapeutic effect over antibody-based drugs.

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.

miR-150 promotes progressive T cell differentiation via inhibiting FOXP1 and RC3H1.

T cells used in immune cell therapy, represented by T cell receptor therapy (TCR-T), are usually activated and proliferated in vitro and are induced to a terminally differentiated phenotype, with limited viability after transfusion back into the body. T cells exhibited a robust proliferative potential and in vivo viability in the early stages of progressive differentiation. In this study, we identified microRNAs that regulate T cell differentiation. After microRNA sequencing of the four subsets: Naïve T cells (TN), stem cell-like memory T cells (TSCM), central memory T cells (TCM）, and effector memory T cells (TEM), miR-150 was identified as the most highly expressed miRNA among the four subsets and was lowly expressed in the TSCM cells. We predicted the target genes of miR-150 miRNA and performed Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes analyses. We observed that the target genes of miR-150 were enriched in pathways associated with T-cell differentiation. FOXP1 and RC3H1 were identified as key target genes of miR-150 in the regulation of T-cell function. We examined the effects of miR-150 on the differentiation and function of healthy donor T-cells. We observed that miR-150 overexpression promoted T-cell differentiation to effector T-cells and effector memory T-cells, enhanced apoptosis, inhibited cell proliferation and increased secretion of pro-inflammatory cytokines such as IFN-γ and TNF-α. In addition, the expressions of early differentiation-related genes (ACTN1, CERS6, BCL2, and EOMES), advanced differentiation-related genes (KLRG1), and effector-function-related genes (PRF1 and GZMB) were significantly decreased after overexpression of miR-150. Collectively, our results suggested that miR-150 can promote progressive differentiation of T cells and the downmodulation of miR-150 expression while performing adoptive immunotherapy may inhibit T-cell differentiation and increase the proliferative potential of T cells.

Targeted inhibition of osteoclastogenesis reveals the pathogenesis and therapeutics of bone loss under sympathetic neurostress.

Sympathetic cues via the adrenergic signaling critically regulate bone homeostasis and contribute to neurostress-induced bone loss, but the mechanisms and therapeutics remain incompletely elucidated. Here, we reveal an osteoclastogenesis-centered functionally important osteopenic pathogenesis under sympatho-adrenergic activation with characterized microRNA response and efficient therapeutics. We discovered that osteoclastic miR-21 was tightly regulated by sympatho-adrenergic cues downstream the ß2-adrenergic receptor (ß2AR) signaling, critically modulated osteoclastogenesis in vivo by inhibiting programmed cell death 4 (Pdcd4), and mediated detrimental effects of both isoproterenol (ISO) and chronic variable stress (CVS) on bone. Intriguingly, without affecting osteoblastic bone formation, bone protection against ISO and CVS was sufficiently achieved by a (D-Asp8)-lipid nanoparticle-mediated targeted inhibition of osteoclastic miR-21 or by clinically relevant drugs to suppress osteoclastogenesis. Collectively, these results unravel a previously underdetermined molecular and functional paradigm that osteoclastogenesis crucially contributes to sympatho-adrenergic regulation of bone and establish multiple targeted therapeutic strategies to counteract osteopenias under stresses.

RNA-binding protein ELAVL4/HuD ameliorates Alzheimer's disease-related molecular changes in human iPSC-derived neurons.

The RNA binding protein ELAVL4/HuD regulates the translation and splicing of multiple Alzheimer's disease (AD) candidate genes. We generated ELAVL4 knockout (KO) human induced pluripotent stem cell-derived neurons to study the effect that ELAVL4 has on AD-related cellular phenotypes. ELAVL4 KO significantly increased the levels of specific APP isoforms and intracellular phosphorylated tau, molecular changes that are related to the pathological hallmarks of AD. Overexpression of ELAVL4 in wild-type neurons and rescue experiments in ELAVL4 KO cells showed opposite effects and also led to a reduction of the extracellular amyloid-beta (Aß)42/40 ratio. All these observations were made in familial AD (fAD) and fAD-corrected neurons. To gain insight into the molecular cascades involved in neuronal ELAVL4 signaling, we conducted pathway and upstream regulator analyses of transcriptomic and proteomic data from the generated neurons. These analyses revealed that ELAVL4 affects multiple biological pathways linked to AD, including those involved in synaptic function, as well as gene expression downstream of APP and tau signaling. The analyses also suggest that ELAVL4 expression is regulated by insulin receptor-FOXO1 signaling in neurons. Taken together, ELAVL4 expression ameliorates AD-related molecular changes in neurons and affects multiple synaptic pathways, making it a promising target for novel drug development.

Time-resolved proteome and transcriptome of paraquat-induced pulmonary fibrosis.

BACKGROUNDS: Pulmonary fibrosis (PF) is a pathological state presenting at the progressive stage of heterogeneous interstitial lung disease (ILD). The current understanding of the molecular mechanisms involved is incomplete. This clinical toxicology study focused on the pulmonary fibrosis induced by paraquat (PQ), a widely-used herbicide. Using proteo-transcriptome analysis, we identified differentially expressed proteins (DEPs) derived from the initial development of fibrosis to the dissolved stage and provided further functional analysis. METHODS: We established a mouse model of progressive lung fibrosis via intratracheal instillation of paraquat. To acquire a comprehensive and unbiased understanding of the onset of pulmonary fibrosis, we performed time-series proteomics profiling (iTRAQ) and RNA sequencing (RNA-Seq) on lung samples from paraquat-treated mice and saline control. The biological functions and pathways involved were evaluated through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analysis. Correlation tests were conducted on comparable groups 7 days and 28 days post-exposure. Differentially expressed proteins and genes following the same trend on the protein and mRNA levels were selected for validation. The functions of the selected molecules were identified in vitro. The protein level was overexpressed by transfecting gene-containing plasmid or suppressed by transfecting specific siRNA in A549 cells. The levels of endothlial-mesenchymal transition (EMT) markers, including E-cadherin, vimentin, FN1, and α-SMA, were determined via western blot to evaluate the fibrotic process. RESULTS: We quantified 1358 DEPs on day 7 and 426 DEPs on day 28 post exposure (Fold change >1.2; Q value < 0.05). The top 5 pathways - drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, complement and coagulation cascades, chemical carcinogenesis, protein digestion and absorption - were involved on both day 7 and day 28. Several pathways, including tight junction, focal adhesion, platelet activation, and ECM-receptor interaction, were more enriched on day 28 than on day 7. Integrative analysis of the proteome and transcriptome revealed a moderate correlation of quantitative protein abundance ratios with RNA abundance ratios (Spearman R = 0.3950 and 0.2477 on days 7 and 28, respectively), indicating that post-transcriptional regulation plays an important role in lung injury and repair. Western blot identified that the protein expressions of FN1, S100A4, and RBM3 were significantly upregulated while that of CYP1A1, FMO3, and PGDH were significantly downregulated on day 7. All proteins generally recovered to baseline on day 28. qPCR showed the mRNA levels of Fn1, S100a4, Rbm3, Cyp1a1, Fmo3, and Hpgd changed following the same trend as the levels of their respective proteins. Further, in vitro experiments showed that RBM3 was upregulated while PGDH was downregulated in an EMT model established in human lung epithelial A549 cells. RBM3 overexpression and PGDH knockout could both induce EMT in A549 cells. RBM3 knockout or PGDH overexpression had no reverse effect on EMT in A549 cells. CONCLUSIONS: Our proteo-transcriptomic study determined the proteins responsible for fibrogenesis and uncovers their dynamic regulation from lung injury to repair, providing new insights for the development of biomarkers for diagnosis and treatment of fibrotic diseases.

Overexpression of lncRNA HCP5 in human umbilical cord mesenchymal stem cell-derived exosomes promoted the proliferation and inhibited the apoptosis of ovarian granulosa cells via the musashi RNA-binding protein 2/oestrogen receptor alpha 1 axis.

HCP5 has been reported to be downregulated in ovarian granulosa cells (OGCs) and to facilitate cell proliferation. Human umbilical cord mesenchymal stem cell exosome (hucMSCs-exo) treatment can prevent OGCs apoptosis in vitro. However, the functional mechanism of HCP5 and hucMSCs-exo requires further exploration. Fluorescence-activated cell sorting (FACS) was performed to measure the expression of markers related to hucMSCs. The osteogenic and adipogenic potential of hucMSCs was measured by alkaline phosphatase (ALP) and Alizarin red and by oil red-O staining, respectively. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to detect the mRNA and protein levels, respectively. Cell proliferation and apoptosis were measured by Cell Counting Kit-8 (CCK-8) assay, colony formation assay and flow cytometry. The interaction of HCP5/musashi RNA-binding protein 2 (MSI2) and oestrogen receptor alpha 1 (ESR1) mRNA was analysed using RNA pulldown and RIP assays. HucMSCs and exosomes were successfully isolated and identified. HucMSC-derived exosomes promoted the proliferation of OGCs and ESR1 expression and inhibited cell apoptosis. HCP5 overexpression in exosomes further enhanced these effects. MSI2 knockdown led to the opposite results. HCP5 targeted MSI2, and MSI2 knockdown reduced the decreases in HCP5 and ESR1 expression. Mechanistically, HCP5 in HucMSC-derived exosomes promoted ESR1 expression by binding to MSI2, which promoted the proliferation of OGCs.