Recombinant Slit2 attenuates tracheal fibroblast activation in benign central airway obstruction by inhibiting the TGF-ß1/Smad3 signaling pathway.

Airway fibrosis is among the pathological manifestations of benign central airway obstruction noted in the absence of effective treatments and requires new drug targets to be developed. Slit guidance ligand 2-roundabout guidance receptor 1 (Slit2-Robo1) is involved in fibrosis and organ development. However, its significance in airway fibrosis has not yet been reported. The study explored how the recombinant protein Slit2 functions in transforming growth factor-ß1 (TGF-ß1)-mediated airway fibrosis in vivo and in vitro. In this study, Slit2 expression initially increased in the tracheal granulation tissues of patients with tracheobronchial stenosis but decreased in the fibrotic tissue. In primary rat tracheal fibroblasts (RTFs), recombinant Slit2 inhibited the expression of extracellular matrices such as Timp1, α-SMA, and COL1A2, whereas recombinant TGF-ß1 promoted the expression of Robo1, α-SMA, and COL1A2. Slit2 and TGF-ß1 played a mutual inhibitory role in RTFs. Slit2 supplementation and Robo1 downregulation inhibited excessive extracellular matrix (ECM) deposition induced by TGF-ß1 in RTFs via the TGF-ß1/Smad3 pathway. Ultimately, exogenous Slit2 and Robo1 knockdown-mediated attenuation of airway fibrosis were validated in a trauma-induced rat airway obstruction model. These findings demonstrate that recombinant Slit2 alleviated pathologic tracheobronchial healing by attenuating excessive ECM deposition. Slit2-Robo1 is an attractive target for further exploring the mechanisms and treatment of benign central airway obstruction.

GDF15 alleviates the progression of benign tracheobronchial stenosis by inhibiting epithelial-mesenchymal transition and inactivating fibroblasts.

Benign tracheobronchial stenosis (BTS) is a fatal and incurable disease. Epithelial repair and matrix reconstruction play an important role in the wound repair process. If the interstitial context is not restored and stabilized in time, it can lead to pathological fibrosis. Here we attempted to identify cytokines that are involved in promoting wound repair. Growth differentiation factor 15 (GDF15) is a cytokine secreted by tracheal epithelial cells, which is indispensable for the growth of epithelial cells and inhibits the overgrowth of fibroblasts. GDF15 can counteract transforming growth factor-ß (TGFß1) stimulation of epithelial-mesenchymal transition (EMT) in tracheal epithelial cells and inhibit fibroblast activation via the TGFß1-SMAD2/3 pathway. In a rat model of tracheal stenosis, GDF15 supplementation alleviated the degree of tracheal stenosis. These results suggest that GDF15 prevents fibroblast hyperactivation and promotes epithelial repair in injured trachea. GDF15 may be a potential therapy to improve benign tracheobronchial stenosis.

Trifluoperazine Synergistically Potentiates Bortezomib-Induced Anti-Cancer Effect in Multiple Myeloma via Inhibiting P38 MAPK/NUPR1.

Multiple myeloma (MM) is a common hematological malignancy. Bortezomib (BTZ) is a traditional medicine for MM treatment, but there are limitations for current treatment methods. Trifluoperazine (TFP) is a clinical drug for acute and chronic psychosis therapy. Lately, researchers have found that TFP can suppress tumor growth in many cancers. We attempted to study the effects of BTZ and TFP on MM in vivo and in vitro. We concentrated on the individual and combined impact of BTZ and TFP on the proliferation and apoptosis of MM cells via Cell Counting kit-8 assay, EdU assay, western blot, and flow cytometry. We found that combination therapy has a strong synergistic impact on MM cells. Combination therapy could induce cell arrest during G2/M phase and induce apoptosis in MM cells. Meanwhile, BTZ combined with TFP could play a better role in the anti-MM effect in vivo through MM.1s xenograft tumor models. Furthermore, we explored the mechanism of TFP-induced apoptosis in MM, and we noticed that TFP might induce MM apoptosis by inhibiting p-P38 MAPK/NUPR1. In summary, our findings suggest that TFP could synergistically enhance the BTZ-induced anti-cancer effect in multiple myeloma, which might be a promising therapeutic strategy for MM treatment.

Altered amygdala-related structural covariance and resting-state functional connectivity in end-stage renal disease patients.

Depression and cognitive control deficits were frequently reported in concurrent end-stage renal disease (ESRD) patients. Neuroimaging studies indicated depression could be a risk factor for cognitive control deficits, and amygdala-related circuitry may play a critical role in this abnormal interaction. To investigate the potential relationship between depressive symptoms and cognitive control reduction in ESRD patients, T1-weighted and resting fMRI images were obtained in 29 ESRD patients and 29 healthy controls. Voxel-based morphometry (VBM), structural covariance (SC) analysis based on grey matter volume (GMV), and functional connectivity (FC) analysis were adopted. All subjects performed the Beck Depression Inventory (BDI) assessment and Stroop test. The patients also underwent blood biochemistry tests (urea, creatinine, phosphate, Ca2+, hematocrit, cystatin, hemoglobin). Compared with controls, GMV reductions were found mainly in the anterior cingulate cortex (ACC) and bilateral amygdala, and decreased SC was found between the amygdala and ACC in ESRD patients. This indicated that structural changes in the amygdala may be related to the GMV alterations in the ACC. Additionally, decreased FC between the amygdala and ACC was revealed in ESRD patients. Negative correlation was found between the FC of the amygdala-ACC and reaction delay during the Stroop test, but this correlation disappeared after controlling BDI. Stepwise regression analysis showed that the low level of hemoglobin was contributed to the reduced FC of the amygdala-ACC in ESRD patients. Our results demonstrated the abnormal interaction between depressive mood and cognitive control deficits in ESRD patients.

Mitochondrial dysfunction is associated with hypertrophic cardiomyopathy in Pompe disease-specific induced pluripotent stem cell-derived cardiomyocytes.

Pompe disease (PD) is a rare autosomal recessive disorder that presents with progressive hypertrophic cardiomyopathy. However, the detailed mechanism remains clarified. Herein, PD patient-specific induced pluripotent stem cells were differentiated into cardiomyocytes (PD-iCMs) that exhibited cardiomyopathic features of PD, including decreased acid alpha-glucosidase activity, lysosomal glycogen accumulation and hypertrophy. The defective mitochondria were involved in the cardiac pathology as shown by the significantly decreased number of mitochondria and impaired respiratory function and ATP production in PD-iCMs, which was partially due to elevated levels of intracellular reactive oxygen species produced from depolarized mitochondria. Further analysis showed that impaired fusion and autophagy of mitochondria and declined expression of mitochondrial complexes underlies the mechanism of dysfunctional mitochondria. This was alleviated by supplementation with recombinant human acid alpha-glucosidase that improved the mitochondrial function and concomitantly mitigated the cardiac pathology. Therefore, this study suggests that defective mitochondria underlie the pathogenesis of cardiomyopathy in patients with PD.

Gene Expression, Morphology, and Electrophysiology During the Dynamic Development of Human Induced Pluripotent Stem Cell-Derived Atrial- and Ventricular-Like Cardiomyocytes.

Background and Objectives: Gene expression, morphology, and electrophysiological combination are essential for assessing the dynamic development of human induced pluripotent stem cell-derived atrial- and ventricular-like cardiomyocytes (iPS-AM and iPS-VM, respectively). Methods: For iPS-AM/VM differentiation, we performed the small molecule-based temporal modulation of the retinoic acid and bone morphogenetic protein signaling pathways. We investigated the gene expression and morphology using immunofluorescence, quantitative real-time polymerase chain reaction, flow cytometry, and transmission electron microscopy as well as registered electrophysiological functions using a whole-cell patch clamp on days 20, 30, and 60 post-differentiations. Results: Pan-cardiomyocyte marker, including troponin T2 (TNNT2) and alpha-actinin-2 (ACTN2), expressions increased both in iPS-AMs and iPS-VMs. Similarly, the mRNA expression of both iPS-AM-specific markers, ie, natriuretic peptide A (NPPA), myosin light chain 7 (MYL7), and K+ channel Kir3.4 (KCNJ5), and iPS-VM-specific markers, ie, gap junction α-1 (GJA1), myosin light chain 2 (MYL2), and alpha-1-subunit of a voltage-dependent L-type calcium channel (CACNA1C), increased from 0 to 20 days, and then decreased from 30 to 60 days. Concerning morphology, cardiac troponin-T (cTnT) arrangement was progressively organized and developed from a disorderly myofibrillar distribution to an organized sarcomere pattern both in iPS-AMs and iPS-VMs. Mitochondrial numbers gradually increased and those of lipid droplets decreased during dynamic development. Regarding physiological function, the resting and action potential amplitudes remained statistically indifferent in both cell types, and the action potential duration was prolonged during the development. Conclusion: IPS-AMs/VMs displayed dynamic development concerning their gene expression, morphology, and electrophysiological function. The discoveries of this study could provide novel insights into heart development and encourage further research.

Icariin inhibits chondrocyte ferroptosis and alleviates osteoarthritis by enhancing the SLC7A11/GPX4 signaling.

BACKGROUND: Chondrocyte ferroptosis plays a critical role in the pathogenesis of osteoarthritis (OA), regulated by the SLC7A11/GPX4 signaling pathway. Icariin (ICA), a flavonoid glycoside, exhibits strong anti-inflammatory and antioxidant activities. This study investigated whether ICA could modulate the SLC7A11/GPX4 signaling to inhibit chondrocyte ferroptosis and alleviate OA. PURPOSE: The objective was to explore the impact of ICA on chondrocyte ferroptosis in OA and its modulation of the SLC7A11/GPX4 signaling pathway. METHODS: The anti-ferroptosis effects of ICA were evaluated in an interleukin-1ß (IL-1ß)-treated SW1353 cell model, using Ferrostatin-1 (Fer-1) and Erastin (Era) as ferroptosis inhibitor and inducer, respectively, along with GPX4 knockdown via lentivirus-based shRNA. Additionally, the therapeutic efficacy of ICA on OA-related articular cartilage damage was assessed in rats through histopathology and immunohistochemistry (IHC). RESULTS: IL-1ß treatment upregulated the expression of OA-associated matrix metalloproteinases (MMP3 and MMP1), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-5), and increased intracellular ROS, lipid ROS, and MDA levels while downregulating collagen II and SOX9 expression in SW1353 cells. ICA treatment countered the IL-1ß-induced upregulation of MMPs and ADAMTS-5, restored collagen II and SOX9 expression, and reduced intracellular ROS, lipid ROS, and MDA levels. Furthermore, IL-1ß upregulated P53 but downregulated SLC7A11 and GPX4 expression in SW1353 cells, effects that were mitigated by ICA or Fer-1 treatment. Significantly, ICA also alleviated Era-induced ferroptosis, whereas it had no effect on GPX4-silenced SW1353 cells. In vivo, ICA treatment reduced articular cartilage damage in OA rats by partially restoring collagen II and GPX4 expression, inhibiting cartilage extracellular matrix (ECM) degradation and chondrocyte ferroptosis. CONCLUSION: ICA treatment mitigated chondrocyte ferroptosis and articular cartilage damage by enhancing the SLC7A11/GPX4 signaling, suggesting its potential as a therapeutic agent for OA interventions.

Profibrotic role of the SOX9-MMP10-ECM biosynthesis axis in the tracheal fibrosis after injury and repair.

Fibroblast activation and extracellular matrix (ECM) deposition play an important role in the tracheal abnormal repair process and fibrosis. As a transcription factor, SOX9 is involved in fibroblast activation and ECM deposition. However, the mechanism of how SOX9 regulates fibrosis after tracheal injury remains unclear. We investigated the role of SOX9 in TGF-ß1-induced fibroblast activation and ECM deposition in rat tracheal fibroblast (RTF) cells. SOX9 overexpression adenovirus (Ad-SOX9) and siRNA were transfected into RTF cells. We found that SOX9 expression was up-regulated in RTF cells treated with TGF-ß1. SOX9 overexpression activated fibroblasts and promoted ECM deposition. Silencing SOX9 inhibited cell proliferation, migration, and ECM deposition, induced G2 arrest, and increased apoptosis in RTF cells. RNA-seq and chromatin immunoprecipitation sequencing (ChIP-seq) assays identified MMP10, a matrix metalloproteinase involved in ECM deposition, as a direct target of SOX9, which promotes ECM degradation by increasing MMP10 expression through the Wnt/ß-catenin signaling pathway. Furthermore, in vivo, SOX9 knockdown ameliorated granulation proliferation and tracheal fibrosis, as manifested by reduced tracheal stenosis. In conclusion, our findings indicate that SOX9 can drive fibroblast activation, cell proliferation, and apoptosis resistance in tracheal fibrosis via the Wnt/ß-catenin signaling pathway. The SOX9-MMP10-ECM biosynthesis axis plays an important role in tracheal injury and repair. Targeting SOX9 and its downstream target MMP10 may represent a promising therapeutic approach for tracheal fibrosis.

GATA6 promotes fibrotic repair of tracheal injury through NLRP3 inflammasome-mediated epithelial pyroptosis.

Tracheal injury is a challenging emergency condition that is characterized by the abnormal repair of the trachea. GATA6, a well-established transcription factor, plays a crucial role in tissue injury and epithelial regenerative repair. This study aims to evaluate the role of GATA6 in NF-κB-mediated NLRP3 inflammasome activation and pyroptosis after tracheal injury. Tracheal tissues and serum samples were collected from clinical patients and a rat model of tracheal injury. Upon GATA6 knockdown or overexpression, BEAS-2B and rat tracheal epithelial (RTE) cells were treated with lipopolysaccharides and nigericin before being co-cultured with primary tracheal fibroblasts. The changes of NLRP3 inflammasome activation and pyroptosis and their underlying mechanisms were detected. Additionally, the role of GATA6 downregulation in tracheal injury was verified in rats. GATA6 expression and NLRP3 inflammasome activation were upregulated following tracheal injury in the epithelium of granulation tissues. GATA6 silencing inhibited NLRP3 priming, NLRP3 inflammasome activation, and pyroptosis in BEAS-2B and RTE cells. Mechanistically, GATA6 was determined to have bound to the promoter region of NLRP3 and synergistically upregulated NLRP3 promoter activity with NF-κB. Furthermore, GATA6 overexpression promoted epithelial-mesenchymal transition via modulating the NF-κB/NLRP3 pathway. Epithelial NLRP3 inflammasome activation triggered ECM production in fibroblasts, which was suppressed by GATA6 knockdown and induced by GATA6 overexpression. Finally, the downregulation of GATA6 alleviated NLRP3 inflammasome-mediated pyroptosis induced by tracheal injury in rats, thereby reducing tracheal stenosis, inflammation, and fibrosis. GATA6 promotes fibrotic repair in tracheal injury through NLRP3 inflammasome-mediated epithelial pyroptosis, making it a potential biological therapeutic target for tracheal injury.

IL-11 drives the phenotypic transformation of tracheal epithelial cells and fibroblasts to enhance abnormal repair after tracheal injury.

Tracheal stenosis (TS) is a multifactorial and heterogeneous disease that can easily lead to respiratory failure and even death. Interleukin-11 (IL-11) has recently received increased attention as a fibrogenic factor, but its function in TS is uncertain. This study aimed to investigate the role of IL-11 in TS regulation based on clinical samples from patients with TS and a rat model of TS produced by nylon brush scraping. Using lentiviral vectors expressing shRNA (lentivirus-shRNA) targeting the IL-11 receptor (IL-11Rα), we lowered IL-11Rα levels in the rat trachea. Histological and immunostaining methods were used to evaluate the effects of IL-11Rα knockdown on tracheal injury, molecular phenotype, and fibrosis in TS rats. We show that IL-11 was significantly elevated in circulating serum and granulation tissue in patients with TS. In vitro, TGFß1 dose-dependently stimulated IL-11 secretion from human tracheal epithelial cells (Beas-2b) and primary rat tracheal fibroblasts (PRTF). IL-11 transformed the epithelial cell phenotype to the mesenchymal cell phenotype by activating the ß-catenin pathway. Furthermore, IL-11 activated the atypical ERK signaling pathway, stimulated fibroblasts proliferation, and transformed fibroblasts into alpha-smooth muscle actin (α-SMA) positive myofibroblasts. IL-11-neutralizing antibodies (IL-11NAb) or ERK inhibitors (U0126) inhibited IL-11 activity and downregulated fibrotic responses involving TGFß/SMAD signaling. In vivo, IL-11Rα knockdown rats showed unobstructed tracheal lumen, relatively intact epithelial structure, and significantly reduced granulation tissue proliferation and collagen fiber deposition. Our findings confirm that IL-11 may be a target for future drug prevention and treatment of tracheal stenosis.

Patient-specific induced pluripotent stem cell properties implicate Ca2+-homeostasis in clinical arrhythmia associated with combined heterozygous RYR2 and SCN10A variants.

We illustrate use of induced pluripotent stem cells (iPSCs) as platforms for investigating cardiomyocyte phenotypes in a human family pedigree exemplified by novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants occurring alone and in combination. The proband, a four-month-old boy, presented with polymorphic ventricular tachycardia. Genetic tests revealed double novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants inherited from his father (F) and mother (M), respectively. His father showed ventricular premature beats; his mother was asymptomatic. Molecular biological characterizations demonstrated greater TNNT2 messenger RNA (mRNA) expression in the iPSCs-induced cardiomyocytes (iPS-CMs) than in the iPSCs. Cardiac troponin Ts became progressively organized but cytoplasmic RYR2 and SCN10A aggregations occurred in the iPS-CMs. Proband-specific iPS-CMs showed decreased RYR2 and SCN10A mRNA expression. The RYR2-A1855D variant resulted in premature spontaneous sarcoplasmic reticular Ca2+ transients, Ca2+ oscillations and increased action potential durations. SCN10A-Q1362H did not confer any specific phenotype. However, the combined heterozygous RYR2-A1855D and SCN10A-Q1362H variants in the proband iPS-CMs resulted in accentuated Ca2+ homeostasis disorders, action potential prolongation and susceptibility to early afterdepolarizations at high stimulus frequencies. These findings attribute the clinical phenotype in the proband to effects of the heterozygous RYR2 variant exacerbated by heterozygous SCN10A modification. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

GATA6 triggers fibroblast activation and tracheal fibrosis through the Wnt/ß-catenin pathway.

Tracheal fibrosis is a key abnormal repair process leading to fatal stenosis, characterized by excessive fibroblast activation and extracellular matrix (ECM) deposition. GATA6, a zinc finger-containing transcription factor, is involved in fibroblast activation, while its role in tracheal fibrosis remains obscure. The present study investigated the potential role of GATA6 as a novel regulator of tracheal fibrosis. It was found that GATA6 and α-smooth muscle actin (α-SMA) were obviously increased in tracheal fibrotic granulations and in TGFß1-treated primary tracheal fibroblasts. GATA6 silencing inhibited TGFß1-stimulated fibroblast proliferation and ECM synthesis, promoted cell apoptosis, and inactivated Wnt/ß-catenin pathway, whereas GATA6 overexpression showed the reverse effects. SKL2001, an agonist of Wnt/ß-catenin signaling, restored collagen1a1 and α-SMA expression which was suppressed by GATA6 silencing. Furthermore, in vivo, knockdown of GATA6 ameliorated tracheal fibrosis, as manifested by reduced tracheal stenosis and ECM deposition. GATA6 inhibition in rat tracheas also impaired granulation proliferation, increased apoptosis, and inactivated Wnt/ß-catenin pathway. In conclusion, our findings indicate that GATA6 triggers fibroblast activation, cell proliferation, and apoptosis resistance in tracheal fibrosis via the Wnt/ß-catenin signaling pathway. Targeting GATA6 may represent a promising therapeutic approach for tracheal fibrosis.

Estradiol inhibits autophagy of Mycobacterium tuberculosis­infected 16HBE cells and controls the proliferation of intracellular Mycobacterium tuberculosis.

Tracheobronchial tuberculosis (TBTB) is most common in young, middle­aged females. Despite adequate anti­tuberculosis therapy, >90% of patients develop tracheobronchial stenosis, which has a high rate of resulting in disability. The present study aimed to explore the effect of estradiol on the development of TBTB. Estrogen receptor (ER) expression in granulomatous tissue was assessed via immunofluorescence. In order to determine whether estrogen affected the proliferation of intracellular Mycobacterium tuberculosis (Mtb), 16HBE cells were infected with Mtb in vitro, followed by estradiol treatment. Intracellular Mtb was quantified via colony counting. The effect of estradiol on autophagy of infected 16HBE cells was determined via western blotting and transmission electron microscopy. Necrosis assays of infected 16HBE cells were analyzed using propidium iodide staining and assessing lactate dehydrogenase (LDH) release. To determine how estradiol affects autophagy, infected 16HBE cells were treated with ER­specific and non­specific modulators. Reactive oxygen species (ROS) levels were analyzed via flow cytometry. Additionally, the protein expression levels of autophagy­associated proteins were determined via western blotting. Mtb could enter human lobar bronchial goblet cells and ciliated cells in patients with TBTB. The results also demonstrated that ERα was expressed in granulomatous tissue from patients with TBTB. Administration of 10­6 M estradiol reduced the number of intracellular Mtb colony­forming units in vitro in the 16HBE human bronchial epithelial cell line at day 3 after infection. Furthermore, cells treated with estradiol and infected with Mtb released less LDH at 72 h and exhibited reduced necrosis levels at 24 h compared with the untreated cells. In addition, autophagy of infected 16HBE cells was inhibited by estradiol. Estradiol and the specific ERα agonist had similar effects on autophagy in infected 16HBE cells. Additionally, treatment with the ERα antagonist abolished the inhibition of autophagy by estradiol in infected 16BHE cells. Compared with the untreated infected 16HBE cells, the ROS levels in the infected 16HBE cells treated with estradiol and the ERα agonist significantly decreased. The levels of phosphorylated (p)­mTOR and p­AKT notably increased in estradiol­ and ERα agonist­treated infected 16HBE cells. In summary, estradiol may serve a key role in the development of TBTB through binding to ERα.

Knockdown of SOX9 alleviates tracheal fibrosis through the Wnt/ß-catenin signaling pathway.

Trachealfibrosis is an important cause of tracheal stenosis without effective treatments, and new drug targets need to be developed. The role of SOX9 in the injury and repair of the trachea is unknown; this study aims to investigate the role of SOX9 in the regulation of tracheal fibrosis based on clinical samples from patients with tracheal injury and a model of tracheal fibrosis produced by tracheal brushing in rats. The results showed that the expressions of SOX9 and mesenchymal and ECM-related indicators were increased in the injury and fibrosis of the trachea in patients and rats. Serum SOX9 levels exhibited a sensitivity of 83.87% and specificity of 90% in distinguishing patients with tracheal fibrosis from healthy volunteers when the cut­off value was 13.24 ng/ml. Knockdown SOX9 can markedly inhibit granulation tissue proliferation, reduce inflammation and ECM deposition, promote epithelial regeneration and granulation tissue apoptosis, and attenuate the tracheal fibrosis after injury. Additionally, RNA sequencing showed that the proliferation, migration, and ECM deposition of tracheal granulation tissue were related to the activation of Wnt pathway, activation of the ß-catenin, and p-GSK3ß after injury can be inhibited by the knockdown of SOX9. In summary, SOX9 is upregulated in tracheas fibrosis and may be a novel factor to promote tracheal fibrosis progression. Inhibiting SOX9 may be used to prevent and treat tracheal fibrosis in the future. KEY MESSAGE : The expression of SOX9 is upregulated the process of injury and repair of the tracheal fibrosis. Knocking down SOX9 can attenuate tracheal fibrosis after injury by inhibiting inflammation response, granulation tissue proliferation, ECM deposition, and promoting granulation tissue apoptosis. The Wnt/ß-catenin-SOX9 axis is activated during tracheal injury and fibrosis, and inhibition of SOX9 can partially alleviate tracheal fibrosis. SOX9 may act as a new diagnostic and therapeutic target in patients with tracheal fibrosis in the future.

microRNA-637 promotes apoptosis and suppresses proliferation and autophagy in multiple myeloma cell lines via NUPR1.

Multiple myeloma (MM) is a heterogeneous disease with poor prognosis. Increasing evidence has revealed that microRNAs (miRNAs) are strongly associated with the pathogenesis and progression of MM. Here, we investigated the role of microRNA-637 (miR-637) in MM to identify potential therapeutic targets. We measured the expression of miR-637 in bone marrow samples of MM patients and MM cell lines by quantitative real-time PCR and western blot. The effect of miR-637 on proliferation and apoptosis of MM primary cells was also investigated. Analyses of four bioinformatics databases showed that miR-637 is associated with nuclear protein 1 (NUPR1) in MM cells, which was confirmed by luciferase reporter assay. We found that the overexpression of miR-637 suppressed the development of MM. miR-637 mimics increased the levels of Bax, cleaved caspase 3, and P62, and decreased the levels of Bcl2 and LC3. Additionally, luciferase reporter assays were performed to demonstrate that NUPR1 is the main target of miR-637 in MM cells. Overexpression of NUPR1 reversed the effects of miR-637 mimics in MM cells. Our results suggest that miR-637 inhibits cell proliferation and autophagy, and promotes apoptosis in MM cells by targeting NUPR1. Our findings also suggest that miR-637 may have potential as a novel molecular therapeutic target for MM treatment.

Establishment of iPSC line from a Chinese infant (XACHi012-A) with Jervell and Lange-Nielsen syndrome carrying combined KCNQ1 frameshift c.431delC(p.I145Sfs*92) and nonsense c.1175G > A (p.W392X) variants and two iPSC lines from the parents (XACHi013-A, XACHi014-A).

Induced pluripotent stem cell lines (iPSCs) were generated from peripheral blood mononuclear cells (PBMCs) isolated from the peripheral blood of a two month-old boy and the parents. Jervell and Lange-Nielsen syndrome (JLNS) was diagnosed in the boy carrying combined KCNQ1 frameshift c.431delC (p.I145Sfs*92) and nonsense c.1175G > A(p.W392X) variants inherited from his mother and father respectively. PBMCs were reprogrammed using non-integrative Sendai viral vectors containing reprogramming factors OCT4, SOX2, KLF4 and C-MYC. IPSCs were shown to express pluripotent markers, have trilineage differentiation potential, carrying identified KCNQ1 variants with corresponding PBMC, and have a normal karyotype. Thus we established three iPSC lines as useful tools for studying the pathophysiological mechanism of JLNS and drug testing.

Trifluoperazine induces cellular apoptosis by inhibiting autophagy and targeting NUPR1 in multiple myeloma.

Multiple myeloma (MM) is the second most common hematologic malignancy of immunoglobulin-secreting plasma cells. Recent modern combination therapies have improved survival rates, but many patients develop resistance to novel drugs, leading to relapse. Trifluoperazine (TFP), a typical antipsychotic drug, has been reported to exert antitumor effects by targeting various pathways. Thus far, the role of TFP in MM has not been elucidated. In the current study, we demonstrated that TFP inhibited cell growth and autophagy activity but induced apoptosis of U266 and RPMI 8226 MM cells. Furthermore, cotreatment of these cell lines with TFP and rapamycin, a potent autophagy inducer, reduced cell apoptosis compared with TFP treatment alone. We also found that TFP inhibited nuclear protein 1 (NUPR1) expression. In the presence of TFP, cells stably overexpressing NUPR1 showed a higher viability than cells treated with the nonspecific control. Autophagy suppression and apoptosis induction caused by TFP were also reversed in MM cells upon NUPR1 overexpression. Overall, our results indicate that in the context of MM, TFP targets NUPR1, inhibiting cell growth and inducing apoptosis by autophagy inhibition. Our results could contribute toward efforts for the development of more effective therapies for MM to be tested in future clinical trials.

NUPR1 Silencing Induces Autophagy-Mediated Apoptosis in Multiple Myeloma Cells Through the PI3K/AKT/mTOR Pathway.

Nuclear protein 1 (NUPR1) is a stress-related small molecule and plays important roles in various tumors, including multiple myeloma (MM). Autophagy is essential for maintaining cellular homoeostasis in response to stress and, together with apoptosis, determines cell fate. Previous studies indicate that NUPR1 is involved in cancer progression of MM, but the underlying mechanisms have not been elucidated. In this study, we confirmed that NUPR1 and basal autophagy markers were highly expressed in the bone marrow of MM patients. The overexpression of NUPR1 was correlated with staging (both by Revised International Staging System [RISS] and Durie-Salmon [D-S] Staging System), levels of hemoglobin and calcium, and bone marrow plasma cell ratio in the MM patients. NUPR1 silencing reduced autophagy activities and induced apoptosis in U266 and RPMI 8226. We further observed a decrease in NUPR1 silencing-induced apoptosis in the presence of rapamycin, while an increase in apoptosis after chloroquine and 3-methyladenine treatment. Analysis of the mechanism indicated that PI3K/AKT/mTOR pathway was involved in autophagy-mediated apoptosis upon NUPR1 knockdown. In summary, our results demonstrate that NUPR1 silencing suppresses autophagy activities and induces autophagy-mediated apoptosis in MM cells through the PI3K/AKT/mTOR pathway, which exhibits potential as a treatment strategy for MM.

Generation of two induced pluripotent stem cell lines (XACHi0010-A, XACHi0011-A) from a Chinese family with combined oxidative phosphorylation deficiency carrying homozygous and heterozygous C1QBP-L275F mutation.

Induced pluripotent stem cell lines (iPSCs) were generated from peripheral blood mononuclear cells (PBMCs) isolated from the peripheral blood of a 14 year-old boy and his mother using same protocols. Diagnosis of combined oxidative phosphorylation deficiency (COXPD) was established after identifying a homozygous c.823C > T(p.L275F) variant in C1QBP gene carried by the boy, inherited from his asymptomatic consanguineous parents carrying this heterozygous variant. PBMCs were reprogrammed using non-integrative sendai viral vectors containing reprogramming factors OCT4, SOX2, KLF4 and C-MYC. iPSCs were shown to express pluripotent markers, have trilineage differentiation potential, carry C1QBP-L275F mutation, have a normal karyotype. These lines are useful tools for studying the pathophysiological mechanism of COXPD.

Generation of induced pluripotent stem cells (iPSCs) from an infant with Pompe disease carrying with compound mutations of R608X and E888X in GAA gene.

Induced pluripotent stem cells (iPSCs) were generated from peripheral blood mononuclear cells (PBMCs) isolated from the peripheral blood of a five months-old boy with glycogen storage disease type II(GSD II, also known as Pompe disease, PD) carries compound mutations R608X E888X in GAA gene. PBMCs were reprogrammed using non-integrative Sendai viral vectors containing reprogramming factors OCT4, SOX2, KLF4 and C-MYC. iPSCs were shown to express pluripotent markers, have trilineage differentiation potential, carry GAA-R608X and GAA-E888X compound mutations, have a normal karyotype. It is useful tool for studying GSDII.