Protein Chip Analysis Research Articles

Anti-TCP1 Antibody Is a Potential Biomarker for Diagnosing Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease caused by autoantibodies. Serum samples from patients with SLE (n = 10) were compared with those from normal controls (NCs, n = 5) using 21K protein chip analysis to identify a biomarker for SLE, revealing 63 SLE-specific autoantibodies. The anti-chaperonin-containing t-complex polypeptide-1 (TCP1) antibody exhibited higher expression in patients with SLE than in NCs. To validate the specificity of the anti-TCP1 antibody in SLE, dot blot analysis was conducted using sera from patients with SLE (n = 100), rheumatoid arthritis (RA; n = 25), Behçet's disease (BD; n = 28), and systemic sclerosis (SSc; n = 30) and NCs (n = 50). The results confirmed the detection of anti-TCP1 antibodies in 79 of 100 patients with SLE, with substantially elevated expression compared to both NCs and patients with other autoimmune diseases. We performed an enzyme-linked immunosorbent assay to determine the relative amounts of anti-TCP1 antibodies; markedly elevated anti-TCP1 antibody levels were detected in the sera of patients with SLE (50.1 ± 17.3 arbitrary unit (AU), n = 251) compared to those in NCs (33.9 ± 9.3 AU), RA (35 ± 8.7 AU), BD (37.5 ± 11.6 AU), and SSc (43 ± 11.9 AU). These data suggest that the anti-TCP1 antibody is a potential diagnostic biomarker for SLE.

Transplantation of human placental chorionic plate-derived mesenchymal stem cells for repair of neurological damage in neonatal hypoxic-ischemic encephalopathy.

JOURNAL/nrgr/04.03/01300535-202409000-00035/figure1/v/2024-01-16T170235Z/r/image-tiff Neonatal hypoxic-ischemic encephalopathy is often associated with permanent cerebral palsy, neurosensory impairments, and cognitive deficits, and there is no effective treatment for complications related to hypoxic-ischemic encephalopathy. The therapeutic potential of human placental chorionic plate-derived mesenchymal stem cells for various diseases has been explored. However, the potential use of human placental chorionic plate-derived mesenchymal stem cells for the treatment of neonatal hypoxic-ischemic encephalopathy has not yet been investigated. In this study, we injected human placental chorionic plate-derived mesenchymal stem cells into the lateral ventricle of a neonatal hypoxic-ischemic encephalopathy rat model and observed significant improvements in both cognitive and motor function. Protein chip analysis showed that interleukin-3 expression was significantly elevated in neonatal hypoxic-ischemic encephalopathy model rats. Following transplantation of human placental chorionic plate-derived mesenchymal stem cells, interleukin-3 expression was downregulated. To further investigate the role of interleukin-3 in neonatal hypoxic-ischemic encephalopathy, we established an in vitro SH-SY5Y cell model of hypoxic-ischemic injury through oxygen-glucose deprivation and silenced interleukin-3 expression using small interfering RNA. We found that the activity and proliferation of SH-SY5Y cells subjected to oxygen-glucose deprivation were further suppressed by interleukin-3 knockdown. Furthermore, interleukin-3 knockout exacerbated neuronal damage and cognitive and motor function impairment in rat models of hypoxic-ischemic encephalopathy. The findings suggest that transplantation of hpcMSCs ameliorated behavioral impairments in a rat model of hypoxic-ischemic encephalopathy, and this effect was mediated by interleukin-3-dependent neurological function.

KCNAB2 overexpression inhibits human non-small-cell lung cancer cell growth in vitro and in vivo

Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases. NSCLC patients often have poor prognosis demanding urgent identification of novel biomarkers and potential therapeutic targets. KCNAB2 (regulatory beta subunit2 of voltage-gated potassium channel), encoding aldosterone reductase, plays a pivotal role in regulating potassium channel activity. In this research, we tested the expression of KCNAB2 as well as its potential functions in human NSCLC. Bioinformatics analysis shows that expression of KCNAB2 mRNA is significantly downregulated in human NSCLC, correlating with poor overall survival. In addition, decreased KCNAB2 expression was detected in different NSCLC cell lines and local human NSCLC tissues. Exogenous overexpression of KCNAB2 potently suppressed growth, proliferation and motility of established human NSCLC cells and promoted NSCLC cells apoptosis. In contrast, CRISPR/Cas9-induced KCNAB2 knockout further promoted the malignant biological behaviors of NSCLC cells. Protein chip analysis in the KCNAB2-overexpressed NSCLC cells revealed that KCNAB2 plays a possible role in AKT-mTOR cascade activation. Indeed, AKT-mTOR signaling activation was potently inhibited following KCNAB2 overexpression in NSCLC cells. It was however augmented by KCNAB2 knockout. In vivo, the growth of subcutaneous KCNAB2-overexpressed A549 xenografts was significantly inhibited. Collectively, KCNAB2 could be a novel effective gene for prognosis prediction of NSCLC. Targeting KCNAB2 may lead to the development of advanced therapies.

Targeted siRNA Delivery by Bioinspired Cancer Cell Membrane-Coated Nanoparticles with Enhanced Anti-Cancer Immunity.

Cell-membrane nanocarriers are usually constructed by modifying the nanoparticle surface with cell membrane extracts, which has a direct benefit in endowing targeting capacity to nanocarriers based on their original cell types. However, delivering nucleic acid cargos by cell membrane-based nanoparticles is difficult owing to the strong negative charge of the cell membrane fraction. In this study, we developed a cancer cell membrane-based drug delivery system, the cMDS, for efficient siRNA delivery. Meanwhile, the cancer-specific immune response stimulated by the gene vector itself could offer synergistic anti-cancer ability. The cMDS was prepared by ultrasound, and its transfection efficiency and anti-cancer ability were examined using cultures of CT26 cells. MTT and red blood cell hemolysis tests were performed to assess the safety of cMDS, while its targeted gene delivery and strong immune stimulation were investigated in a subcutaneous tumor model. Moreover, the detailed anti-cancer immune stimulation mechanisms of cMDS are uncovered by protein chip analysis. The cMDS was spherical core-shell structure. It showed high transfection efficiency and anti-cancer ability in vitro. In animal experiments, intravenously administered cMDS/siStat3 complex efficiently suppress the growth of colon cancer. Moreover, the result of protein chip analysis suggested that cMDS affect the migration and chemotaxis of immune cells. The cMDS shows obvious tumor tissue-specific accumulation properties and strong immune stimulation ability. It is an advanced targeted gene delivery system with potent immunotherapeutic properties.

Paracrine effects of mir-210-3p on angiogenesis in hypoxia-treated c-kit-positive cardiac cells

Objective: Treatment with c-kit-positive cardiac cells (CPCs) has been shown to improve the prognosis of ischemic heart disease. MicroRNAs (miRNAs) confer protection by enhancing the cardiac repair process, but their specific functional mechanisms remain unclear. This study aimed to screen for differentially expressed miRNAs in CPCs under hypoxia and explore their effects on the function of CPCs. Methods: We harvested CPCs from C57 adult mice and later performed a high-throughput miRNA sequencing for differential expression profiling analysis. Subsequently, we intervened with the differentially expressed gene miR-210-3p in CPCs and detected changes in the secretion of angiogenesis-related factors through a protein-chip analysis. Finally, we applied CPC supernatants of different groups as conditioned medium to treat mouse cardiac microvascular endothelial cells (CMECs) and further investigated the functional effects of miR-210-3p on c-kit+CPCs under ischemia and hypoxia conditions. Results: The miR-210-3p was highly increased in hypoxia-treated CPCs. Protein-chip detection revealed that CPCs expressed cytokines such as FGF basic, angiogenin, and vascular endothelial growth factor (VEGF) and that hypoxia enhanced their release. Silencing miR-210-3p resulted in a reduction in the release of these angiogenesis-related factors. In addition, the conditioned medium of hypoxia-treated CPCs promoted the proliferation, migration, and tube-forming capabilities of CMECs. In contrast, the conditioned media of CPCs with silenced miR-210-3p after hypoxia decreased the proliferation, migration, and tube-forming ability of CMEC. Conclusions: The CPCs exert proangiogenic effects via paracrine pathways mediated by miR-210-3p. Upregulation of miR-210-3p in hypoxia-treated CPCs may enhance their paracrine function by regulating the secretion of angiogenic factors, thereby promoting angiogenesis in ischemic heart disease.

Identification of matrix-remodeling associated 5 as a possible molecular oncotarget of pancreatic cancer

Pancreatic cancer has an extremely poor prognosis. Here we examined expression, potential functions and underlying mechanisms of MXRA5 (matrix remodeling associated 5) in pancreatic cancer. Bioinformatics studies revealed that MXRA5 transcripts are significantly elevated in pancreatic cancer tissues, correlating with the poor overall survival, high T-stage, N1 and pathologic stage of the patients. MXRA5 mRNA and protein expression is significantly elevated in microarray pancreatic cancer tissues and different pancreatic cancer cells. In primary and immortalized (BxPC-3 and PANC-1 lines) pancreatic cancer cells, shRNA-induced MXRA5 silencing or CRISPR/Cas9-mediated MXRA5 knockout suppressed cell survival, proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT), while provoking cell apoptosis. Conversely, forced overexpression of MXRA5 further promoted pancreatic cancer cell progression and EMT. Bioinformatics studies and the protein chip analyses revealed that differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) in MXRA5-overexpressed primary pancreatic cancer cells were enriched in the PI3K-Akt-mTOR cascade. Indeed, Akt-mTOR activation in primary human pancreatic cancer cells was inhibited by MXRA5 shRNA or knockout, but was augmented following MXRA5 overexpression. In vivo, the growth of MXRA5 KO PANC-1 xenografts was largely inhibited in nude mice. Moreover, intratumoral injection of adeno-associated virus-packed MXRA5 shRNA potently inhibited primary pancreatic cancer cell growth in nude mice. Akt-mTOR activation was also largely inhibited in the MXRA5-depleted pancreatic cancer xenografts. Contrarily MXRA5 overexpression promoted primary pancreatic cancer cell growth in nude mice. Together, overexpressed MXRA5 is important for pancreatic cancer cell growth possibly through promoting EMT and Akt-mTOR activation. MXRA5 could be a potential therapeutic oncotarget for pancreatic cancer.

Intravitreal slow-release dexamethasone alleviates traumatic proliferative vitreoretinopathy by inhibiting persistent inflammation and Müller cell gliosis in rabbits.

To evaluate the effects of intravitreal slow-release dexamethasone on traumatic proliferative vitreoretinopathy (PVR) and Müller cell gliosis and preliminarily explored the possible inflammatory mechanism in a rabbit model induced by penetrating ocular trauma. Traumatic PVR was induced in the right eyes of pigmented rabbits by performing an 8-mm circumferential scleral incision placed 2.5 mm behind the limbus, followed by treatment with a slow-release dexamethasone implant (Ozurdex) or sham injection. Left eyes were used as normal controls. The intraocular pressure (IOP) was monitored using an iCare tonometer. PVR severity was evaluated via anatomical and histopathological examinations every week for 6wk; specific inflammatory cytokine and proliferative marker levels were measured by quantitative real-time polymerase chain reaction, Western blot, protein chip analysis, or immunofluorescence staining. During the observation period, PVR severity gradually increased. Intense Müller cell gliosis was observed in the peripheral retina near the wound and in the whole retina of PVR group. Ozurdex significantly alleviated PVR development and Müller cell gliosis. Post-traumatic inflammation fluctuated and was persistent. The interleukin-1β (IL-1β) mRNA level was significantly upregulated, peaking on day 3 and increasing again on day 21 after injury. The expression of nod-like receptor family pyrin domain containing 3 (NLRP3) showed a similar trend that began earlier than that of IL-1β expression. Ozurdex suppressed the expression of IL-1β, NLRP3, and phosphorylated nuclear factor-kappa B (NF-κB). The average IOP after treatment was within normal limits. The present study demonstrates chronic and fluctuating inflammation in a traumatic PVR rabbit model over 6wk. Ozurdex treatment significantly inhibites inflammatory cytokines expression and Müller cell gliosis, and thus alleviates PVR severity. This study highlights the important role of IL-1β, and Ozurdex inhibites inflammation presumably via the NF-κB/NLRP3/IL-1β inflammatory axis. In summary, Ozurdex provides a potential therapeutic option for traumatic PVR.

AIMP1 promotes multiple myeloma malignancy through interacting with ANP32A to mediate histone H3 acetylation

Multiple myeloma (MM) is the second most common hematological malignancy. An overwhelming majority of patients with MM progress to serious osteolytic bone disease. Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1) participates in several steps during cancer development and osteoclast differentiation. This study aimed to explore its role in MM. The gene expression profiling cohorts of MM were applied to determine the expression of AIMP1 and its association with MM patient prognosis. Enzyme-linked immunosorbent assay, immunohistochemistry, and Western blotting were used to detect AIMP1 expression. Protein chip analysis, RNA-sequencing, and chromatin immunoprecipitation and next-generation sequencing were employed to screen the interacting proteins and key downstream targets of AIMP1. The impact of AIMP1 on cellular proliferation was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro and a xenograft model in vivo. Bone lesions were evaluated using tartrate-resistant acid phosphatase staining in vitro. A NOD/SCID-TIBIA mouse model was used to evaluate the effect of siAIMP1-loaded exosomes on bone lesion formation in vivo. AIMP1 expression was increased in MM patients and strongly associated with unfavorable outcomes. Increased AIMP1 expression promoted MM cell proliferation in vitro and in vivo via activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Protein chip assays and subsequent experiments revealed that AIMP1 interacted with acidic leucine-rich nuclear phosphoprotein 32 family member A (ANP32A) to regulate histone H3 acetylation. In addition, AIMP1 increased histone H3 acetylation enrichment function of GRB2-associated and regulator of MAPK protein 2 (GAREM2) to increase the phosphorylation of extracellular-regulated kinase 1/2 (p-ERK1/2). Furthermore, AIMP1 promoted osteoclast differentiation by activating nuclear factor of activated T cells c1 (NFATc1) in vitro. In contrast, exosome-coated small interfering RNA of AIMP1 effectively suppressed MM progression and osteoclast differentiation in vitro and in vivo. Our data demonstrate that AIMP1 is a novel regulator of histone H3 acetylation interacting with ANP32A in MM, which accelerates MM malignancy via activation of the MAPK signaling pathway.

FBXL17/spastin axis as a novel therapeutic target of hereditary spastic paraplegia

BackgroundSpastin significantly influences microtubule regulation in neurons and is implicated in the pathogenesis of hereditary spastic paraplegia (HSP). However, post-translational regulation of the spastin protein remains nebulous. The association between E3 ubiquitin ligase and spastin provides a potential therapeutic strategy.ResultsAs evidenced by protein chip analysis, FBXL17 inversely correlated with SPAST-M1 at the protein level in vitro and, also in vivo during embryonic developmental stage. SPAST-M1 protein interacted with FBXL17 specifically via the BTB domain at the N-terminus of SPAST-M1. The SCFFBXL17 E3 ubiquitin ligase complex degraded SPAST-M1 protein in the nuclear fraction in a proteasome-dependent manner. SPAST phosphorylation occurred only in the cytoplasmic fraction by CK2 and was involved in poly-ubiquitination. Inhibition of SCFFBXL17 E3 ubiquitin ligase by small chemical and FBXL17 shRNA decreased proteasome-dependent degradation of SPAST-M1 and induced axonal extension. The SPAST Y52C mutant, harboring abnormality in BTB domain could not interact with FBXL17, thereby escaping protein regulation by the SCFFBXL17 E3 ubiquitin ligase complex, resulting in loss of functionality with aberrant quantity. Although this mutant showed shortening of axonal outgrowth, low rate proliferation, and poor differentiation capacity in a 3D model, this phenotype was rescued by inhibiting SCFFBXL17 E3 ubiquitin ligase.ConclusionsWe discovered that a novel pathway, FBXL17-SPAST was involved in pathogenicity of HSP by the loss of function and the quantitative regulation. This result suggested that targeting FBXL17 could provide new insight into HSP therapeutics.

Vascular toxicity of multi-walled carbon nanotubes targeting vascular endothelial growth factor

Multiwalled carbon nanotubes (MWCNTs) are currently widely used and are expected to be used as drug carriers and contrast agents in clinical practice. Previous studies mainly focused on their lung toxicity; therefore, their effects on the vascular endothelium are unclear. In this study, a human angiogenesis array was used to determine the effect of MWCNTs on the expression profile of angiogenic factors in endothelial cells and to clarify the role of vascular endothelial growth factor (VEGF) in MWCNT-induced endothelial cell injury at the cellular and animal levels. The results indicated that MWCNTs (20–30 nm and 30–50 nm) could enter endothelial cells and disrupt human umbilical vein endothelial cell (HUVECs) activity in a concentration-dependent manner. MWCNTs disrupted the tube formation ability and cell migration function of HUVECs. The results from a Matrigel Plug experiment in mice showed that angiogenesis in the MWCNT experimental group was significantly reduced. The results of a protein chip analysis indicated that VEGF expression in the MWCNT treatment group was decreased, a finding that was validated by ELISA results. The protein expression levels of AKT and eNOS in the MWCNT treatment group were significantly decreased; the administration of recombinant VEGF significantly alleviated the migration ability and tube formation ability of endothelial cells injured by MWCNTs, upregulated the protein expression of AKT and eNOS, and increased the number of neovascularization in mice in the MWCNT treatment group. This study demonstrated that MWCNTs affect angiogenesis via the VEGF-Akt-eNOS axis which can be rescued by VEGF endothelial treatment.

Shenlijia Attenuates Doxorubicin-Induced Chronic Heart Failure by Inhibiting Cardiac Fibrosis.

Application of the anticancer drug doxorubicin (DOX) is restricted due to its adverse, cardiotoxic side effects, which ultimately result in heart failure. Moreover, there are a limited number of chemical agents for the clinical prevention of DOX-induced cardiotoxicity. Based on the theories of traditional Chinese medicine (TCM) on chronic heart failure (CHF), Shenlijia (SLJ), a new TCM compound, has been developed to fulfill multiple functions, including improving cardiac function and inhibiting cardiac fibrosis. In the present study, the protective effects and molecular mechanisms of SLJ on DOX-induced CHF rats were investigated. The CHF rat model was induced by intraperitoneal injection of DOX for six weeks with the cumulative dose of 15 mg/kg. All rats were then randomly divided into the control, CHF, CHF + SLJ (3.0 g/kg per day), and CHF + captopril (3.8 mg/kg per day) groups and treated for further four weeks. Echocardiography and the assessment of hemodynamic parameters were performed to evaluate heart function. A protein chip was applied to identify proteins with diagnostic values that were differentially expressed following SLJ treatment. The data from these investigations showed that SLJ treatment significantly improved cardiac function by increasing the left ventricular ejection fraction, improving the hemodynamic index, and inhibiting interstitial fibrosis. Protein chip analysis revealed that SLJ upregulated MCP-1, MDC, neuropilin-2, TGF-β3, thrombospondin, TIE-2, EG-VEGF/PK1, and TIMP-1/2/3 expressions and downregulated that of MMP-13. In addition, immunohistochemistry and western blot results further confirmed that SLJ promoted TIMP-1/2/3 and inhibited MMP-13 expression. The results of the present study suggest that SLJ was effective against DOX-induced CHF rats and is related to the improvement of heart function and ultrastructure and the inhibition of myocardial fibrosis.

MiR-30c-5p regulates adventitial progenitor cells differentiation to vascular smooth muscle cells through targeting OPG

BackgroundAs the most important component of the vascular wall, vascular smooth muscle cells (VSMCs) participate in the pathological process by phenotype transformation or differentiation from stem/progenitor cells. The main purpose of this study was to reveal the role and related molecular mechanism of microRNA-30c-5p (miR-30c-5p) in VSMC differentiation from adventitial progenitor cells expressing stem cell antigen-1(Sca-1).MethodsIn this study, we detected the expression of miR-30c-5p in human normal peripheral arteries and atherosclerotic arteries. In vitro, a stable differentiation model from adventitial Sca-1+ progenitor cells to VSMCs was established using transforming growth factor-β1 (TGF-β1) induction and the expression of miR-30c-5p during the process was observed. Then, we explored the effect of miR-30c-5p overexpression and inhibition on the differentiation from adventitial Sca-1+ progenitor cells to VSMCs. The target genes of miR-30c-5p were identified by protein chip and biological analyses and the expression of these genes in the differentiation process were detected. Further, the relationship between the target gene and miR-30c-5p and its effect on differentiation were evaluated. Finally, the co-transfection of miR-30c-5p inhibitor and small interfering RNA (siRNA) of the target gene was implemented to verify the functional target gene of miR-30c-5p during the differentiation from adventitial Sca-1+ progenitor cells to VSMCs, and the dual-luciferase reporter gene assay was performed to detect whether the mRNA 3′untranslated region (UTR) of the target gene is the direct binding site of miR-30c-5p.ResultsThe expression of miR-30c-5p in the human atherosclerotic arteries was significantly lower than that in the normal arteries. During the differentiation from adventitial Sca-1+ progenitor cells to VSMCs, the expression of VSMC special markers including smooth muscle α-actin (SMαA), smooth muscle-22α (SM22α), smooth muscle myosin heavy chain (SMMHC), and h1-caponin increased accompanied with cell morphology changing from elliptic to fusiform. Meanwhile, the expression of miR-30c-5p decreased significantly. In functional experiments, overexpression of miR-30c-5p inhibited SMαA, SM22α, SMMHC, and h1-caponin at the mRNA and protein levels. In contrast, inhibition of miR-30c-5p promoted the expression of SMαA, SM22α, SMMHC, and h1-caponin. The target gene, osteoprotegerin (OPG), was predicted through protein chip and bioinformatics analyses. Overexpression of miR-30c-5p inhibited OPG expression while inhibition of miR-30c-5p had an opposite effect. Co-transfection experiments showed that low expression of OPG could weaken the promotion effect of miR-30c-5p inhibitor on the differentiation from adventitial Sca-1+ progenitor cells to VSMCs and the dual-luciferase reporter gene assay demonstrated that miR-30c-5p could target the mRNA 3′UTR of OPG directly.ConclusionsThis study demonstrates that miR-30c-5p expression was significantly decreased in atherosclerotic arteries and miR-30c-5p inhibited VSMC differentiation from adventitial Sca-1+ progenitor cells through targeting OPG, which may provide a new target for the treatment of VSMCs-associated diseases.

Renalase improves pressure overload-induced heart failure in rats by regulating extracellular signal-regulated protein kinase 1/2 signaling.

Renalase, a novel flavoprotein that is mainly expressed in the kidney and heart, plays a crucial role in hypertension. Recent studies have shown that renalase is expressed at low levels in the serum of patients with heart failure, while the role of renalase and its mechanism in cardiac failure is unclear. Adult Sprague-Dawley (SD) rats were used to investigate the role and function of renalase in the pathological process of transverse aortic constriction (TAC)-induced heart failure. Renalase-human protein chip analysis showed that renalase was directly associated with P38 and extracellular signal-regulated protein kinase 1/2 (ERK1/2) signaling. We further used lentivirus-mediated RNA interference to study the role of renalase in the progression of pathological ventricular hypertrophy and found that renalase inhibition attenuated the noradrenaline-induced hypertrophic response in vitro or the pressure overload-induced hypertrophic response in vivo. Recombinant renalase protein significantly alleviated pressure overload-induced cardiac failure and was associated with P38 and ERK1/2 signaling. These findings demonstrate that renalase is a potential biomarker of hypertrophy and that exogenous recombinant renalase is a potential and novel drug for heart failure.

Cyclin-dependent kinase like 3 promotes triple-negative breast cancer progression via inhibiting the p53 signaling pathway.

It has been reported that cyclin-dependent kinase like 3 (CDKL3) plays a crucial role in cell proliferation and migration in several cancers. However, the function of CDKL3 in triple-negative breast cancer (TNBC) is still unclear. In the present study, immunohistochemistry (IHC) was conducted to detect the CDKL3 expression. CCK-8, flow cytometry, Transwell assays, and mice xenograft models, were performed to explore the roles of CDKL3 on the proliferation and migration of TNBC in vitro and in vivo. Besides, protein chip analysis was used to screen the potential pathways, which was further confirmed by promoter activity assay, western blotting, and CCK-8 assay. Our findings reveal a high expression of CDKL3 in TNBC tissues, which is closely related to a poor prognosis of patients with TNBC. In TNBC cells, CDKL3 knockdown inhibits cell proliferation and migration, whereas CDKL3 overexpression has exactly the opposite effect. Consistently, CDKL3 knockdown induces cell apoptosis in vitro but suppresses tumor growth in vivo. Furthermore, CDKL3 knockdown increases p53 expression and reduces cell viability, and these effects are significantly weakened by the p53 inhibitor, PFT-α. In conclusion, the current study highlights that CDKL3 promotes TNBC progressions via regulating the p53 signaling pathway, suggesting that CDKL3 is a novel therapeutic target for TNBC treatment.

Treatment with Minocycline Suppresses Microglia Activation and Reverses Neural Stem Cells Loss after Simulated Microgravity.

The present study aimed to investigate the effect of microglia on simulated microgravity-induced hippocampal neurogenesis reduction and the possible mechanism underlying. Adult rats were treated with tail suspension for different times and the changes of neural stem cells (NSCs) were examined by immunohistochemistry. Then, minocycline was used to inhibit the activation of microglia, and the numbers of microglia and NSCs were detected after microgravity. Additionally, liquid protein chip analysis was applied to detect proinflammatory factors in hippocampus in order to find out the cytokines responsible for microglia activation after microgravity. The results revealed that microgravity increased the numbers of Iba1+ cells and decreased the numbers of BrdU+ and DCX+ cells in hippocampus but did not affect the ratio of NeuN+/BrdU+ cells to the total number of BrdU+ cells. After treated with minocycline, activated microglia were suppressed and the reduction of NSCs induced by microgravity recovered. Besides, compared with the control, higher concentrations of INF-γ and TNF-α were detected in the rats treated with microgravity. Our study provides the first evidence that microglia-mediated inflammation plays an important part in microgravity-induced neurogenesis reduction in hippocampus, and INF-γ and TNF-α secreted by microglia might be the key factors in this process.

The kinase inhibitor BX795 suppresses the inflammatory response via multiple kinases

BX795, a small molecule with an aminopyrimidine backbone, is a potent ATP-competitive inhibitor of phosphoinositide-dependent kinase 1 (PDK1) and TANK-binding kinase 1 (TBK1). BX795 has significant functions in various immune responses and cancer. Few reports on the anti-inflammatory effect of BX795 are available, and its molecular mechanisms have not been fully elucidated. In this study, lipopolysaccharide (LPS)-treated macrophages (RAW264.7 cells), luciferase reporter gene assay, knock-down and overexpression strategies, kinase assay, protein chip, immunoprecipitation, and immunoblotting analyses were employed to clarify the anti-inflammatory mechanism of BX795. BX795 was found to dose-dependently inhibit the production of pro-inflammatory mediators without exhibiting cytotoxicity. Luciferase assay and immunoblotting analysis with nuclear fractions showed that activator protein-1 (AP-1), signal transducer and activator of transcription 1 (STAT1), and interferon regulatory factor 3 (IRF3) are targeted by BX795 rather than nuclear factor (NF)-κB. Moreover, TBK1 and AKT, transforming growth factor activated kinase (TAK)-1/c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase kinase 4 (MKK4) for AP-1 activation, and Janus kinase 2 (JAK2)/STAT1 were inhibited by BX795. Consistent with these findings, BX795 strongly ameliorated inflammatory symptoms in colitis models. These results suggest that BX795 can suppress inflammatory responses triggered by Gram-positive bacteria by suppressing multiple pathways.

Calpastatin participates in the regulation of cell migration in BAP1-deficient uveal melanoma cells.

To detect how BRCA-associated protein 1 (BAP1) regulates cell migration in uveal melanoma (UM) cells. Wound healing and transwell assays were performed to detect UM cell migration abilities. Protein chip, immunoprecipitations and surface plasmon resonance analyses were applied to identify BAP1 protein partners. Western blot and calpain activity assays were used to test the expression and function of calpastatin (CAST). CAST protein was confirmed as a new BAP1 protein partner, and loss of BAP1 reduced the expression and function of CAST in UM cells. The overexpression of CAST rescued the cell migration phenotype caused by BAP1 loss. BAP1 interacts with CAST in UM cells, and CAST and its subsequent calpain pathway may mediate BAP1-related cell migration regulation.

Changes of cytokines in serum and cerebrospinal fluid of newborns infected with human cytomegalovirus

Objective To investigate the molecules of cytokine in the serum and cerebrospinal fluid of newborns infected with human cytomegalovirus (HCMV) by using protein chip technology and to analyze the changes of specific cytokine in serum and cerebrospinal fluid caused by HCMV infection, in order to provide a reliable index for predicting nervous system injury caused by HCMV infection. Methods Serum and cerebrospinal fluid in 4 newborns with HCMV infection and central nervous system injury (HCMV-infected group), and 4 newborns without HCMV infection and central nervous system infection (control group) were collected in Shengjing Hospital of China Medical University from June 2016 to December 2017, and protein chip was used to screen the differentially expressed cytokines in newborns serum and cerebrospinal fluid.The samples were further expanded to collect cerebrospinal fluid from 30 newborns HCMV infection group and 30 newborns in the control group, and the expression of differentially proteins was verified by adopting enzyme linked immunosorbent assay(ELISA) method. Results The results of protein chip analysis showed that newborns in HCMV infection group, compared with the control group, had 3 differentially expressed cytokines in the cerebrospinal fluid sample: adipocyte complement-related protein of 30 kD(Acrp30), interleukin-1 alpha(IL-1α), and matrix metallo protein-3(MMP3) (all P<0.05). Newborns in the HCMV-infected group, compared with the control group, had no differential cytokine expression in the serum.The results of ELISA showed that expression of Acrp30 was significantly higher in the cerebrospinal fluid of newborns with HCMV infection and central nervous system injury [(39.76±2.01) ng/L vs.(7.75±0.10) ng/L, t=87.09, P<0.001], and MMP3 expression was higher than that of control group [(1.40±2.13) ng/L vs.(0.18±0.45) ng/L, t=3.07, P=0.003], while the expression of IL-1α was significantly lower than that of the control group [(2.36±0.99) ng/L vs.(2.91±0.78) ng/L, t=2.39, P=0.020], and the differences were statistically significant. Conclusions The changes of cytokine in cerebrospinal fluid of HCMV infected newborn children may provide a reliable index for predicting injury degree of central nervous system in HCMV, and may further assist clinicians to give timely and appropriate treatment to newborns, and further assist clinicians to improve the prognosis for newborns. Key words: Human cytomegalovirus; Cerebrospinal fluid; Cytokine; Protein chip; Central nervous system injury

A Novel Role of VEGFC in Cerebral Ischemia With Lung Injury.

Cerebral ischemia (CI) is a severe brain injury resulting in a variety of motor impairments combined with secondary injury in remote organs, especially the lung. This condition occurs due to insufficient blood supply to the brain during infancy. However, it has a molecular linkage that needs to be thoroughly covered. Here, we report on the role of vascular endothelial growth factor C (VEGFC) in lung injury induced by CI. The middle cerebral artery occlusion (MCAO) was depended to establish the animal model of CI. Rats were used and brain ischemia was confirmed through TTC staining. Serum was used for protein chip analysis to study the proteomic interaction. Immunohistochemistry analyses were used to quantify and locate the VEGFC in the lung and brain. The role of VEGFC was detected by siVEGFC technology in SY5Y, HUCEV, and A549 cell lines, under normal and oxygen glucose deprivation (OGD) conditions in vitro. As a result, the TTC staining demonstrated that the model of brain ischemia was successfully established, and MPO experiments reported that lung damage was induced in MCAO rats. VEGFC levels were up-regulated in serum. On the other hand, immunohistochemistry showed that VEGFC increased significantly in the cytoplasm of neurons, the endothelium of small trachea and the lung cells of CI animals. On a functional level, siVEGFC effectively inhibited the proliferation of SY5Y cells and decreased the viability of HUVEC cells in normal cell lines. But under OGD conditions, siVEGFC decreased the growth of HUVEC and increased the viability of A549 cells, while no effect was noticed on SYSY cells. Therefore, we confirmed the different role of VEGFC played in neurons and lung cells in cerebral ischemia-reperfusion injury. These findings may contribute to the understanding the molecular linkage of brain ischemia and lung injury, which therefore provides a new idea for the therapeutic approach to cerebral ischemia-reperfusion.

Selective Serotonin Reuptake Inhibitors Aggravate Depression-Associated Dry Eye Via Activating the NF-κB Pathway.

Our study aimed to evaluate the side effects of selective serotonin reuptake inhibitors (SSRIs) on the ocular surface. Twenty patients with depression and dry eye disease (DED) were randomly picked to receive SSRI treatment, whereas another 20 patients received placebo treatment. The serotonin, inflammatory cytokine, and proapoptotic protein levels were determined by using protein chip, qRT-PCR, and ELISA analyses. A rat depression model was established, and SSRIs were applied for 3 or 6 weeks. Tear production and corneal epithelial barrier function were evaluated. The serotonin and inflammatory cytokine levels were analyzed by qRT-PCR, immunohistochemical staining, and ELISA. Human corneal epithelial cells were subjected to serotonin, a HTR antagonist, and/or an NF-κB signaling inhibitor. The inflammatory cytokine and proapoptotic protein levels were determined by qRT-PCR, Western blot analysis, and ELISA. The cell apoptosis rate was assessed by using flow cytometry. The SSRI group had higher tear serotonin levels and more serious inflammation and cell apoptosis on the ocular surface. In the rat depression model, depression decreased tear secretion and increased IL-1β and TNF-α production, whereas the serotonin, TLR2, and TLR4 levels were not increased. SSRI aggravated DED, disrupted the corneal epithelial barrier, and promoted an inflammatory response on the ocular surface by increasing the tear serotonin levels. In addition, serotonin induced an inflammatory response and cell apoptosis in corneal epithelial cells by activating NF-κB signaling. SSRIs aggravate depression-associated DED via activating the NF-κB pathway. The antagonist of HTRs or the inhibitor of NF-κB signaling presents a potential therapeutic strategy for depression-associated DED. (Trial registration number, ChiCTR1800015592).