MMP-9 Protein Levels Research Articles

TNFα induces matrix metalloproteinase-9 expression in monocytic cells through ACSL1/JNK/ERK/NF-kB signaling pathways

Studies have established the association between increased plasma levels of matrix metalloproteinase (MMP)-9 and adipose tissue inflammation. Tumor necrosis factor α (TNFα) was elevated in obesity and is involved in the induction of MMP-9 in monocytic cells. However, the underlying molecular mechanism was incompletely understood. As per our recent report, TNFα mediates inflammatory responses through long-chain acyl-CoA synthetase 1 (ACSL1). Therefore, we further investigated the role of ACSL1 in TNFα-mediated MMP-9 secretion in monocytic cells. THP-1 cells and primary monocytes were used to study MMP-9 expression. mRNA and protein levels of MMP-9 were determined by qRT-PCR and ELISA, respectively. Signaling pathways were studied using Western blotting, inhibitors, and NF-kB/AP1 reporter cells. We found that THP-1 cells and primary human monocytes displayed increased MMP-9 mRNA expression and protein secretion after incubation with TNFα. ACSL1 inhibition using triacsin C significantly reduced the expression of MMP-9 in the THP-1 cells. However, the inhibition of β-oxidation and ceramide biosynthesis did not affect the TNFα-induced MMP-9 production. Using small interfering RNA-mediated ACSL1 knockdown, we further confirmed that TNFα-induced MMP-9 expression/secretion was significantly reduced in ACSL1-deficient cells. TNFα-mediated MMP-9 expression was also significantly reduced by the inhibition of ERK1/ERK2, JNK, and NF-kB. We further observed that TNFα induced phosphorylation of SAPK/JNK (p54/46), ERK1/2 (p44/42 MAPK), and NF-kB p65. ACSL1 inhibition reduced the TNFα-mediated phosphorylation of SAPK/JNK, c-Jun, ERK1/2, and NF-kB. In addition, increased NF-κB/AP-1 activity was inhibited in triacsin C treated cells. Altogether, our findings suggest that ACSL1/JNK/ERK/NF-kB axis plays an important role in the regulation of MMP-9 induced by TNFα in monocytic THP-1 cells.

Serelaxin inhibits LPS-induced inflammatory response in cardiac fibroblasts by activating PPAR-γ and suppressing NF-κB signaling pathway.

Serelaxin has an inhibitory effect on fibrosis. However, whether the anti-fibrotic effects of serelaxin are achieved by inhibiting the inflammatory response has not been clarified. This study aimed to investigate the role of serelaxin in lipopolysaccharide (LPS)-induced inflammation in cardiac fibroblasts (CFs) and elucidate the underlying mechanisms.CFs were isolated from adult rat hearts. The effect of serelaxin on the inhibition of the inflammatory response after LPS induction was examined. Cell viability was measured by MMT assay. Cell proliferation was determined using the cell-counting kit-8. The levels of inflammatory cytokines IL-1β, IL-6, TNF-α, and IL-10 were measured using an enzyme-linked immunosorbent assay (ELISA). The mRNA levels of α-SMA, collagen I/III, MMP-2, MMP-9, IL-1β, IL-6,TNF-α, IL-10, IκBα, p-IκBα, p65 subunit of nuclear factor-kappa B (NF-κB) and peroxisome proliferator-activated receptor-γ (PPAR-γ) were assessed by real-time quantitative PCR (RT-qPCR). The protein levels of α-SMA, collagen I/III, MMP-2, MMP-9, IκBα, p-IκBα, p65, p-p65 and PPAR-γ were examined by western blotting.Serelaxin inhibited LPS-induced IL-1β, IL-6, TNF-α, α-SMA, and collagen I/III, and elevated the expression of IL-10, MMP-2 and MMP-9. Moreover, LPS-induced activation of NF-κB pathway was suppressed by serelaxin treatment. Further studies showed that serelaxin did not significantly increase the expression of PPAR-γ mRNA and protein, but activated PPAR-γ activity, and the PPAR-γ inhibitor GW9662 reversed the inhibitory effect of serelaxin on IL-1β, IL-6, and TNF-α production. These results suggest that serelaxin alleviates cardiac fibrosis by stimulating PPAR-γ via a ligand-independent mechanism which subsequently abolish the expression of NF-κB signalling pathway.

Matrix Metalloproteinase-9, Neuron-specific Enolase, S100 B and Tau Protein Levels in the Patients with Carbon monoxide Poisoning

Background: S100B, NSE, MMP-9, and Tau protein levels increase in cases causing hypoxic cell damage. The diagnosis of the severity of carbon monoxide (CO) poisoning in the early period of these parameters was studied. Material and Method: COHb level measurement was made using a signal capture CO-pulse oximeter (Masimo's SET Rainbow, Masimo's Co, USA) at the first admission of the patients. Then, COHb levels were confirmed by arterial blood gas(ABG) analysis. The patients were divided into two groups as mild and moderate-severe, according to their Glasgow coma scores (GCS) [Mild (14–15); Moderate (9–13) or Severe (3–8)]. The control group was composed of 16 healthy and non-smoking volunteers. Results: The serum S100B protein and MMP-9 values at 0 hr of admission in the hospital and 3hr of treatment were not significantly different in the patient group as compared to the control group. Tau protein levels were significantly higher in the patient group at 0 and 3 hours (p> 0.05) as compared to healthy person. Conclusion: There was no relationship between CO poisoning and MMP-9 and S100B protein levels. NSE and Tau protein were significantly higher in the patient group than the control group. Tau protein may be more useful marker as compared to neuron-specific enolase.

Sesamin attenuates UVA-induced keratinocyte injury via inhibiting ASK-1-JNK/p38 MAPK pathways.

Ultraviolet (UV) exposure-stimulated reactive oxygen species (ROS) formation in keratinocytes is a crucial factor in skin aging. Phytochemicals have become widely popular for protecting the skin from UV-induced cell injury. Sesamin (SSM) has been shown to play a role in extensive pharmacological activity and exhibit photoprotective effects. To assess the protective effect of SSM on UVA-irradiated keratinocytes and determine its potential antiphotoaging effect. HaCaT keratinocytes pretreated with SSM were exposed to UVA radiation at 8 J/cm2 for 10 min. Cell viability and oxidative stress indicators were evaluated using a cell counting kit-8 and lactate dehydrogenase (LDH), malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) assay kits. Apoptosis and intracellular ROS levels were analyzed using annexin V-fluorescein isothiocyanate/propyridine iodide and dichlorodihydrofluorescein diacetate staining, respectively. Protein levels of matrix metalloprotein-1 (MMP-1), MMP-9, Bax/Bcl-2, and mitogen-activated protein kinase (MAPK) pathway proteins, phospho-apoptosis signal-regulating kinase-1 (p-ASK-1)/ASK-1, phospho-c-Jun N-terminal protein kinase (p-JNK)/JNK, and p-p38/p38 were determined using western blotting. Sesamin showed no cytotoxicity until 160 μmol/L on human keratinocytes. Sesamin pretreatment (20 and 40 μM) reversed the suppressed cell viability, increased LDH release and MDA content, decreased cellular antioxidants GSH and SOD, and elevated intracellular ROS levels, which were induced by UVA irradiation. Additionally, SSM inhibited the expression of Bax, MMP-1, and MMP-9 and stimulated Bcl-2 expression. In terms of the regulatory mechanisms, we demonstrated that SSM inhibits the phosphorylation of ASK-1, JNK, and p38. The results suggest that SSM attenuates UVA-induced keratinocyte injury by inhibiting the ASK-1-JNK/p38 MAPK pathways.

EGF-expressed human mesenchymal stem cells inhibit collagenase1 expression in keratinocytes

Mesenchymal stem cells (MSCs) repair tissue injury by upregulating the paracrine secretion of cytokines and growth factors. Human MSC has been recognized as a promising therapeutic material for treatment of various human diseases. Even though the effect of epidermal growth factor (EGF) has been well investigated, the synergetic effect of EGF and MSC has not been studied. Therefore, we expect our basic study to contribute to developing new therapeutic reagents for skin diseases or innovative cosmetics. In this study, we examined the effect of human epidermal growth factor-transfected MSCs (hEGF MSCs) on human keratinocyte HaCaT cell proliferation and the mechanisms that regulate matrix metalloproteinase (MMP)-1 expression in HaCaT cells. To identify the hEGF plasmid and its transfection into MSCs, we performed gel electrophoresis and quantitative PCR. Proliferation and migration of HaCaT cells were examined using water Soluble Tetrazolium (WST-1) and wound-healing assays, respectively. Zymography was performed to investigate the correlation between hEGF MSC-conditioned medium (CM)-treated HaCaT cells and MMP-1 expression. We found that cell proliferation and wound-healing rates were increased in hEGF MSC-CM-treated HaCaT cells compared to those in MSC-CM-treated cells, and conversely collagenase activity was decreased. The mRNA and protein levels of MMP-1 were also decreased in hEGF MSC-CM-treated HaCaT cells. 2-DE analysis showed that the expression of carboxypeptidase, which promotes growth factors and wound healing, was increased in hEGF MSC-CM-treated HaCaT cells. Finally, western blot was used to determine whether MMP-1 expression was reduced via the mitogen-activated protein kinase (MAPK) pathway; the results showed that the levels of MAPK pathway-related proteins (pErk, pJNK, and p-p38) and the levels of transcription factors (pCREB, NFκB, and p-c-Fos) were decreased. In addition, pAkt expression was found to be elevated. The results of our study suggest that hEGF MSCs promote cell proliferation and reduce MMP-1 expression via the MAPK pathway in human keratinocyte HaCaT cells.

Low-dose rosmarinic acid and thymoquinone accelerate wound healing in retinal pigment epithelial cells.

Thymoquinone (TQ) and rosmarinic acid (RA) are two biologically active compounds found in plants and that possess remarkable anti-oxidant and anti-inflammatory properties. The present study aimed to investigate the potential protective effects of RA and TQ, which have known anti-inflammatory and anti-oxidant effects, on retinal damage by establishing a wound healing model for retinal pigment epithelial cells (ARPE-19). To this end, IC50 doses of RA and TQ in ARPE-19 cells were calculated by MTT assay. Both agents were administered at IC50, IC50/2 and IC50/4 doses for wound healing assay, and wound closure percentages were analyzed. Since the best wound healing was found at IC50/4 dose (low dose) for both agents, other biochemical and molecular analyses were planned to be performed using these doses. Following low dose RA and TQ treatments, the cells were lysed and TGF-β1 and MMP-9 levels were analyzed by ELISA technique from the cell lysates obtained. In addition, the mRNA expression levels of TLR3, IFN-γ and VEGF were calculated by RT-PCR technique. Low dose of RA and TQ dramatically increased wound healing. RA may have achieved this by increasing levels of MMP-9 and TLR-3. In contrast, the mRNA expression level of VEGF remained unchanged. TQ accelerated wound healing by increasing both the protein levels of TGF-β1 and MMP-9. Furthermore, low dose of TQ decreased both TLR3 and IFN-γ mRNA expression levels. Low doses of RA and TQ were clearly demonstrated to have protective properties against possible damage to retinal pigment epithelial cells.

LED irradiation at 630nm alleviates collagen-induced arthritis in mice by inhibition of NF-κB-mediated MMPs production.

Matrix metallopreteinase (MMP), a family of matrix degrading enzyme, plays a significant role in persistent and irreversible joint damage in rheumatoid arthritis (RA). Photobiomodulatory therapy (PBMT) has become an emerging adjunct therapy for RA. However, the molecular mechanism of PBMT on RA remains unclear. The purpose of this study is to explore the effect of 630nm light emitting diode (LED) irradiation on RA and its underly molecular mechanism. Arthritis clinic scores, histology analysis and micro-CT results show that 630nm LED irradiation ameliorates collagen-induced arthritis (CIA) in mice with the reduction of the extents of paw swelling, inflammation and bone damage. 630nm LED irradiation significantly reduces MMP-3 and MMP-9 levels and inhibits p65 phosphorylation level in the paws of CIA mice. Moreover, 630nm LED irradiation significantly inhibits the mRNA and protein levels of MMP-3 and MMP-9 in TNF-α-treated MH7A cells, a human synovial cell line. Importantly, 630nm LED irradiation reduces TNF-α-induced the phosphorylated level of p65 but not alters STAT1, STAT3, Erk1/2, JNK and p38 phosphorylation levels. Immunofluorescence result showed that 630nm LED irradiation blocks p65 nuclear translocation in MH7A cells. In addition, other MMPs mRNA regulated by NF-κB were also significantly inhibited by LED irradiation in vivo and in vitro. These results indicates that 630nm LED irradiation reduces the MMPs levels to ameliorate the development of RA by inhibiting the phosphorylation of p65 selectively, suggesting that 630nm LED irradiation may be a beneficial adjunct therapy for RA.Graphical abstract.

E2F6 promotes cell proliferation and invasion in gastric cancer through the regulation of lncRNA TUSC7

Purpose: To examine the regulatory effects of E2F6/long noncoding ribonucleic acid (lncRNA) TUSC7 on the progression of gastric cancer (GC), and the underlying mechanism of action.
 Methods: Relative levels of E2F6 and TUSC7 in GC tissues were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The influence of TUSC7 on the overall survival of patients with GC was analyzed using Kaplan-Meier method. Interaction between E2F6 and TUSC7 was analyzed by Spearman correlation test and dual-luciferase reporter gene assay. Changes in proliferation, invasion and apoptosis of BGC823 and SGC7901 cells regulated by E2F6 and TUSC7 were assessed. The protein levels of MMP-2 and the activity of caspase-3 were evaluated in GC cells treated with E2F6 and TUSC7 using western blot and caspase-3 kit.
 Results: TUSC7 was downregulated while E2F6 was upregulated in GC tissues and cells. A negative correlation was observed between the expression levels of TUSC7 and E2F6, and a low level of TUSC7 predicted poor prognosis in GC patients. The knockdown of E2F6 attenuated cell viability and invasion, and stimulated the apoptosis of GC cells, but these were reversed by co-knockdown of TUSC7. In addition, knockdown of E2F6 downregulated MMP-2 protein levels, and upregulated caspase-3 GC cells, but these changes were partially reversed by co-transfection of si-TUSC7.
 Conclusion: TSUC7 is downregulated in GC, and its low level predicts poor prognosis in GC patients. E2F6 promotes proliferation and invasion of GC cells by negatively regulating TUSC7. Therefore, E2F6/TUSC7 may be utilized as potential therapeutic targets for GC.

CircCRIM1 mediates proliferation, migration, and invasion of trophoblast cell through regulating miR-942-5p/IL1RAP axis.

Preeclampsia (PE) is a severe complication that occurs during pregnancy and a main cause of perinatal mortality of mothers as well as infants, which is characterized by abnormal placental trophoblast. Previous study reported that aberrant circular RNA (circRNA) was involved in the pathogenesis and progression of PE. Herein, we aimed to investigate the role of circCRIM1 and explore the mechanism of circCRIM1 in PE. The quantitative real-time PCR (qRT-PCR) was conducted to determine the relative expression of circCRIM1, miR-942-5p, and IL1RAP in tissues and cells. Cell proliferation viability was assessed by both MTT and EdU assays. Cell cycle distribution was analyzed using flow cytometry. Transwell assay was performed to test the cell migration and invasion. The protein levels of CyclinD1, MMP9, MMP2, and IL1RAP were measured by western blot. The putative binding sites between miR-942-5p and circCRIM1 or IL1RAP 3'UTR were verified by dual-luciferase reporter gene assay. Rescue experiment was performed to confirm that miR-942-5p/IL1RAP axis was functional target of circCRIM1 in trophoblast cells. CircCRIM1 was upregulated in placenta tissues of PE and its expression was inversely related to infant weight. Overexpression of circCRIM1 suppressed proliferation, migration, and invasion and reduced the protein levels of CyclinD1, MMP9, MMP2 of trophoblast cells, whereas its knockdown exerted the opposite effect. CircCRIM1 could interact with miR-942-5p, and introduction of miR-942-5p partially abated the inhibitory effect of circCRIM1 on trophoblast cell behaviors. IL1RAP was directly targeted and negatively regulated by miR-942-5p. miR-942-5p played its regulatory role on cell proliferation, migration, and invasion of trophoblast by IL1RAP. Further analysis showed that circCRIM1 modulated IL1RAP expression via sponging miR-942-5p. The results of the present study demonstrated that circCRIM1 inhibited the proliferation, migration, and invasion of trophoblast cells through sponging miR-942-5p and up-regulating IL1RAP, providing a possible new mechanism of PE.

ANTIFIBROTIC POTENTIAL OF ANGIOTENSIN (1-7) IN HEMODYNAMICALLY OVERLOADED RAT HEART

Objective: Heart failure is a clinical syndrome characterized by cardiac dysfunction due to structural abnormalities of the myocardium that results in the inability of the heart to eject sufficient volume of the blood to the circulation. HF is a major cause of morbidity and mortality in the world. The ECM is a highly dynamic structure controlling the proper functioning of heart muscle. ECM remodelling with enhanced deposition of collagen due to hemodynamic overload impairs cardiomyocyte adhesion and electrical coupling that contributes to cardiac mechanical dysfunction and arrhythmias. Design and method: We aimed to explore ECM and connexin-43 (Cx43) signalling pathways in hemodynamically overloaded rat heart as well as the possible implication of Ang(1-7) to prevent/attenuate adverse myocardial remodelling. Males, 8 weeks-old, normotensive Hannover Spraque-Dawley rats (HSD), hypertensive Ren-2 transgenic rats (TGR) and Ang(1-7)-transgenic rats (TGR(A1-7)) underwent aortocaval fistula (ACF) to produce volume overload. 5 weeks later, were right and left ventricular tissue samples used for biometric, biochemical, and proteomic analyzes. Results: Cardiac hypertrophy in response to volume overload was significantly less pronounced in TGR(A1-7) comparing to HSD rats. Moreover, a marker of fibrosis, hydroxyproline, was increased in both ventricles of volume overloaded TGR while reduced in Ang(1-7) right heart ventricle. Protein level and activity of MMP-2 were reduced in both ventricles of volume overloaded TGR/TGR(A1-7) compared to HSD. SMAD2/3 protein levels were decreased in the right ventricle of TGR(A1-7) compared to HSD/TGR in response to volume overload. In parallel, Cx43 and pCx43 implicated in electrical coupling were increased in TGR(A1-7) versus HSD/TGR. PKC↑ and PKC↗ showed a decrease in the TGR group compared to HSD and a decrease in the TGR(A1-7) ACF group compared to HSD and TGR after ACF in both chambers. Conclusions: It can be concluded that Ang(1-7) exhibits cardioprotective and anti-fibrotic potential in condition of cardiac volume overload. If we look at the results comprehensively, it can be concluded that Ang(1-7) exhibits cardioprotective and antifibrotic potential in condition of cardiac volume overload. Supported by grants VEGA 2/0006/23, APVV 21-0410.

CircKDM1B promotes hepatocellular carcinoma progression through regulating miR-1322/PRC1 axis.

Circular RNA (circRNA) is considered an important molecular marker for the early diagnosis of tumors and a potential therapeutic target. Herein, we investigated the role and regulatory mechanism of circKDM1B in hepatocellular carcinoma (HCC). The expression of circKDM1B, miR-1322 and Protein regulator of cytokinesis 1 (PRC1) mRNA were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Cell counting kit-8 (CCK8) and 5-ethynyl-2'-deoxyuridine (EdU) staining assays were performed to assess cell proliferation activity. Cell migration and invasion were detected by wound-healing scratch and transwell assay. Flow cytometry was used to analyze cell apoptosis. The protein levels of PCNA, MMP9, C-caspase3 and PRC1 were examined using western blot. The binding of circKDM1B and miR-1322 was verified by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) and RNA pull down assay. CircKDM1B was overexpressed in HCC tissues and cells, and its overexpression was related to tumor stage and poor prognosis of HCC patients. Functionally, knockdown of circKDM1B suppressed proliferation, migration, invasion and promoted apoptosis of HCC cells. Mechanistically, circKDM1B functioned as ceRNA of miR-1322 to upregulate PRC1 in HCC cells. Overexpression of miR-1322 inhibited proliferation, migration, invasion and facilitated apoptosis of HCC cells, which was partly reversed by PRC1 overexpression. CircKDM1B knockdown impeded HCC tumor growth in vivo. CircKDM1B played a critical role in HCC progression by regulating cell proliferation, migration, invasion and apoptosis. CircKDM1B/miR-1322/PRC1 axis might be a novel therapeutic target of HCC patients.

Analysis of the potential biological mechanisms of diosmin against renal fibrosis based on network pharmacology and molecular docking approach

BackgroundInterstitial fibrosis is involved in the progression of various chronic kidney diseases and renal failure. Diosmin is a naturally occurring flavonoid glycoside that has antioxidant, anti-inflammatory, and antifibrotic activities. However, whether diosmin protects kidneys by inhibiting renal fibrosis is unknown.MethodsThe molecular formula of diosmin was obtained, targets related to diosmin and renal fibrosis were screened, and interactions among overlapping genes were analyzed. Overlapping genes were used for gene function and KEGG pathway enrichment analysis. TGF-β1 was used to induce fibrosis in HK-2 cells, and diosmin treatment was administered. The expression levels of relevant mRNA were then detected.ResultsNetwork analysis identified 295 potential target genes for diosmin, 6828 for renal fibrosis, and 150 hub genes. Protein–protein interaction network results showed that CASP3, SRC, ANXA5, MMP9, HSP90AA1, IGF1, RHOA, ESR1, EGFR, and CDC42 were identified as key therapeutic targets. GO analysis revealed that these key targets may be involved in the negative regulation of apoptosis and protein phosphorylation. KEGG indicated that pathways in cancer, MAPK signaling pathway, Ras signaling pathway, PI3K-Akt signaling pathway, and HIF-1 signaling pathway were key pathways for renal fibrosis treatment. Molecular docking results showed that CASP3, ANXA5, MMP9, and HSP90AA1 stably bind to diosmin. Diosmin treatment inhibited the protein and mRNA levels of CASP3, MMP9, ANXA5, and HSP90AA1. Network pharmacology analysis and experimental results suggest that diosmin ameliorates renal fibrosis by decreasing the expression of CASP3, ANXA5, MMP9, and HSP90AA1.ConclusionsDiosmin has a potential multi-component, multi-target, and multi-pathway molecular mechanism of action in the treatment of renal fibrosis. CASP3, MMP9, ANXA5, and HSP90AA1 might be the most important direct targets of diosmin.

Palmitic acid promotes human retinal pigment epithelial cells migration by upregulating miR-222 expression and inhibiting NUMB.

High glucose promotes retinal pigment epithelial cell (RPEC) migration. However, the underlying molecular mechanisms explaining how high fatty acid levels affect RPEC migration remain largely unknown. We investigated whether and how palmitic acid (PA) impacts the migration of human RPEC cell line ARPE-19. ARPE-19 cells were treated with varying doses of palmitic acid, and the RPEC migration was evaluated by scratch and transwell migration assays. Cell viability was determined by the CCK-8 method. The levels of epithelial-mesenchymal transition (EMT)-associated proteins, including E-cadherin, vimentin, MMP2, and MMP3, were evaluated by western blot. The microRNAs and mRNAs levels were assessed by quantitative PCR. miRNA targets were predicted with online tools and validated with the luciferase reporter assay. miRNA mimics, inhibitors, and siRNA oligos were used to perform gain-of-function and loss-of-function studies. We found that PA increased viability of ARPE-19 cells, promoted their migration and EMT. PA decreased E-cadherin protein expression, and increased vimentin, MMP2, and MMP3 protein levels. Additionally, PA increased miR-222 expression in ARPE-19 cells, and functionally blocking miR-222 suppressed the PA-induced RPEC migration and EMT. NUMB was identified as a downstream target of miR-222, and NUMB knockdown abolished the effects of PA on promoting the migration and EMT of ARPE-19 cells. Therefore, PA promotes human RPEC migration by upregulating miR-222 expression and downregulating NUMB. This study unravels a novel PA-miR-222-NUMB axis that can be potentially targeted for therapy of high fat acid-related ocular diseases.

ALIX promotes cell migration and invasion of head and neck squamous cell carcinoma by regulating the expression of MMP9, MMP14, VEGF-C

ObjectiveThe poor survival rate of head and neck squamous cell carcinoma (HNSCC), one of the most prevalent human cancer, is attributed to frequent locoregional recurrence and lymph node metastases. Though it is reported that the expression of ALG-2 interacting protein X (ALIX) closely correlates with the progression of various tumors, its role in HNSCC remains unclear. The present study aims to investigate the role of ALIX in the development of HNSCC. DesignWith immunohistochemical staining, the expression levels of ALIX and series of related functional proteins were compared in normal mucosal (n = 18), HNSCC tissues (n = 54), and metastatic lymph nodes (n = 11). Further, the correlation analysis was performed among the proteins detected. By knocking down ALIX in HNSCC cell lines, the correlation of ALIX with the proteins was verified in vitro. The role of ALIX in proliferation, migration, and invasion of HNSCC cells was further studied by flow cytometry, wounding healing, and transwell assays, respectively. ResultsHigher expression level of ALIX was revealed in HNSCC samples, especially in metastatic lymph nodes, than in normal mucosal tissues. Accordingly, increasing levels of MMP9, MMP14, and VEGF-C were also discovered in metastatic lymph nodes and significantly correlated with the expression of ALIX. In vitro assays demonstrated that the knockdown of ALIX reduced both the transcriptional and protein levels of MMP9, MMP14, and VEGF-C, together with suppressed migration and weakened invasion of HNSCC cell lines. ConclusionsALIX up-regulated the expression of MMP9, MMP14 and VEGF-C, and promoted migration and invasion of HNSCC cells.

Effect of mir-92a-3p on hydroquinone induced changes in human lymphoblastoid cell cycle and apoptosis.

Hydroquinone (HQ), one of the metabolites of benzene in humans, has significant hepatotoxic properties. Chronic exposure to HQ can lead to leukemia. In a previous study by this group, we constructed a model of malignant transformation of human lymphoblastoid cells (TK6) induced by chronic exposure to HQ with significant subcutaneous tumorigenic capacity in nude mice. miR-92a-3p is a tumor factor whose role in HQ-induced malignant transformation is not yet clear. In the present study, raw signal analysis and dual-luciferase reporter gene results suggested that miR-92a-3p could target and regulate TOB1, and the expression level of miR-92a-3p was significantly upregulated in the long-term HQ-induced TK6 malignant transformation model, while the anti-proliferative factor TOB1 was significantly downregulated. To investigate the mechanism behind this, we inhibited miR-92a-3p in a malignant transformation model and found a decrease in cell viability, a decrease in MMP-9 protein levels, a G2/M phase block in the cell cycle, and an upregulation of the expression of G2/M phase-related proteins cyclinB1 and CDK1. Inhibition of miR-92a-3p in combination with si-TOB1 restored cell viability, inhibited cyclin B1 and CDK1 protein levels, and attenuated the G2/M phase block. Taken together, miR-92a-3p reduced the cell proliferation rate of HQ19 and caused cell cycle arrest by targeting TOB1, which in turn contributed to the altered malignant phenotype of the cells. This study suggests that miR-92a-3p is likely to be a biomarker for long-term HQ-induced malignant transformation of TK6 and could be a potential therapeutic target for leukemia caused by long-term exposure to HQ.

Differential bone remodeling mechanism in hindlimb unloaded and hibernating Daurian ground squirrels: a comparison between artificial and natural disuse within the same species.

Loss of bone mass can occur in mammals after prolonged disuse but the situation for hibernators that are in a state of torpor for many months of the year is not yet fully understood. The present study assesses the bone remodeling mechanisms present in Daurian ground squirrels (Spermophilus dauricus) during hibernation as compared with a model of hindlimb disuse. Differences in microstructure, mechanical properties, bone remodeling-related proteins (Runx2, OCN, ALP, RANKL, CTK and MMP-9) and key proteins of Wnt/β-catenin signaling pathway (GSK-3β and phospho-β-catenin) were evaluated in ground squirrels under 3 conditions: summer active (SA) vs. hibernation (HIB) vs. hindlimb unloaded (HLU). The results indicated that the body weight in HLU ground squirrels was lower than the SA group, and the middle tibia diameter in the HLU group was lower than that in SA and HIB groups. The thickness of cortical and trabecular bone in femurs from HLU ground squirrels was lower than in SA and HIB groups. Most parameters of the tibia in the HLU group were lower than those in SA and HIB groups, which indicated cortical bone loss in ground squirrels. Moreover, our data showed that the changes in microscopic parameters in the femur were more obvious than those in the tibia in HLU and HIB ground squirrels. The levels of Runx2 and ALP were lower in HLU ground squirrels than SA and HIB groups. The protein levels of OCN were unchanged in the three groups, but the protein levels of ALP were lower in the HLU group than in SA and HIB groups. RANKL, CTK and MMP-9 protein levels were significantly decreased in tibia of HLU ground squirrels as compared with SA and HIB groups. In addition, the protein expression levels of RANKL, CTK and MMP-9 showed no statistical difference between SA and HIB ground squirrels. Thus, the mechanisms involved in the balance between bone formation and resorption in hibernating and hindlimb unloading ground squirrels may be different. The present study showed that in femur, the Wnt signaling pathway was inhibited, the protein level of GSK-3β was increased, and the protein expression of phospho-β-catenin was decreased in the HIB group as compared with the SA group, which indicates that the Wnt signaling pathway has a great influence on the femur of the HIB group. In conclusion, the natural anti-osteoporosis properties of Daurian ground squirrels are seasonal. The squirrels do not experience bone loss when they are inactive for a long time during hibernation, but the mechanisms of anti-osteoporosis did not work in HLU summer active squirrels.

MicroRNA-150-5p inhibits the proliferation and invasion of human larynx epidermiod cancer cells though regulating peptidyl-prolyl cis/trans isomerase

ObjectiveThis study aimed to investigate the molecular mechanism of miR-150-5p regulating the malignant biological behavior of Human Epidermoid cancer cell (HEp-2) by targeting peptidyl-prolyl cis/trans isomerase NIMA-Interacting-1 (PIN1). MethodsFirstly, qRT-PCR and Western blot were adopted to detect the expression levels of miR-150-5p and PIN1 in cancer tissue and paracancerous tissues of patients with LSCC, and those in human bronchial epithelial cells (16 HBE) and HEp-2. Next, the targeted relationship between miR-150-5p and PIN1 was assessed by bioinformatics website and dual-luciferase reporter assay, followed by their correlation analysis. Besides, after interfering with miR-150-5p or PIN1 expression in HEp-2 cells, CCK-8, cell colony formation assay, and transwell assay were utilized to detect the proliferation, viability, and invasion of cells, respectively. Subsequently, the protein levels of MMP-2, MMP-9, and EMT-related proteins in HEp-2 cells were checked by Western blot. ResultsExpression of miR-150-5p was down-regulated in LSCC tissues and HEp-2 cells. Moreover, miR-150-5p suppressed proliferation and invasion of HEp-2 cells, affected protein expression related to MMP and EMT, thereby inhibiting development of cancer. The expression of PIN1 was significantly increased in cancer tissues and HEp-2 cells, and there was a targeted relationship and negative correlation between miR-150-5p and PIN1 in cancer tissue. However, overexpression of PIN1 could reverse the effect of miR-150-5p on the proliferation and invasion of HEp-2 cells. ConclusionIn a nutshell, there is a targeted relationship between PIN1 and miR-150-5p. Besides, miR-150-5p can inhibit the proliferation and invasion of HEp-2 cells by regulating the expression of PIN1. Level of evidence3.

Zinc Finger Protein 90 Knockdown Promotes Cisplatin Sensitivity via Nrf2/HO-1 Pathway in Ovarian Cancer Cell.

Our study discussed the role of Zfp90 in ovarian cancer (OC) cell lines' sensitivity to cisplatin. We used two OC cell lines, SK-OV-3 and ES-2, to evaluate their role in cisplatin sensitization. The protein levels of p-Akt, ERK, caspase 3, Bcl-2, Bax, E-cadherin, MMP-2, MMP-9 and other drug resistance-related molecules, including Nrf2/HO-1, were discovered in the SK-OV-3 and ES-2 cells. We also used a human ovarian surface epithelial cell to compare the effect of Zfp90. Our outcomes indicated that cisplatin treatment generates reactive oxygen species (ROS) that modulate apoptotic protein expression. The anti-oxidative signal was also stimulated, which could hinder cell migration. The intervention of Zfp90 could greatly improve the apoptosis pathway and block the migrative pathway to regulate the cisplatin sensitivity in the OC cells. This study implies that the loss of function of Zfp90 might promote cisplatin sensitization in OC cells via regulating the Nrf2/HO-1 pathway to enhance cell apoptosis and inhibit the migrative effect in both SK-OV-3 and ES-2 cells.

Therapeutic Potential of Zeolites/Vitamin B12 Nanocomposite on Complete Freund's Adjuvant-Induced Arthritis as a Bone Disorder: In Vivo Study and Bio-Molecular Investigations.

Rheumatoid arthritis (RA) is a long-term autoimmune disease. As nanotechnology has advanced, a growing number of nanodrugs have been used in the treatment of RA due to their unique physical and chemical properties. The purpose of this study was to assess the therapeutic potential of a novel zeolite/vitamin B12 nanocomposite (Nano ZT/Vit B12) formulation in complete Freund's adjuvant (CFA)-induced arthritis. The newly synthesized Nano ZT/Vit B12 was fully characterized using various techniques such as XRD, FT-IR, BET analysis, HERTEM, SEM, practical size, zeta potential, XRF, and EDX. The anti-arthritic, anti-inflammatory, and antioxidant activities as well as the immunomodulation effect of Nano ZT/Vit B12 on the CFA rat model of arthritis were examined. Histopathologic ankle joint injuries caused by CFA intrapedal injection included synovium hyperplasia, inflammatory cell infiltration, and extensive cartilage deterioration. The arthritic rats' Nano ZT/Vit B12 supplementation significantly improved these effects. Furthermore, in arthritic rats, Nano ZT/Vit B12 significantly reduced serum levels of RF and CRP, as well as the levels of IL-1β, TNF-α, IL-17, and ADAMTS-5, while increasing IL-4 and TIMP-3 levels. Nano-ZT/Vit B12 significantly declined the LPO level and increased antioxidant activities, such as GSH content and GST activity, in the arthritic rats. In arthritic rats, Nano ZT/Vit B12 also reduced TGF-β mRNA gene expression and MMP-13 protein levels. Collectively, Nano ZT/Vit B12 seems to have anti-arthritic, anti-inflammatory, and antioxidant properties, making it a promising option for RA in the future.

Anti-Fibrotic Potential of Angiotensin (1-7) in Hemodynamically Overloaded Rat Heart

The extracellular matrix (ECM) is a highly dynamic structure controlling the proper functioning of heart muscle. ECM remodeling with enhanced collagen deposition due to hemodynamic overload impairs cardiomyocyte adhesion and electrical coupling that contributes to cardiac mechanical dysfunction and arrhythmias. We aimed to explore ECM and connexin-43 (Cx43) signaling pathways in hemodynamically overloaded rat heart as well as the possible implication of angiotensin (1-7) (Ang (1-7)) to prevent/attenuate adverse myocardial remodeling. Male 8-week-old, normotensive Hannover Spraque-Dawley rats (HSD), hypertensive (mRen-2)27 transgenic rats (TGR) and Ang (1-7) transgenic rats (TGR(A1-7)3292) underwent aortocaval fistula (ACF) to produce volume overload. Five weeks later, biometric and heart tissue analyses were performed. Cardiac hypertrophy in response to volume overload was significantly less pronounced in TGR(A1-7)3292 compared to HSD rats. Moreover, a marker of fibrosis hydroxyproline was increased in both ventricles of volume-overloaded TGR while it was reduced in the Ang (1-7) right heart ventricle. The protein level and activity of MMP-2 were reduced in both ventricles of volume-overloaded TGR/TGR(A1-7)3292 compared to HSD. SMAD2/3 protein levels were decreased in the right ventricle of TGR(A1-7)3292 compared to HSD/TGR in response to volume overload. In parallel, Cx43 and pCx43 implicated in electrical coupling were increased in TGR(A1-7)3292 versus HSD/TGR. It can be concluded that Ang (1-7) exhibits cardio-protective and anti-fibrotic potential in conditions of cardiac volume overload.