Western Blot Assay Research Articles

Inhibition of SENP5 reduces brain injury in TBI rats by regulating NEDD4L/TCF3 axis.

Background: The underlying mechanism of SENP5 influences neuronal regeneration and apoptosis in the context of TBI remains largely unexplored.Methods: In the present study, PC12 cells treated with scratch for 24 h were regarded as a TBI cell model. The expression of SENP5 in PC12 cells was measured via Quantitative Real-Time PCR (qRT-PCR) and western blot assays. Cell Counting Kit 8 (CCK-8) and Flow cytometry assays were used to evaluate the activity of TBI cells. In addition, we assessed the effect of inhibiting SENP5 in vivo on neurological function deficits and apoptosis in the hippocampal tissues of TBI rats. The relationship between SENP5 and NEDD4L/TCF3 axis was proved via immunoprecipitation (IP) and double luciferase assays.Results: Following TBI cell modeling, an increase in SENP5 expression has been found. Moreover, TBI modeling resulted in reduced cell viability and increased apoptosis, which was rescue by inhibition of SENP5. In vivo experiments demonstrated that SENP5 inhibition could mitigate TBI-induced brain injury in rats. Specifically, this inhibition led to lower neurological impairment scores, improved neuronal morphology and structure, and decreased neuronal apoptosis. In addition, NEDD4L has been proved to be relevant to the enhanced stability of the transcription factor TCF3, which in turn promoted the expression of SENP5.Conclusions: This study reveals that inhibiting SENP5 can alleviate brain injury following TBI. NEDD4L/TCF3 axis can regulate the expression of SENP5 to affect the development of TBI. However, SENP5 regulates downstream targets of TBI and important mechanisms need to be further explored.

Crosstalk between cancer cells and macrophages promotes OSCC cell migration and invasion through a CXCL1/EGF positive feedback loop.

C-X-C motif chemokine ligand 1 (CXCL1) and epithelial growth factor (EGF) are highly secreted by oral squamous cell carcinoma (OSCC) cells and tumor-associated macrophages, respectively. Recent studies have shown that there is intricate "cross-talk" between OSCC cells and macrophages. However, the underlying mechanisms are still poorly elucidated. The expression of CXCL1 was detected by immunohistochemistry in OSCC clinical samples. CXCL1 levels were evaluatedby RT‒PCR and ELISA in an OSCC cell line and a normal epithelial cell line. The expression of EGF was determined by RT‒PCR and ELISA. The effect of EGF on the proliferation of OSCC cells was evaluated by CCK-8 and colony formation assays. The effect of EGF on the migration and invasion ability and epithelial-mesenchymal transition (EMT) of OSCC cells was determined by wound healing, Transwell, RT‒PCR, Western blot and immunofluorescence assays. The polarization of macrophages was evaluated by RT‒PCR and flow cytometry. Western blotting was used to study the molecular mechanism in OSCC. The expression of C-X-C motif chemokine ligand 1 (CXCL1) was higher in the OSCC cell line (Cal27) than in immortalized human keratinocytes (Hacat cells). CXCL1 derived from Cal27 cells upregulates the expression of epithelial growth factor (EGF) in macrophages. Paracrine stimulation mediated by EGF further facilitates the epithelial-mesenchymal transition (EMT) of Cal27 cells and initiates the upregulation of CXCL1 in a positive feedback-manner. Mechanistically, EGF signaling-induced OSCC cell invasion and migration can be ascribed to the activation of NF-κB signaling mediated by the epithelial growth factor receptor (EGFR), as determined by western blotting. OSCC cell-derived CXCL1 can stimulate the M2 polarization of macrophages and the secretion of EGF. Moreover, EGF significantly activates NF-κB signaling and promotes the migration and invasion of OSCC cells in a paracrine manner. A positive feedback loop between OSCC cells and macrophages was formed, contributing to the promotion of OSCC progression.

Atractylenolide II regulates the proliferation, ferroptosis, and immune escape of hepatocellular carcinoma cells by inactivating the TRAF6/NF-κB pathway.

Hepatocellular carcinoma (HCC) is a common and lethal tumor worldwide. Atractylenolide II (AT-II) is a natural sesquiterpenoid monomer, with anti-tumor effect. To address the effect and mechanisms of AT-II on HCC. The role and mechanisms of AT-II were assessed through cell counting kit-8, flow cytometry, enzyme-linked immunosorbent assay, immunofluorescence, and western blot experiments in Hep3B and Huh7 cells. In vivo experiments were conducted in BALB/c nude mice using immunohistochemistry and western blot assays. AT-II decreased the cell viability of Hep3B and Huh7 cells with a IC50 of 96.43 µM and 118.38 µM, respectively. AT-II increased relative Fe2+ level, which was further promoted with the incubation of erastin and declined with the ferrostatin-1 in Hep3B and Huh7 cells. AT-II enhanced the level of ROS and MDA, but reduced the GSH level, and the expression of xCT and GPX4. AT-II elevated the percent of CD8+ T cells and the IFN-γ contents, and declined the IL-10 concentrations and the expression of PD-L1 in Hep3B and Huh7 cells. AT-II downregulated the relative protein level of TRAF6, p-p65/p-65, and p-IkBα/IkBα, which was rescued with overexpression of TRAF6. Upregulation of TRAF6 also reversed the effect of AT-II on proliferation, ferroptosis, and immune escape in Hep3B cells. In vivo, AT-II reduced tumor volume and weight, the level of GPX4, xCT, and PD-L1, and the expression of TRAF6, p-p65/p-65, and p-IkBα/IkBα, with the increased expression of CD8. AT-II modulated the proliferation, ferroptosis, and immune escape of HCC cells by downregulating the TRAF6/NF-κB pathway.

Inhibition of Liver Cancer Cell Viability by Triazole through Up-regulation of p38 Phosphorylation and Targeting the Activation of p-ERK1/2 and Akt Protein Expression.

The present study was aimed to explore the effect of triazole on growth and viability of liver cancer cells. Cell growth was examined using the MTT test and expression of several proteins was assessed by western blotting assay. The Matrigel-coated Transwell assay was employed to examine the infiltration of cells. The data from MTT assay showed that MHCC97H and H4TG liver cancer cell viability was inhibited by triazole in a concentration-dependent manner. After treatment with 0.5, 1.0, 2.0, 4, 8, and 16 µM doses of triazole, the rate of H4TG cell viability was decreased to 96, 73, 58, 39, 29, and 28%, respectively. Treatment of MHCC97H cells with 0.5, 1.0, 2.0, 4, 8, and 16 µM doses of triazole resulted in a reduction in cell viability to 94, 70, 53, 35, 22, and 21%, respectively. Triazole treatment also led to a significant reduction in MHCC97H cell invasiveness compared to the control cells. In MHCC97H cells treated with triazole, there was a noticeable decrease in the levels of p-ERK1/2, and p-Akt protein expression. Treatment of MHCC97H cells with triazole resulted in a prominent increase in p-p38 level. In summary, triazole inhibits growth and viability of liver cancer cells through targeting the activation of p-ERK1/2 and Akt proteins. Therefore, triazole may be investigated further as a therapeutic agent for the treatment of liver cancer.

Abstract PO4-08-01: Methylsterol monooxygenase 1 (MSMO1) modulates endoplasmic reticulum stress through cholesterol metabolic reprogramming to alter chemotherapy sensitivity in breast cancer

Abstract Background Endoplasmic reticulum stress (ER stress) is a process in which cells activate unfolded protein response and other signaling pathways in response to misfolded and unfolded protein aggregation and calcium ion balance disturbance in the endoplasmic reticulum cavity. Depending on the intensity of endoplasmic reticulum stress, it can not only induce chaperone expression to play a protective effect, but also induce apoptosis independently. Metabolic reprogramming is one of the characteristics of tumor, which refers to the metabolic changes that cells make in response to various stimuli. Here, we demonstrate that methylsterol monooxygenase 1 (MSMO1) regulates cellular cholesterol metabolism and influences breast cancer susceptibility to chemotherapy by regulating the relative intracellular T-MAS content. Methods RNA-seq was performed on core needle biopsy samples from 26 patients receiving neoadjuvant chemotherapy for breast cancer to screen for genes that may influence neoadjuvant chemotherapy sensitivity. Cell proliferation, CCK8 assay and apoptosis assay were performed for MSMO1 phenotype study. Transmission electron microscopy (TEM) was conducted to visualize the structure of endoplasmic reticulum. Western Blot was used to detect the activation level of the unfolded protein response pathway. The relative content of sterols in MSMO1 knockdown cells was detected by targeted lipid mass spectrometry. AO/PI staining of breast cancer organoids was performed to verify the effect of MSMO1 metabolic substrate T-MAS on chemotherapy drug sensitivity. Results RNA-seq of core needle biopsy samples from breast cancer patients receiving neoadjuvant chemotherapy suggested that MSMO1 was highly expressed in non-pCR patients, and clinical survival data suggested that MSMO1 was associated with poor prognosis in patients with all types of breast cancer, suggesting that MSMO1 might be related to chemotherapy sensitivity of breast cancer. Phenotypic experiments revealed that MSMO1 regulates chemotherapy sensitivity of breast cancer both in vivo and in vitro. Ectopic overexpression of MSMO1 promoted cancer cell resistance to chemotherapy. To uncover the underlying mechanisms, RNA-seq of MSMO1 knock-down cells were carried out and identified that MSMO1 was associated with ER stress. TEM and western blot assay indicated that downregulation of MSMO1 expression leads to endoplasmic reticulum swelling and activation of the unfolded protein response. Considering that MSMO1 is a cholesterol metabolizing enzyme, targeted lipids mass spectrometry was performed to detect the changes in the contents of various intermediate metabolites in the cholesterol pathway, which indicated that the relative content of T-MAS, a metabolic substrate of MSMO1, was significantly up-regulated in MSMO1 knockdown cells. Then, in order to verify whether this metabolite can independently regulate ER stress, breast cancer cells were given T-MAS externally, and TEM and Western Blot analysis indicated that T-MAS could induce ER stress and activate the unfolded protein response independently. Apoptosis assay and AO/PI staining of breast cancer organoids revealed that T-MAS can improve the sensitivity of breast cancer to chemotherapy drugs. Conclusion In summary, these results shed light on the role of MSMO1 in cholesterol metabolism reprogramming, which adjust the endoplasmic reticulum stress state of cells by changing the relative content of intracellular T-MAS, and thereby influencing the sensitivity of breast cancer chemotherapy. As an intermediate metabolite of cholesterol metabolism, T-MAS has potential clinical application value of chemotherapy sensitization. Citation Format: Hengyu Ren, Liren Wangxu, Yayun Chi, Jiong Wu. Methylsterol monooxygenase 1 (MSMO1) modulates endoplasmic reticulum stress through cholesterol metabolic reprogramming to alter chemotherapy sensitivity in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-08-01.

Whole transcriptome profiling reveals a lncMDP1 that regulates myogenesis by adsorbing miR-301a-5p targeting CHAC1

Myoblast proliferation and differentiation are essential for skeletal muscle development. In this study, we generated the expression profiles of mRNAs, long noncoding RNAs (lncRNAs), and microRNAs (miRNAs) in different developmental stages of chicken primary myoblasts (CPMs) using RNA sequencing (RNA-seq) technology. The dual luciferase reporter system was performed using chicken embryonic fibroblast cells (DF-1), and functional studies quantitative real-time polymerase chain reaction (qPCR), cell counting kit-8 (CCK-8), 5-Ethynyl-2′-deoxyuridine (EdU), flow cytometry cycle, RNA fluorescence in situ hybridization (RNA-FISH), immunofluorescence, and western blotting assay. Our research demonstrated that miR-301a-5p had a targeted binding ability to lncMDP1 and ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1). The results revealed that lncMDP1 regulated the proliferation and differentiation of myoblasts via regulating the miR-301a-5p/CHAC1 axis, and CHAC1 promotes muscle regeneration. This study fulfilled the molecular regulatory network of skeletal muscle development and providing an important theoretical reference for the future improvement of chicken meat performance and meat quality.

FBXO28 reduces high-fat diet-induced hyperlipidemia in mice by alleviating abnormal lipid metabolism and inflammatory responses.

Hyperlipidemia is a lipid metabolism disorder with increasing incidence and prevalence worldwide. Abnormal lipid metabolism and inflammation are two significant characteristics of hyperlipidemia. The purpose of this study was to explore the role and mechanism of F-box only protein 28 (FBXO28) in hyperlipidemia. Mice were fed with high-fat diet (HFD) to elicit obesity, and 3T3-L1 preadipocytes were stimulated with MDI cocktail (IBMX, DEX and insulin) to evoke differentiation. In vivo and in vitro role of FBXO28 in hyperlipidemia was investigated by hematoxylin-eosin and oil Red O staining, the lipid biochemistry measurement, enzyme-linked immunosorbent assay, reverse transcription quantitative polymerase chain reaction and western blotting assays. The mechanism of FBXO28 explored by co-immunoprecipitation, immunofluorescence, ubiquitination and cycloheximide assays. Low expression of FBXO28 was found in hyperlipidemia in silico, in vivo and in vitro. Upregulation of FBXO28 declined the body weight, fat accumulation, and serum lipid content in HFD-fed mice. Abnormal lipid accumulation, and the level of liposynthetic genes and beta-oxidation related genes were improved by overexpression of FBXO28 both in HFD-elicited mice and MDI-treated 3T3-L1 preadipocytes. Besides, overexpression of FBXO28 declined HFD-induced the level of proinflammatory factors and F4/80. Mechanically, FBXO28 directly bound RAB27A and promoted its ubiquitinated degradation. Thus, upregulation of RAB27A inverted the improved role of FBXO28 in abnormal lipid metabolism and inflammation in vivo and in vitro. FBXO28 ameliorated abnormal lipid metabolism and inflammation through the ubiquitinated degradation of RAB27A, thereby attenuating HFD-induced hyperlipidemia. The results could promote the treatment of hyperlipidemia, and the relevant diseases.

Scutellarein alleviates osteoarthritis progression through the PI3K/Akt/NF-kappaB signaling pathway: In vitro and in vivo studies.

Osteoarthritis (OA), a joint disease that is associated with inflammatory processes is involved in joint destruction. Scutellarein (Scu), a component of the medicinal herbs Scutellaria barbata D. Don and Erigeron breviscapus (vant) Hand Mass, has anti-inflammatory effects. We explored the role of Scu in the development of OA and the underlying mechanisms. CCK-8 assays, Calcein-AM/PI and EdU staining were used to determine chondrocyte viability after Scu exposure. Western blot, qPCR, as well as ELISA were utilized to measure extracellular matrix (ECM) degradation and inflammation. Immunofluorescence (IF), western blot and luciferase assays were used to examine the NF-kappaB (NF-κB) pathway. Scu interacting proteins were predicted using network pharmacology analysis and molecular docking. X-ray, H&E, Safranin O-Fast Green(S-O), toluidine blue, and immunohistochemistry analysis were used to examine the therapeutic effects of Scu in OA using destabilization of medial meniscus (DMM) models. Scu demonstrated inhibitory effects on ECM degradation and pro-inflammatory factor levels in chondrocytes treated with IL-1β. Mechanistically, Scu inhibited the IL-1β-induced activation of the PI3K/Akt/ NF-κB signaling pathway cascades. Furthermore, Scu has been shown to have significant binding capacities to PI3K. Additionally, Scu ameliorated the OA progression in DMM models. Our findings suggest that Scu may contribute to the amelioration of OA progression by targeting the PI3K/Akt/NF-κB signaling pathway, implying Scu possesses promising therapeutic potential for the treatment of OA.

Abstract PO2-05-09: Beyond endocrine resistance: estrogen receptor (ESR1) activating mutations mediate chemotherapy resistance through the JNK/c-Jun MDR1 pathway in breast cancer

Abstract Purpose: All patients with metastatic breast cancer (MBC) expressing estrogen receptor-α (ESR1) will eventually develop resistance to endocrine therapies. In up to 40% of patients, this resistance is caused by activating mutations in the ligand-binding domain (LBD) of ESR1. Accumulating clinical evidence indicate adverse outcomes for these patients, beyond that expected by resistance to endocrine therapy. We hypothesized that ESR1 mutations may also confer resistance to chemotherapy. Experimental Design: MCF-7 cells harboring Y537S and D538G ESR1 mutations (mut-ER) were employed to study response to chemotherapy using viability and apoptotic assay in vitro, and tumor growth in vivo. JNK/c-Jun/MDR1 pathway was studied using qRT-PCR, western-blot, gene-reporter and ChIP assays. MDR1 expression was analyzed in clinical samples using IHC. Results: Cell harboring ESR1 mutations displayed relative chemoresistance, evidenced by higher viability and reduced apoptosis as well as resistance to paclitaxel in vivo. To elucidate the underlying mechanism, MDR1 expression was examined and elevated levels were observed in mut-ER cells, and in clinical BC samples. MDR1 is regulated by the JNK/cJun pathway, and indeed, we detected higher JNK/cJun expression and activity in mut-ER cells, as well as increased occupancy of c-Jun in MDR1 promoter. Importantly, JNK inhibition decreased MDR1 expression, particularly of D538G-cells, and reduced viability in response to chemotherapy. Conclusions: Taken together, these data indicate that ESR1 mutations confer chemoresistance in BC through activation of the JNK/MDR1 axis. Targeting this pathway may restore sensitivity to chemotherapy and serve as a novel treatment strategy for MBC patients carrying ESR1 mutations. Citation Format: Marwa Taya, Keren Merenbakh-Lamin, Asia Zubkov, Zohar Honig, Ori Mayer, Noam Shomron, Wolf Ido, Tami Rubinek. Beyond endocrine resistance: estrogen receptor (ESR1) activating mutations mediate chemotherapy resistance through the JNK/c-Jun MDR1 pathway in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-05-09.

MiR-92b-3p Protects against Myocardial Ischemia-Reperfusion Injury by Inhibiting MAP3K2 in a Mouse Model.

MicroRNAs are well-known RNA regulators modulating biological functions in complex signaling networks. This work aims to explore the impact of microRNA-92b-3p (miR-92b-3p) on myocardial ischemia-reperfusion (I/R) injury. The I/R model was established by left anterior descending coronary artery ligation in mice. The hemodynamic parameters were detected through a multichannel physiological recorder. Myocardial injury markers: serum cardiac troponin I, myocardial kinase isoenzyme (creatine kinase-MB), and serum inflammatory factors (tumor necrosis factor-α, interleukin [IL]-1β, and IL-6) were evaluated by enzyme-linked immunosorbent assay. Cardiac tissue oxidative stress-related factors (malondialdehyde, glutathione peroxidase, total antioxidation capability, and superoxide dismutase) were assessed by colorimetry, myocardial pathology was observed by hematoxylin-eosin staining, and cardiomyocyte apoptosis was measured by triphosphate nick end-labeling staining, as well as the expression of miR-92b-3p and mitogen-activated protein kinase kinase kinase 2 (MAP3K2) in cardiac tissues were determined by reverse transcription quantitative polymerase chain reaction or western blot assay. The targeting relationship between miR-92b-3p and MAP3K2 was verified by bioinformatics, RNA immunoprecipitation, and luciferase reporter assays. miR-92b-3p was lowly expressed and MAP3K2 was highly expressed in myocardial I/R injury mice. Upregulation of miR-92b-3p improved hemodynamic indices, decreased serum levels of myocardial injury biomarkers, inhibited serum inflammatory response, alleviated cardiac tissue oxidative stress, relieved myocardial pathology, and reduced cardiomyocyte apoptosis during the myocardial I/R injury in mice. MAP3K2 was a direct target gene of miR-92b-3p. This research suggests that miR-92b-3p protects against myocardial I/R injury by inhibiting MAP3K2, which may provide novel candidates for treatment of myocardial I/R injury.

Subchronic co-exposure of polystyrene nanoplastics and 3-BHA significantly aggravated the reproductive toxicity of ovaries and uterus in female mice.

Both nanoplastics (NPs) and 3-tert-butyl-4-hydroxyanisole (3-BHA) are environmental contaminants that can bio-accumulate through the food chain. However, the combined effects of which on mammalian female reproductive system remain unclear. Here, the female ICR-CD1 mice were used to evaluate the damage effects of ovaries and uterus after NPs and 3-BHA co-treatment for 35 days. Firstly, co-exposure significantly reduced the body weight and organ index of ovaries and uterus in mice. Secondly, combined effects of NPs and 3-BHA exacerbated the histopathological abnormalities to the ovaries and uterus and decreased female sex hormones such as FSH and LH while increased antioxidant activities including CAT and GSH-Px. Moreover, the apoptotic genes, inflammatory cytokines and the key reproductive development genes such as FSTL1 were significantly up-regulated under co-exposure conditions. Thirdly, through transcriptional and bioinformatics analysis, immunofluorescence and western blotting assays, together with molecular docking simulation, we determined that co-exposure up-regulated the FSTL1, TGF-β and p-Smad1/5/9 but down-regulated the expression of BMP4. Finally, the pharmacological rescue experiments further demonstrated that co-exposure of NPs and 3-BHA mainly exacerbated the female reproductive toxicity through FSTL1-mediated BMP4/TGF-β/SMAD signaling pathway. Taken together, our studies provided the theoretical basis of new environmental pollutants on the reproductive health in female mammals.

LINC00115 aggravates thyroid cancer progression by targeting miR-489-3p, which downregulates EVA1A to regulate the Hippo signaling pathway

LINC00115 has been documented to regulate many different cancers; however, its function in thyroid cancer (THCA) remains unexplored. Therefore, we examined the effects of LINC00115 on THCA and the associated molecular mechanisms. In THCA cell lines and tumor samples, the expression levels of LINC00115, miR-489-3p, and EVA1A were analyzed by qRT-PCR along with respective controls. Cell viability, migration, and apoptosis were analyzed by employing CCK-8, transwell, and western blotting assays, respectively. Xenograft experiments were done to assess in vivo tumor growth. The interaction among LINC00115, miR-489-3p, and EVA1A was tested using RNA-binding protein immunoprecipitation and luciferase assays. Key proteins of the Hippo signaling pathway were ascertained by western blotting. The outcomes elucidated that LINC00115 was overexpressed in THCA cell lines and tumor tissues. LIN00115 knockdown reduced in vitro proliferation and migration but facilitated apoptosis in THCA cells and inhibited in vivo tumor growth. The target of LINC00115 was miR-489-3p, which binds to EVA1A in THCA. Functional assays revealed that miR-489-3p inhibition boosted THCA cell proliferation and migration, but hindered apoptosis. However, EVA1A knockdown resulted in the opposite effects via the Hippo signaling pathway. Additionally, miR-489-3p inhibition partially negated the effects of LINC00115 knockdown in THCA cells, and EVA1A knockdown remarkably impeded the effects of miR-489-3p inhibition in THCA cells. Thus, LINC00115 knockdown suppressed THCA carcinogenesis via targeting miR-489-3p, which regulates EVA1A expression and affects the Hippo signaling pathway.

DOCK4 is a novel prognostic biomarker and correlated with immune infiltrates in colon adenocarcinoma.

Dedicator for cytokinesis 4 (DOCK4) is a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. However, the functions of DOCK4 concerning the tumor microenvironment (TME) in colon adenocarcinoma (COAD) remain uncertain. The TIMER and GEPIA databases were used to analyze the DOCK4 expression between COAD tissues and adjunct normal tissues. The PrognoScan database was used to assess the prognosis of DOCK4 expression in COAD. The co-expression networks of DOCK4 in COAD were constructed by the LinkedOmics website. Furthermore, the correlation between DOCK4 expression and TME of COAD was explored using TIMER and TISIDB databases. Finally, the clone formation assay was used to further verify the function of DOCK4 in COAD. The Western blotting assay was used to confirm the mechanism related to DOCK4 in COAD. The DOCK4 expression was different significantly in COAD tissues and paracancerous tissues. The DOCK4 was found to play a poor role in the prognosis of patients with COAD. The DOCK4 was found to participate in the TME by promoting immune evasion of COAD. The reduction of DOCK4 expression inhibited the clone formation and Ras-associated protein 1A (Rap1A) expression of HCT116 cells. DOCK4 potentially plays an important role in the regulation of TME in COAD. DOCK4 facilitates the development through the Rap1A pathway, thus becoming a novel prognostic biomarker in COAD.

Denosumab Induces Neoplastic Stromal Cell Apoptosis via p62 Downregulation Dependent on Autophagy Pathway in Giant Cell Tumour of Bone.

As the only humanized monoclonal antibody against receptor activator of nuclear factor-κB ligand (RANKL) for giant cell tumour of bone (GCTB) therapy, denosumab has limited antitumour effect on neoplastic stromal cells. Nevertheless, its mechanism of action has not yet been clarified. A previous study has revealed that p62 may play an important role in the antitumour activity of denosumab. The study aimed to investigate if the mechanism by which denosumab inhibits GCTB neoplastic stromal cells growth is via p62 modulation and other related mechanisms. p62 expression before and after denosumab therapy was analysed by RT‒qPCR, western blot, ELISA, and immunohistochemical assays. Two primary neoplastic stromal cells were isolated from fresh GCTB tumour tissue (L cell) and metastatic tissue (M cell). Cell proliferation, migration, apoptosis, and autophagy were investigated in p62 knockdown neoplastic stromal cells transfected by short hairpin RNA lentivirus in vitro. Tumor growth was evaluated in the chick chorioallantoic membrane model in vivo. p62 expression was found to be downregulated following denosumab therapy. The patients with a decrease in p62 expression had lower recurrence-free survival rates. The proliferation of M cells was not inhibited by denosumab therapy, but it was restored by p62 knockdown. Moreover, p62 knockdown inhibited tumour growth in vivo. Denosumab induced M cell apoptosis and arrested the cell cycle at the G1/G0 transition and these effects were also enhanced by p62 knockdown. Autophagic flux assays revealed p62 modulation to be dependent on autophagy following denosumab incubation. Denosumab induced neoplastic stromal cells apoptosis via p62 downregulation dependent on autophagy pathway. The combination of p62 and RANKL knockdown might be a better strategy than RANKL knockdown alone for GCTB targeted therapy.

3D cell culture of nanofiber hydrogel promotes the growth and osteogenesis of bone marrow mesenchymal stem cells (BMSC)

Hydrogels, as a type of polymer network material with high water content, find wide application in various tissue repair procedures. In addition, a three-dimension (3D) cell culture platform similar to natural extracellular matrix can be constructed in vitro by modifying natural polymer chains. Therefore, this study based on natural polymer hydrogel, the introduction of inorganic nanoparticles POSS to prepare GelMA-POSS nanohydrogel. The physical and chemical properties of the hydrogel were evaluated through SEM, swelling test and degradation analysis. Subsequently, the GelMA-POSS nano-hydrogel was used as matrix material to culture rBMSCs in vitro. CCK-8 and flow cytometry were used to detect the proliferation ability and apoptosis rate of rBMSCs. The expression of osteogenic markers in rBMSCs was detected by qPCR, Western Blotting and ELISA assay, and the osteogenic differentiation of rBMSCs was detected by ALP staining and alizarin red staining. The results indicate that compared with pure gelatin (Gel) hydrogel, GelMA-POSS nanohydrogel has slower degradation rate and better water retention capacity. Furthermore, in vitro cell culture experiments showed that GelMA-POSS nano-hydrogel had good biocompatibility, and could promote the proliferation of rBMSCs and increase the expression level of osteogenic markers in cells, and induce osteogenic differentiation of rBMSCs. GelMA-POSS nano-hydrogel based on POSS significantly improves the physical and chemical properties of Gel hydrogel, and has good biocompatibility and the ability to induce osteogenic differentiation of rBMSCs, Thus, it holds great promise as a potential material for repairing bone defects.

Circ_PABPC1 promotes the malignancy of gastric cancer through interacting with ILK to activate NF-κB pathway

BackgroundGastric cancer (GC) is a common cancer type with both high incidence and mortality. Recent studies have revealed an important role of circRNA in the development of GC. However, more experiments are needed to reveal the precise molecular mechanisms of circRNA in GC development. MethodsBioinformatics analysis was conducted to predict the potential role of circ_PABPC1 in GC and the target proteins of circ_PABPC1. Quantitative RT-PCR, western blot and immunohistochemistry assays were conducted to detect the levels of circ_PABPC1, NF-κB p65, NF-κB p65 (Ser536) and ILK. MTT, Edu staining, cell scratch-wound and trans-well assays were carried out to detect cell proliferation, migration and invasion. The interaction between ILK and circ_PABPC1 was confirmed by RNA immunoprecipitation (RIP), RNA pull-down and fluorescence in situ hybridization assays. Genetically modified GC cells were injected into mice to evaluate the tumor growth performance. ResultsThis study found that the high expression of circ_PABPC1 was associated with a poor prognosis of GC. The up-regulation of circ_PABPC1 promoted the proliferation, migration and invasion of GC cells. Circ_PABPC1 bound to ILK protein, thereby preventing the degradation of ILK. ILK mediated the effect of circ_PABPC1 on GC cells through activating NF-κB. Conclusioncirc_PABPC1 promotes the malignancy of GC cells through binding to ILK to activate NF-κB signaling pathway .

Antibodies to Aedes aegypti D7L salivary proteins as a new serological tool to estimate human exposure to Aedes mosquitoes

IntroductionAedes spp. are the most prolific mosquito vectors in the world. Found on every continent, they can effectively transmit various arboviruses, including the dengue virus which continues to cause outbreaks worldwide and is spreading into previously non-endemic areas. The lack of widely available dengue vaccines accentuates the importance of targeted vector control strategies to reduce the dengue burden. High-throughput tools to estimate human-mosquito contact and evaluate vector control interventions are lacking. We propose a novel serological tool that allows rapid screening of human cohorts for exposure to potentially infectious mosquitoes. MethodsWe tested 563 serum samples from a longitudinal pediatric cohort study previously conducted in Cambodia. Children enrolled in the study were dengue-naive at baseline and were followed biannually for dengue incidence for two years. We used Western blotting and enzyme-linked immunosorbent assays to identify immunogenic Aedes aegypti salivary proteins and measure total anti-Ae. aegypti IgG. ResultsWe found a correlation (rs=0.86) between IgG responses against AeD7L1 and AeD7L2 recombinant proteins and those to whole salivary gland homogenate. We observed seasonal fluctuations of AeD7L1+2 IgG responses and no cross-reactivity with Culex quinquefasciatus and Anopheles dirus mosquitoes. The baseline median AeD7L1+2 IgG responses for young children were higher in those who developed asymptomatic versus symptomatic dengue. DiscussionThe IgG response against AeD7L1+2 recombinant proteins is a highly sensitive and Aedes specific marker of human exposure to Aedes bites that can facilitate standardization of future serosurveys and epidemiological studies by its ability to provide a robust estimation of human-mosquito contact in a high-throughput fashion.

Atorvastatin combined with imipenem alleviates lung injury in sepsis by inhibiting neutrophil extracellular trap formation via the ERK/NOX2 signaling pathway.

Sepsis is a systemic inflammatory response syndrome caused by the invasion of pathogenic microorganisms. Despite major advances in diagnosis and technology, morbidity and mortality remain high. The level of neutrophil extracellular traps (NETs) is closely associated with the progression and prognosis of sepsis, suggesting the regulation of NET formation as a new strategy in sepsis treatment. Owing to its pleiotropic effects, atorvastatin, a clinical lipid-lowering drug, affects various aspects of sepsis-related inflammation and immune responses. To align closely with clinical practice, we combined it with imipenem for the treatment of sepsis. In this study, we used a cecum ligation and puncture-induced lung injury mouse model and employed techniques including western blot, immunofluorescence, and enzyme-linked immunosorbent assay to measure the levels of NETs and other sepsis-related lung injury indicators. Our findings indicate that atorvastatin effectively inhibited the formation of NETs. When combined with imipenem, it significantly alleviated lung injury, reduced systemic inflammation, and improved the 7-day survival rate of septic mice. Additionally, we explored the inhibitory mechanism of atorvastatin on NET formation in vitro, revealing its potential action through the ERK/NOX2 pathway. Therefore, atorvastatin is a potential immunomodulatory agent that may offer new treatment strategies for patients with sepsis in clinical settings.

Unveiling the Molecular Mechanisms of Size-Dependent Effect of Polystyrene Micro/nano-plastics on Chlamydomonas reinhardtii through Proteomic Profiling.

Microplastics have become a prevalent environmental pollutant due to widespread release and production. Algae, as primary producers, play a crucial role in maintaining the ecological balance of freshwater environments. Despite reports on the inhibition of microalgae by microplastics, the size-dependent effects on microalgae and associated molecular mechanism remain poorly understood. This study investigates the impacts of three polystyrene micro/nano-plastics (PS-MNPs) with different sizes (100 nm, 350 nm, and 6 μm) and concentrations (25 ∼ 200 mg/L) on Chlamydomonas reinhardtii (C. reinhardtii) throughout its growth period. Results reveal size- and concentration-dependent growth inhibition and induction of oxidative stress by PS-MNPs, with microalgae exhibiting increased vulnerability to smaller-sized and higher-concentration PS-MNPs. Proteomics analysis elucidates the size-dependent suppression of proteins involved in the photosynthesis process by PS-MNPs. Photosynthetic activity assays demonstrate that smaller PS-MNPs more significantly reduce chlorophyll content and the maximal photochemical efficiency of photosystem II. Finally, electron microscope and Western blot assays collectively confirm the size effect of PS-MNPs on microalgae growth is attributable to suppressed protein expression rather than shading effects. This study contributes to advancing our understanding of the intricate interactions between micro/nano-plastics and algae at the molecular level, emphasizing the efficacy of proteomics in dissecting the mechanistic aspects of microplastics-induced biological effects on environmental indicator organisms.

MEG3 shuttled by exosomes released from human bone marrow mesenchymal stem cells promotes TP53 stability to regulate MCM5 transcription in keloid fibroblasts.

Despite the interest in mesenchymal stem cells (MSC), their potential to treat abnormal scarring, especially keloids, is yet to be described. The present study aimed to investigate the therapeutic potential of exosomes derived from human bone marrow MSCs (hBMSC-Exos) in alleviating keloid formation. Exosomes were isolated from hBMSC, and keloid fibroblasts (KFs) were treated with hBMSC-Exos. Cell counting kit-8, wound healing, transwell invasion, immunofluorescence, and western blot assays were conducted to study the malignant phenotype of KFs. Mice were induced with keloids and treated with hBMSC-Exos. The effect of hBMSC-Exos on keloid formation in vivo was evaluated by hematoxylin and eosin staining, Masson staining, immunohistochemistry, and western blotting. The GSE182192 dataset was screened for differentially expressed long non-coding RNA during keloid formation. Next, maternally expressed gene 3 (MEG3) was knocked down in hBMSC to obtain hBMSC-Exossh-MEG3. The molecular mechanism of MEG3 was investigated by bioinformatic screening, and the relationship between MEG3 and TP53 or MCM5 was verified. hBMSC-Exos inhibited the malignant proliferation, migration, and invasion of KFs at same time as promoting their apoptosis, Moreover, hBMSC-Exos reduced the expression of fibrosis- and collagen-related proteins in the cells and the formation of keloids caused by KFs. The reduction in MEG3 enrichment in hBMSC-Exos weakened the inhibitory effect of hBMSC-Exos on KF activity. hBMSC-Exos delivered MEG3 to promote MCM5 transcription by TP53 in KFs. Overexpression of MCM5 in KFs reversed the effects of hBMSC-Exossh-MEG3, leading to reduced KF activity. hBMSC-Exos delivered MEG3 to promote the protein stability of TP53, thereby activating MCM5 and promoting KF activity.