Migration-related Proteins Research Articles

Splicing dysregulation in glioblastoma alters the function of cell migration-related genes.

Glioblastoma (GBM) has a poor prognosis with a high recurrence and low survival rate. Previous RNA-seq analyses have revealed that alternative splicing (AS) plays a role in GBM progression. Here, we present a novel AS analysis method (Semi-Q) and describe its use to identify GBM-specific AS events. We analyzed RNA-seq data from normal brain (NB), normal human astrocytes (NHAs) and GBM samples, and found that comparison between NHA and GBM was especially informative. Importantly, this analysis revealed that genes encoding cell migration-related proteins, including filamins (FLNs) and actinins (ACTNs), were among those most affected by differential AS. Functional assays revealed that dysregulated AS of FLNA, B and C transcripts produced protein isoforms that not only altered transcription of cell proliferation-related genes but also led to enhanced cell migration, resistance to cell death and/or mitochondrial respiratory function, while a dysregulated AS isoform of ACTN4 enhanced cell migration. Together, our results indicate that cell migration and actin cytoskeleton-related genes are differentially regulated by AS in GBM, supporting a role for AS in facilitating tumor growth and invasiveness.

Quercetin regulates pulmonary vascular remodeling in pulmonary hypertension by downregulating TGF-β1-Smad2/3 pathway

BackgroundPulmonary arterial hypertension (PAH) is a worldwide challenging disease characterized by progressive elevation of pulmonary artery pressure. The proliferation, migration and phenotypic transformation of pulmonary smooth muscle cells are the key steps of pulmonary vascular remodeling. Quercetin (3,3', 4', 5, 6-pentahydroxyflavone, Que) is a natural flavonol compound that has antioxidant, anti-inflammatory, anti-tumor and other biological activities. Studies have shown that Que has therapeutic effects on PAH. However, the effect of quercetin on pulmonary vascular remodeling in PAH and its mechanism remain unclear.Methods and resultsIn vivo, PAH rats were constructed by intraperitoneal injection of monocrotaline (MCT) at 60 mg/kg. Human pulmonary artery smooth muscle cells (HPASMCs) were treated with platelet-derived growth factor BB (PDGF-BB) 20 ng/mL to construct PAH cell model in vitro. The results showed that in vivo studies, MCT could induce right ventricular wall hyperplasia, narrow the small and medium pulmonary artery cavity, up-regulate the expression of proliferating and migration-related proteins proliferating cell nuclear antigen (PCNA) and osteopontin (OPN), and down-regulate the expression of alpha-smooth muscle actin (α-SMA). Que reversed the MCT-induced results. This process works by down-regulating the transforming growth factor-β1 (TGF-β1)/ Smad2/3 signaling pathway. In vitro studies, Que had the same effect on PDGF-BB-induced proliferation and migration cell models.ConclusionsQue inhibits the proliferation, migration and phenotypic transformation of HPASMCs by down-regulating TGF-β1/Smad2/Smad3 pathway, thereby reducing right ventricular hyperplasia (RVH) and pulmonary vascular remodeling, providing potential pharmacological and molecular explanations for the treatment of PAH.

Activin A inhibits the migration of human lung adenocarcinoma A549 cells induced by EGF

Activin A, a member of the transforming growth factor β (TGF-β) superfamily, is involved in tumorigenesis and tumor progression. However, it remains unclear whether activin A can affect the migration of lung adenocarcinoma (LUAD) cells. In this study, the results of differentially expressed genes (DEGs) identification revealed that lung adenocarcinoma tissues exhibited lower expression of activin βA mRNA, but higher expression of epidermal growth factor (EGF) and MMP9 mRNA compared to nontumor tissues. Moreover, we found that activin A inhibited human LUAD A549 cell proliferation promoted by EGF. Additionally, EGF induced A549 cell migration in microfluidic device, while activin A attenuated EGF actions. Simultaneously, EGF increased the levels of migration-related proteins, but activin A played the opposite role. Furthermore, the study revealed that EGF upregulated the ratio of p-ERK/ERK in A549 cells, which was weakened by activin A, and A549 cell migration regulated by activin A was not related to calcium signaling. In addition, the inhibitory effect of activin A on EGF-induced A549 cell migration was attenuated by the ERK inhibitor FR180204. These findings demonstrate that activin A effectively hinders the migration of A549 cells induced by EGF through ERK1/2 signaling, suggesting that targeting activin A may hold promise in the treatment of EGF-dependent LUAD growth and metastasis.

Activin A plays an essential role in migration and proliferation of hepatic stellate cells via Smad3 and calcium signaling

Activin A and hepatic stellate cells (HSCs) are involved in tissue repair and fibrosis in liver injury. This study investigated the impact of activin A on HSC activation and migration. A microfluidic D4-chip was used for examining the cell migration of mouse hepatic stellate cell line MHSteC. The analysis of differentially expressed genes revealed that activin βA (Inhba), activin receptor type 1A (Acvr1a) and type 2A (Acvr2a) mRNAs were more significantly expressed in human HSCs than in the hepatocytes. Moreover, activin A promoted MHSteC proliferation and induced MHSteC migration. Furthermore, the MHSteCs treated with activin A exhibited increased levels of migration-related proteins, N-cadherin, Vimentin, α-SMA, MMP2 and MMP9, but a decreased level of E-cadherin. Additionally, activin A treatment significantly increased the p-Smad3 levels and p-Smad3/Smad3 ratio in the MHSteCs, and the Smad3 inhibitor SIS3 attenuated activin A-induced MHSteC proliferation and migration. Simultaneously, activin A increased the calcium levels in the MHSteCs, and the migratory effects of activin A on MHSteCs were weakened by the intracellular calcium ion-chelating agent BAPTA-AM. These data indicate that activin A can promote MHSteC activation and migration through the canonical Smad3 signaling and calcium signaling.

NK-92 cells activated by IL-2 inhibit the progression of endometriosis in vitro

Background Interleukin (IL)-2 is a key cytokine capable of modulating the immune response by activating natural killer (NK) cells. This study was recruited to explore the therapeutic potential of IL-2-activated NK-92 cells in endometriosis in vitro. Methods Ectopic endometrial stromal cells (EESCs) were isolated and co-cultured with IL-2-activated NK-92 cells at varying effector-to-target (E:T) ratios (1:0 [Control], 1:1, 1:3, and 1:9). The viability, cytotoxicity, and cell surface antigen expression of IL-2-activated NK-92 cells were assessed. The viability, apoptosis, invasion, and migration ability of EESCs co-cultured with NK-92 cells at different ratios were evaluated. The apoptosis-related proteins, invasion and migration-related proteins as well as MEK/ERK pathway were examined via western blot. Each experiment was repeated three times. Results IL-2 activation enhanced NK-92 cytotoxicity in a concentration-dependent manner. Co-culturing EESCs with IL-2-activated NK-92 cells at E:T ratios of 1:1, 1:3, and 1:9 reduced EESC viability by 20%, 45%, and 70%, respectively, compared to the control group. Apoptosis rates in EESCs increased in correlation with the NK-92 cell proportion, with the highest rate observed at a 1:9 ratio. Moreover, EESC invasion and migration were significantly inhibited by IL-2-activated NK-92 cells, with a 60% reduction in invasion and a 50% decrease in migration at the 1:9 ratio. Besides, the MEK/ERK signalling pathway was down-regulated in EESCs by IL-2-activated NK-92 cells. Conclusion IL-2-activated NK-92 cells exhibit potent cytotoxic effects against EESCs. They promote EESC apoptosis and inhibit viability, invasion, and migration through modulating the MEK/ERK signalling pathway.

N-desmethyldauricine from Menispermum dauricum DC suppresses triple-negative breast cancer growth in 2D and 3D models by downregulating the NF-κB signaling pathway

Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, for which targeted therapy regimens are lacking. The traditional Chinese medicine Menispermum dauricum DC (M. dauricum) and its compounds have been reported to have antitumor activity against various cancers; however, their anti-TNBC activity is unknown. In this work, dauricine and N-desmethyldauricine from M. dauricum were separated and identified to have anti-TNBC via a multi-component bioactivity and structure-guided method. The cell counting kit 8 assay showed that dauricine and N-desmethyldauricine inhibited the proliferation of four tested TNBC cell lines, with half maximal inhibitory concentration values ranging from 5.01 μM to 13.16 μM. Further research suggested that N-desmethyldauricine induced cell apoptosis, arrested cell cycle progression in the G0/G1 phase, and inhibited cell migration. Western blot analysis revealed that the proapoptotic protein cleaved-poly-ADP-ribose polymerase 1 was upregulated, and the G0/G1 phase-related proteins cyclin-dependent kinase 2 and cyclin D1 and the migration-related protein matrix metallopeptidase 9 were downregulated. Furthermore, N-desmethyldauricine decreased the protein expression of p65, an important subunit of nuclear factor kappa-beta (NF-κB). Moreover, an antiproliferation assay of three-dimensional (3D) tumor spheroids showed that N-desmethyldauricine diminished cell‒cell adhesion and suppressed the growth of TNBC 3D spheroids. Taken together, these findings indicate that N-desmethyldauricine inhibited the proliferation of TNBC cells and decreased the expression of p65 in the NF-κB pathway.

Mechanism of salidroside in inhibiting proliferation, migration and promoting phenotypic switching of arterial smooth muscle cells

This study aims to examine the effect of salidroside(SAL) on the phenotypic switching of human aortic smooth muscle cells(HASMC) induced by the platelet-derived growth factor-BB(PDGF-BB) and investigate the pharmacological mechanism. Firstly, the safe concentration of SAL was screened by the lactate dehydrogenase release assay. HASMC were divided into control, model, and SAL groups, and the cells in other groups except the control group were treated with PDGF-BB for the modeling of phenotypic switching. Cell proliferation and migration were detected by the cell-counting kit(CCK-8) assay and Transwell assay, respectively. The cytoskeletal structure was observed by F-actin staining with fluorescently labeled phalloidine. The protein levels of proliferating cell nuclear antigen(PCNA), migration-related protein matrix metalloprotein 9(MMP-9), fibronectin, α-smooth muscle actin(α-SMA), and osteopontin(OPN) were determined by Western blot. To further investigate the pharmacological mechanism of SAL, this study determined the expression of protein kinase B(Akt) and mammalian target of rapamycin(mTOR), as well as the upstream proteins phosphatase and tensin homologue(PTEN) and platelet-derived growth factor receptor β(PDGFR-β) and the downstream protein hypoxia-inducible factor-1α(HIF-1α) of the Akt/mTOR signaling pathway. The results showed that the HASMCs in the model group presented significantly increased proliferation and migration, the switching from a contractile phenotype to a secretory phenotype, and cytoskeletal disarrangement. Compared with the model group, SAL weakened the proliferation and migration of HASMC, promoted the expression of α-SMA(a contractile phenotype marker), inhibited the expression of OPN(a secretory phenotype marker), and repaired the cytoskeletal disarrangement. Furthermore, compared with the control group, the modeling up-regulated the levels of phosphorylated Akt and mTOR and the relative expression of PTEN, HIF-1α, and PDGFR-β. Compared with the model group, SAL down-regulated the protein levels of phosphorylated Akt and mTOR, PTEN, PDGFR-β, and HIF-1α. In conclusion, SAL exerts a protective effect on the HASMCs exposed to PDGF-BB by regulating the PDGFR-β/Akt/mTOR/HIF-1α signaling pathway.

Muscone restores anoikis sensitivity in TMZ-resistant glioblastoma cells by suppressing TOP2A via the EGFR/Integrin β1/FAK signaling pathway

BackgroundTemozolomide (TMZ) resistance is the main obstacle faced by glioblastoma multiforme (GBM) treatment. Muscone, one of the primary active pharmacological ingredients of Shexiang (Moschus), can cross the blood-brain barrier (BBB) and is being investigated as an antineoplastic medication. However, muscone treatment for GBM has received little research, and its possible mechanisms are still unclear. PurposeThis study aims to evaluate the effect and the potential molecular mechanism of muscone on TMZ-resistant GBM cells. MethodsThe differentially expressed genes (DEGs) between TMZ-resistant GBM cells and TMZ-sensitive GBM cells were screened using GEO2R. By progressively raising the TMZ concentration, a relatively stable TMZ-resistant human GBM cell line was established. The drug-resistance traits of U251-TR cells were assessed via the CCK-8 assay and Western Blot analysis of MGMT and TOP2A expression. Cell viability, cell proliferation, cell migration ability, and drug synergism were detected by the CCK-8 assay, colony formation assay, wound healing assay, and drug interaction relationship test, respectively. Anoikis was quantified by Calcein-AM/EthD-1 staining, MTT assay, and flow cytometry. Measurements of cell cycle arrest, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were performed using cell cycle staining, Annexin V-FITC/PI labeling, JC-1 assay, and ROS assay, respectively. DNA damage was measured by TUNEL assay, alkaline comet assay, and γ-H2AX foci assay. GEPIA was used to investigate the link between the anoikis marker (FAK)/drug resistance gene and critical proteins in the EGFR/Integrin β1 signaling pathway. Molecular docking was used to anticipate the probable targets of muscone. The intracellular co-localization and expression of EGFR and FAK were shown using immunofluorescence. The U251-TR cell line stably overexpressing EGFR was constructed using lentiviral transduction to assess the involvement of EGFR-related signaling in anoikis resistance. Western Blot was employed to detect the expression of migration-related proteins, cyclins, anoikis-related proteins, DNA damage/repair-related proteins, and associated pathway proteins. ResultsDEGs analysis identified 97 deregulated chemotherapy-resistant genes and 3779 upregulated genes in TMZ-resistant GBM cells. Subsequent experiments verified TMZ resistance and the hyper-expression of DNA repair-related genes (TOP2A and MGMT) in continuously low-dose TMZ-induced U251-TR cells. Muscone exhibited dose-dependent inhibition of U251-TR cell migration and proliferation, and its co-administration with TMZ showed the potential for enhanced therapeutic efficacy. By downregulating FAK, muscone reduced anoikis resistance in anchorage-independent U251-TR cells. It also caused cell cycle arrest in the G2/M phase by upregulating p21 and downregulating CDK1, CDK2, and Cyclin E1. Muscone-induced anoikis was accompanied by mitochondrial membrane potential collapse, ROS production, an increase in the BAX/Bcl-2 ratio, as well as elevated levels of Cytochrome c (Cyt c), cleaved caspase-9, and cleaved caspase-3. These findings indicated that muscone might trigger mitochondrial-dependent anoikis via ROS generation. Moreover, significant DNA damage, DNA double-strand breaks (DSBs), the formation of γ-H2AX foci, and a reduction in TOP2A expression are also associated with muscone-induced anoikis. Overexpression of EGFR in U251-TR cells boosted the expression of Integrin β1, FAK, β-Catenin, and TOP2A, whereas muscone suppressed the expression levels of EGFR, Integrin β1, β-Catenin, FAK, and TOP2A. Muscone may influence the expression of the key DNA repair enzyme, TOP2A, by suppressing the EGFR/Integrin β1/FAK pathway. ConclusionWe first demonstrated that muscone suppressed TOP2A expression through the EGFR/Integrin β1/FAK pathway, hence restoring anoikis sensitivity in TMZ-resistant GBM cells. These data suggest that muscone may be a promising co-therapeutic agent for enhancing GBM treatment, particularly in cases of TMZ-resistant GBM with elevated TOP2A expression.

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

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

CD36 inhibition reduces non-small-cell lung cancer development through AKT-mTOR pathway

Lung cancer is the most common cause of cancer-related deaths worldwide and is caused by multiple factors, including high-fat diet (HFD). CD36, a fatty acid receptor, is closely associated with metabolism-related diseases, including cardiovascular disease and cancer. However, the role of CD36 in HFD-accelerated non-small-cell lung cancer (NSCLC) is unclear. In vivo, we fed C57BL/6J wild-type (WT) and CD36 knockout (CD36−/−) mice normal chow or HFD in the presence or absence of pitavastatin 2 weeks before subcutaneous injection of LLC1 cells. In vitro, A549 and NCI-H520 cells were treated with free fatty acids (FFAs) to mimic HFD situation for exploration the underlying mechanisms. We found that HFD promoted LLC1 tumor growth in vivo and that FFAs increased cell proliferation and migration in A549 and NCI-H520 cells. The enhanced cell or tumor growth was inhibited by the lipid-lowering agent pitavastatin, which reduced lipid accumulation. More importantly, we found that plasma soluble CD36 (sCD36) levels were higher in NSCLC patients than those in healthy ones. Compared to that in WT mice, the proliferation of LLC1 cells in CD36−/− mice was largely suppressed, which was further repressed by pitavastatin in HFD group. At the molecular level, we found that CD36 inhibition, either with pitavastatin or plasmid, reduced proliferation- and migration-related protein expression through the AKT/mTOR pathway. Taken together, we demonstrate that inhibition of CD36 expression by pitavastatin or other inhibitors may be a viable strategy for NSCLC treatment.Graphical abstract1) Pitavastatin reduces NSCLC progression by inhibiting CD36.2) Inhibition of CD36 can improve HFD- or FFA-induced NSCLC.3) AKT/mTOR pathway is involved in CD36-regulated NSCLC.4) Inhibition of CD36 by pitavastatin or other inhibitors may be a strategy for NSCLC treatment.

PGC-1α affects cochlear pericytes migration in noise-exposed mice

ObjectiveThe study aimed to observe the effects of noise exposure on the pericytes of the cochlear stria vascularis (SV) in mice and to investigate its molecular mechanism. MethodMale C57BL/6J mice aged 6–8 weeks were used as the subjects. Auditory Brainstem Response (ABR) was used to assess hearing loss. Hematoxylin and Eosin (HE) staining was conducted to observe morphological alterations in the SV. Immunofluorescence combined with transmission electron microscopy (TEM) was used to scrutinize changes in pericytes following acoustic injury. Western blotting (WB) was used to assess the expression variations of the migration-related protein Osteopontin (OPN). Evans Blue assay was performed to evaluate the permeability of the blood labyrinth barrier (BLB). 4-Hydroxynonenal (4-HNE) staining, in conjunction with measurements of Superoxide Dismutase (SOD), Malondialdehyde (MDA), and Catalase (CAT) content, was used to ascertain whether oxidative stress injury occurred in the SV. WB, combined with immunofluorescence, was used to examine alterations in the expression of proliferator-activated receptor-gamma coactivator 1α (PGC-1α) in the SV and pericytes. ResultsNoise exposure resulted in permanent hearing loss in C57BL/6J mice, accompanied by SV swelling, migration of pericytes from their vascular attachments, BLB leakage, elevated oxidative stress levels in the SV, and reduced expression of PGC-1α on both the SV and migrating pericytes. ConclusionNoise exposure may potentially increase oxidative stress levels in the SV, downregulate the expression levels of PGC-1α, promote pericytes migration, and subsequently lead to an elevation in BLB permeability.

MicroRNA-378a-3p plays a regulatory role in trophoblast cell function in preeclampsia by targeting CMTM3

BackgroundPreeclampsia (PE) is a potential multisystemic disease in the middle and late pregnancy. Although its precise etiology and pathogenesis remain unknown, it is a significant cause of morbidity and mortality in both pregnant women and newborns. This study explored the effects of the miR-378a-3p/CKLF-like MARVEL transmembrane domain containing 3 (CMTM3) upon the trophoblast biological functions in PE. MethodsThe placental pathology of PE were identified by hematoxylin-eosin (HE) staining, and miR-378a-3p expression in placental tissues of PE was verified by RT-qPCR. Trophoblast cells (HTR-8/SVneo and JEG-3) were treated with lipopolysaccharide (LPS), and cell counting kit-8 (CCK-8) assay, flow cytometry, scratch assay, and Transwell assay were carried out to measure cell viability, apoptosis, migratory and invasive capacities, respectively. Western blot was performed to determine the expression levels of the cell migration-related proteins. The binding of miR-378a-3p to CMTM3 was verified through a dual-luciferase reporter gene assay. ResultsmiR-378a-3p expression levels were down-regulated in placental tissues and primary trophoblast cells from women with PE compared to the control group. The overexpression of miR-378a-3p promoted the capabilities of LPS-treated trophoblast cells to proliferate, migrate and invade. In contrast, it impeded cell apoptosis, promoted matrix metallopeptidase (MMP)-2 and MMP-9 expression and inhibiting TIMP metallopeptidase inhibitor (TIMP)-1 and TIMP-2 expression. Regarding the molecular mechanism, miR-378a-3p was chosen as the target to modulate the expression level of CMTM3. CMTM3 expression was increased in placental tissues and primary trophoblast cells from women with PE compared to the control group. CMTM3 overexpression could partially neutralize the effects of the overexpressed miR-378a-3p on trophoblast cell function and the expression levels of migration-associated proteins. ConclusionOur study provides a foundation for miRNA-targeted therapy for preeclampsia by establishing for the first time a potential role for the miR-378a-3p/CMTM3 axis in regulating trophoblast cell activities by altering the expression of migration-related proteins.

Quercetin induces MGMT+ glioblastoma cells apoptosis via dual inhibition of Wnt3a/β-Catenin and Akt/NF-κB signaling pathways

BackgroundSurgical resection combined with radiotherapy and chemotherapy remains a common clinical treatment for glioblastoma multiforme (GBM). However, the therapeutic outcomes have not been satisfying due to drug resistance and other factors. Quercetin, a phytoingredient capable of crossing the blood-brain barrier, has shown effectiveness in the treatment of various solid tumors. Nevertheless, the potential of quercetin in GBM treatment has not been adequately explored. PurposeThis study aims to investigate the effects and mechanisms of quercetin on MGMT+GBM cells. MethodsThe potential targets and mechanisms of quercetin in glioma treatment were predicted based on network pharmacology and molecular docking. The effects of quercetin on cell inhibition rate, cell migration ability, cell cycle arrest, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), Mitochondrial superoxide formation and apoptosis were measured by the CCK8 assay, wound healing assay, PI/RNase staining, JC-1 assay, DCFH-DA assay, MitoSOX staining and Annexin V-FITC/PI double staining, respectively. The methylation status of the MGMT promoter was assessed through methylation-specific polymerase chain reaction (MS-PCR). DNA damage was quantified by alkaline/neutral comet assay and TUNEL assay. The intracellular localization and expression of NF-κB and MGMT were revealed by immunofluorescence. The expression of migration-related proteins, matrix metalloproteinases, apoptosis-related proteins, cyclins, DNA damage/repair enzymes and related pathway proteins was detected by Western blot. ResultsNetwork pharmacology identified 96 targets and potential molecular mechanisms of quercetin in glioma treatment. Subsequent experiments confirmed the synergistic effect of quercetin in combination with temozolomide (TMZ) on T98G cells. Quercetin significantly suppressed the growth and migration of human GBM T98G cells, induced apoptosis, and arrested cells in the S-phase cell cycle. The collapse of mitochondrial membrane potential, ROS generation, enhanced Bax/Bcl-2 ratio, and strengthened cleaved-Caspase 9 and cleaved-Caspase 3 suggested the involvement of ROS-mediated mitochondria-dependent apoptosis in the process of quercetin-induced apoptosis. In addition, quercetin-induced apoptosis was accompanied by intense DNA double-strand breaks (DSBs), γH2AX foci formation, methylation of MGMT promoter, increased cleaved-PARP, and reduced MGMT expression. Quercetin may influence the expression of the key DNA repair enzyme, MGMT, by dual suppression of the Wnt3a/β-Catenin and the Akt/NF-κB signaling pathways, thereby promoting apoptosis. Inhibition of Wnt3a and Akt using specific inhibitors hindered MGMT expression. ConclusionOur study provides the first evidence that quercetin may induce apoptosis in MGMT+GBM cells via dual inhibition of the Wnt3a/β-Catenin pathway and the Akt/NF-κB signaling pathway. These findings suggest that quercetin could be a novel agent for improving GBM treatment, especially in TMZ-resistant GBM with high MGMT expression.

Upturn Strategies for Arachidonic Acid-Induced MC3T3-E1—625 nm Irradiation in Combination with NSAIDs: Dissipating Inflammation and Promoting Healing

Oral surgery, such as tooth extractions and dental implantations, can cause inflammation in the surrounding tissue, especially in bones. Anti-inflammatory drugs are crucial for pain relief and wound healing. Nonsteroidal anti-inflammatory drugs (NSAIDs) and light-emitting diode irradiation (LEDI) at 625 nm have been used as therapies to reduce inflammation, which ultimately promotes wound healing. The mechanism of these two methods, however, is different, which possibly makes the combined use of the two approaches effective. Therefore, the efficacy of 625 nm LEDI, NSAIDs, or a combination of both on anti-inflammatory and wound healing effects were analyzed in MC3T3-E1. In this study, piroxicam, ibuprofen, indomethacin, and celecoxib were selected as the NSAIDs. The effect of LEDI at 625 nm was investigated by cell viability, prostaglandin E2 (PGE2) release, and the expression of inflammation-related proteins and cell migration-related proteins were evaluated. Additionally, alkaline phosphatase staining with activity, cell migration assay and BrdU cell proliferation assays were performed. Both LEDI and NSAIDs reduced cyclooxygenase-2 (COX-2) and PGE2. Additionally, LEDI promoted cell migration, proliferation, and bone formation as well, but not by NSAIDs. Thus, a combination of LEDI and NSAIDs can benefits the cells in inflammation, which provides upturn strategies for bone healing after tooth extraction.

Inhibition of ATF4-mediated elevation of both autophagy and AKT/mTOR was involved in antitumorigenic activity of curcumin.

Curcumin, a natural hydrophobic polyphenol, carries significant anticancer activity. The protein kinase B (AKT)/the mammalian target of the rapamycin (mTOR) pathway and autophagy are well known to be involved in carcinogenesis, and usually, inhibition of mTOR is the main reason to promote autophagy. In this study, however, autophagy and mTOR were found to be inhibited simultaneously by curcumin treatments, and both of them played an important role in the effect of curcumin on suppressing the growth of A549 cells. Tunicamycin (TM), the activator of Endoplasmic Reticulum (ER) stress, increased both autophagy and AKT/mTOR, while curcumin could significantly decrease TM-induced autophagy and AKT/mTOR. Furthermore, curcumin could inhibit TM-induced aerobic glycolysis in A549 cells, and decrease the level of cycle-related and migration-related proteins. Blocking activating transcription factor 4 (ATF4) by siRNA strongly reduced both the expression of autophagy-related proteins and AKT/mTOR. ChIP assay illustrated that ATF4 protein could bind to the promotor sequence of either ATG4B or AKT1. The transplantation tumor experiment showed that the weight and volume of the transplanted tumors were reduced significantly in the BALB/c mice subcutaneously injected with A549 cells treated with curcumin. Moreover, intranasal administration of curcumin decreased the protein level of autophagy, AKT/mTOR and ER stress in lung tissues of BALB/c mice. Taken together, our results demonstrated that inhibition of ER stress-dependent ATF4-mediated autophagy and AKT/mTOR pathway plays an important role in anticancer effect of curcumin.

ACTN1 interacts with ITGA5 to promote cell proliferation, invasion and epithelial-mesenchymal transformation in head and neck squamous cell carcinoma.

The aim of this study was to detect the expression levels of α-Actinin 1 (ACTN1) and ITGA5 in HNSCC and to explore how ACTN1/ITGA5 regulated the proliferative and invasive abilities, as well as the EMT of Head and neck squamous cell carcinoma (HNSCC) cells. The viability, proliferative, invasive and migrative abilities of HNSCC cells after transfection were, in turn, detected by CCK8 assay, colony formation assay, EdU staining, transwell, as well as wound healing. E-cadherin in transfected cells was assessed utilizing immunofluorescence. RT-qPCR confirmed the transfection effect of ACTN1 and ITGA5 in HNSCC cells and the interaction between ACTN1 and ITGA5 in HNSCC cells was determined by co-immunoprecipitation (Co-IP). With Western blot application, the contents of ACTN1, ITGA5, proliferation-, invasion- and migration-related proteins were estimated. A xenograft model based on nude mice was conducted and Ki-67 content in tumor tissues was evaluated employing immunohistochemistry (IHC) staining. ACTN1 interacted with ITGA5. The contents of ACTN1 and ITGA5 were found to be abundant in HNSCC tissues and cells and associated with poor prognosis. ACTN1 depletion imparted suppressive impacts on cell proliferative, invasive and migrative abilities as well as EMT of HNSCC cells, which were reversed by ITGA5 overexpression. In addition, ACTN1 deficiency repressed the growth and metastasis of tumor tissues in tumor xenografts of nude mice. ACTN1 positively interacts with ITGA5 to promote proliferation, invasion and EMT of HNSCC cells. Also, ACTN1 promotes tumor growth and metastasis.

MiR-134-3p driven by anisomycin impairs ovarian cancer stem cell activity through inhibiting GPR137 expression.

Background: Ovarian cancer recurrence and metastasis are predominantly attributed to ovarian cancer stem cells; however, the mechanism by which anisomycin regulates human ovarian cancer stem cells (HuOCSCs) remains unclear. Methods: cDNA microArray was used to screen microRNAs (miRNAs) targeted by anisomycin, and RT-qPCR validated the miRNA targets. TargetScan database, GO enrichment analysis, and RT-qPCR, accompanied by a fluorescent reporter system, were employed to verify the miRNA target genes. In vitro experimental cell proliferation inhibition assay, flow cytometry, Transwell, angiogenesis assay, and in vivo transplantation tumor assay were implemented to assess the ability of the overexpressed miRNAs to hinder HuOCSC activity. Western blot, RT-qPCR, and immunofluorescence were applied to measure the transcriptional and protein-level expression of the miRNA target genes and their related genes. Bioinformatic analysis predicted and deciphered the role of the miRNA target genes and related genes in the development and prognosis of ovarian cancer. Results: The expression levels of multiple DLK1-DIO3 imprinted microRNA cluster members were altered by anisomycin, among which miR-134-3p expression was most significantly elevated. miR-134-3p overexpression significantly suppressed HuOCSC activity. The screening and validation of target genes uncovered that miR-134-3p was able to markedly suppress GPR137 expression. Additionally, miR-134-3p regulated the cytoskeleton, migration-related protein in the NDEL1/DYNEIN/TUBA1A axis through targeting GPR137. Bioinformatics prediction unveiled a close association of GPR137, NDEL1, DYNC1H1, and TUBA1A with ovarian cancer development and prognosis. Conclusions: The activity of HuOCSCs may be compromised by anisomycin through the regulation of miR-134-3p, which inhibits the GPR137/NDEL1/DYNEIN/TUBA1A axis.

Follistatin is a crucial chemoattractant for mouse decidualized endometrial stromal cell migration by JNK signalling

Follistatin (FST) and activin A as gonadal proteins exhibit opposite effects on follicle-stimulating hormone (FSH) release from pituitary gland, and activin A-FST system is involved in regulation of decidualization in reproductive biology. However, the roles of FST and activin A in migration of decidualized endometrial stromal cells are not well characterized. In this study, transwell chambers and microfluidic devices were used to assess the effects of FST and activin A on migration of decidualized mouse endometrial stromal cells (d-MESCs). We found that compared with activin A, FST exerted more significant effects on adhesion, wound healing and migration of d-MESCs. Similar results were also seen in the primary cultured decidual stromal cells (DSCs) from uterus of pregnant mouse. Simultaneously, the results revealed that FST increased calcium influx and upregulated the expression levels of the migration-related proteins MMP9 and Ezrin in d-MESCs. In addition, FST increased the level of phosphorylation of JNK in d-MESCs, and JNK inhibitor AS601245 significantly attenuated FST action on inducing migration of d-MESCs. These data suggest that FST, not activin A in activin A-FST system, is a crucial chemoattractant for migration of d-MESCs by JNK signalling to facilitate the successful uterine decidualization and tissue remodelling during pregnancy.

Proteome-Wide Identification of RNA-Dependent Proteins in Lung Cancer Cells.

Following the concept of RNA dependence and exploiting its application in the R-DeeP screening approach, we have identified RNA-dependent proteins in A549 lung adenocarcinoma cells. RNA-dependent proteins are defined as proteins whose interactome depends on RNA and thus entails RNA-binding proteins (RBPs) as well as proteins in ribonucleoprotein complexes (RNPs) without direct RNA interaction. With this proteome-wide technique based on sucrose density gradient ultracentrifugation and fractionation followed by quantitative mass spectrometry and bioinformatic analysis, we have identified 1189 RNA-dependent proteins including 170 proteins which had never been linked to RNA before. R-DeeP provides quantitative information on the fraction of a protein being RNA-dependent as well as it allows the reconstruction of protein complexes based on co-segregation. The RNA dependence of three newly identified RNA-dependent proteins, DOCK5, ELMO2, also known as CED12A, and ABRAXAS1, also known as CCDC98, was validated using western blot analysis, and the direct RNA interaction was verified by iCLIP2 for the migration-related protein DOCK5 and the mitosis-related protein ABRAXAS1. The R-DeeP 2.0 database provides proteome-wide and cell line-specific information from A549 and HeLa S3 cells on proteins and their RNA dependence to contribute to understanding the functional role of RNA and RNA-binding proteins in cancer cells.

Follistatin Is a Novel Chemoattractant for Migration and Invasion of Placental Trophoblasts of Mice.

Follistatin (FST) as a gonadal protein is central to the establishment and maintenance of pregnancy. Trophoblasts' migration and invasion into the endometrium are critical events in placental development. This study aimed to elucidate the role of FST in the migration and invasion of placental trophoblasts of mice. We found that FST increased the vitality and proliferation of primary cultured trophoblasts of embryonic day 8.5 (E8.5) mice and promoted wound healing of trophoblasts. Moreover, FST significantly induced migration of trophoblasts in a microfluidic device and increased the number of invasive trophoblasts by Matrigel-coated transwell invasion assay. Being treated with FST, the adhesion of trophoblasts was inhibited, but intracellular calcium flux of trophoblasts was increased. Western blotting results showed that FST had no significant effects on the level of p-Smad3 or the ratio of p-Smad3/Smad3 in trophoblasts. Interestingly, FST elevated the level of p-JNK; the ratio of p-JNK/JNK; and expression of migration-related proteins N-cadherin, vimentin, ezrin and MMP2 in trophoblasts. Additionally, the migration of trophoblasts and expression of N-cadherin, vimentin, and MMP2 in trophoblasts induced by FST were attenuated by JNK inhibitor AS601245. These findings suggest that the elevated FST in pregnancy may act as a chemokine to induce trophoblast migration and invasion through the enhanced JNK signaling to maintain trophoblast function and promote placental development.