α5-nAChR modulates melanoma growth through the Notch1 signaling pathway

The roles of α5-nicotinic acetylcholine receptors (α5-nAChRs) in various types of solid cancer have been reported; however, its role in melanoma remains unknown. We knocked down α5-nAChR expression in melanoma cells to investigate the role of α5-nAChR in the proliferation, migration, and invasion of melanoma cells, and its effect on downstream signaling pathways. Using immunohistochemical analysis, we determined that α5-nAChR expression is significantly increased in human melanoma tissues and cell lines compared with normal human skin tissues. Knocking down α5-nAChR expression in melanoma cells in culture significantly inhibited the proliferation, migration, and invasiveness of melanoma cell lines. Specifically, knockdown of α5-nAChR inhibited PI3K-AKT and ERK1/2 signaling activity. Moreover, we confirmed that the Notch1 signaling pathway is the downstream target of α5-nAChR in melanoma. Our findings suggest that α5-nAChR plays a critical role in melanoma development and progression, and that targeting α5-nAChR may be a strategy for melanoma treatment.     

Knockdown of circular RNA VANGL1 inhibits TGF-β-induced epithelial-mesenchymal transition in melanoma cells by sponging miR-150-5p

Melanoma is one of the most aggressive and life-threatening skin cancers, and in this research, we aimed to explore the functional role of circular RNA VANGL1 (circVANGL1) in melanoma progression. The expression levels of circVANGL1 were observed to be significantly increased in clinical melanoma tissues and cell lines. Moreover, circVANGL1 knockdown suppressed, while circVANGL1 overexpression promoted the proliferation, migration and invasion abilities of melanoma cells. Further investigations confirmed the direct binding relation between circVANGL1 and miR-150-5p in melanoma, and restoration of miR-150-5p blocked the effects of circVANGL1 overexpression in melanoma cells. We further found that circVANGL1 was up-regulated by TGF-β treatment, and the enhanced EMT of TGF-β-treated melanoma cells was blocked by circVANGL1 knockdown. In conclusion, these results indicated that circVANGL1 might serve as a promising therapeutic target for melanoma.     

Regulatory Factors of Lymphocyte-Lymphocyte Interaction: I. Con A-Induced Mitogenic Factor Acts on the Late G1 Stage of T-Cell Proliferation

DNA synthesis in murine lymphocytes was augmented by a soluble factor in the supernatant of serum-free cultures of syngeneic spleen cells activated with concanavalin A (Con A). This so-called mitogenic factor (MF), which is probably identical with interleukin II, partially purified by DEAE-cellulose and Sephadex G-75 chromatography, is a fairly homogeneous molecule of 17–25 × 10³ daltons. By using partially purified MF, the role of MF in lymphocyte proliferation was investigated. Pretreatment of lymphocytes with Con A for the first 3 hr of culture, which does not in itself induce cell proliferation, markedly augmented the effect of MF. The presence of MF, however, is necessary only in a restricted stage (s) of lymphocyte proliferation. The addition and removal of MF at various times during culture showed that MF exerts its effect on a process which occurs 3–6 hr before the beginning of DNA synthesis. These results strongly suggest that MF regulates the proliferation of mitogen-stimulated lymphocytes by acting on a restricted stage (s) of the cell cycle.     

miR-210 promotes lung adenocarcinoma proliferation,migration, and invasion by targeting lysyl oxidase-like 4

Accumulating evidence has revealed that various microRNAs are deregulated and involved in lung cancer development and metastasis. miR-210 is implicated in several cancer progression. However, the detailed biological function and role of miR-210 in lung adenocarcinoma remains unclear. Our current study was aimed to investigate the mechanism of miR-210 in lung adenocarcinoma progression. We observed that miR-210 was significantly upregulated in lung cancer cell lines (A549 and H1650) in comparison to BEAS-2B cells. In addition, we found that miR-210 was greatly elevated in lung adenocarcinoma tissues. Then, it was shown that overexpression of miR-210 was able to promote lung cancer cell proliferation and colony formation ability while inhibitors of miR-210 exhibited a reversed phenomenon. Subsequently, A549 and H1650 cell migration and invasion capacity were obviously restrained by miR-210 inhibition whereas induced by miR-210 mimics. Lysyl oxidase-like 4 (LOXL4), a member of the secreted copper-dependent amine oxidases has been found to be increased or decreased in different cancer types. Here, we confirmed that LOXL4 could serve as a downstream target of miR-210 and miR-210 promoted lung cancer progression via targeting LOXL4. In A549 and H1650 cells, knockdown of LOXL4 dramatically repressed lung cancer cell proliferation, migration, and invasion. In conclusion, our study implied that miR-210 might indicate a new perspective for lung cancer.     

Downregulation of lncRNA HCP5 has inhibitory effects on gastric cancer cells by regulating DDX21 expression

LncRNA HCP5 has been confirmed to play crucial roles in many types of cancers. However, the role of lncRNA HCP5 in regulating the occurrence and development of gastric cancer (GC) remains unknown. In the current study, we aimed to investigate the precise effects of lncRNA HCP5 on cell proliferation, migration and invasion and molecular mechanisms in gastric cancer. Using RT-qPCR analysis, we found that lncRNA HCP5 was differentially expressed in GC cell lines. CCK-8, wound healing and transwell assay indicated that the proliferation, migration and invasion of gastric cancer cells were inhibited by downregulation of lncRNA HCP5 and lncRNA HCP5 overexpression exhibited the opposite effects in gastric cancer cells. Mechanistically, RNA binding protein immunoprecipitation and dual luciferase reporter assay confirmed the interaction between lncRNA HCP5 and DDX21. The effects of lncRNA HCP5 overexpression the proliferation, migration and invasion of GC cells were partly rescued by DDX21 silencing. Taken together, downregulation of lncRNA HCP5 exerted inhibitory effects on GC cell proliferation, migration and invasion through modulation of DDX21 expression, demonstrating the function of lncRNA HCP5 and DDX21 in GC progression.     

Up-Regulation of RFC3 Promotes Triple Negative Breast Cancer Metastasis and is Associated With Poor Prognosis Via EMT

Triple-negative breast cancer (TNBC) was regarded as the most aggressive and mortal subtype of breast cancer (BC) since the molecular subtype system has been established. Abundant studies have revealed that epithelial-mesenchymal transition (EMT) played a pivotal role during breast cancer metastasis and progression, especially in TNBC. Herein, we showed that inhibition the expression of replication factor C subunit 3 (RFC3) significantly attenuated TNBC metastasis and progression, which was associated with EMT signal pathway. In TNBC cells, knockdown of RFC3 can down-regulate mesenchymal markers and up-regulate epithelial markers, significantly attenuated cell proliferation, migration and invasion. Additionally, silencing RFC3 expression can decrease nude mice tumor volume, weight and relieve lung metastasis in vivo. Furthermore, we also demonstrated that overexpression of RFC3 in TNBC showed increased metastasis, progression and poor prognosis. We confirmed all of these results by immunohistochemistry analysis in 127 human TNBC tissues and found that RFC3 expression was significantly associated with poor prognosis in TNBC. Taken all these findings into consideration, we can conclude that up-regulation of RFC3 promotes TNBC progression through EMT signal pathway. Therefore, RFC3 could be an independent prognostic factor and therapeutic target for TNBC.     

CircABCB10 promotes nonsmall cell lung cancer cell proliferation and migration by regulating the miR-1252/FOXR2 axis

Circular RNA (circRNA) is a key regulator in the development and progression of human cancers. Previous studies confirmed circRNA-0008717 (circABCB10) as an oncogene in osteosarcoma, but the regulatory effect of circABCB10 in nonsmall cell lung cancer (NSCLC) is still unclear. In the current study, we examined the expression of circABCB10 in different NSCLC cell lines. Bioinformatics analysis, Cell Counting Kit-8 assays, Transwell migration, fluorescein reporting experiments, and xenografts in mice were used to detect the effect of circABCB10 on NSCLC cell proliferation and migration in vitro and tumor growth in vivo. The results showed that the expression of circABCB10 in NSCLC cell lines was increased. Downregulation of circABCB10 suppressed NSCLC cell proliferation and migration by promoting microRNA miR-1252 expression and suppressing Forkhead box 2 (FOXR2). Fluorescein reporting experiments confirmed that circABCB10 expression increased FOXR2 levels by sponging miR-1252, and in vivo experiments found that knockdown of circABCB10 decreased tumor growth. These data suggested that circABCB10 acted as a tumor promoter through a novel miR-1252/FOXR2 axis, providing potential biomarkers and therapeutic targets for the management of NSCLC.     

Neutrophil extracellular traps activate lung fibroblast to induce polymyositis‐related interstitial lung diseases via TLR9‐miR‐7‐Smad2 pathway

Excessive neutrophil extracellular trap (NET) formation may contribute to polymyositis (PM)‐associated interstitial lung diseases (ILD), but the underlying mechanism is not fully revealed. In this study, we found that NET accelerated the progression of ILD and promoted pulmonary fibrosis (PF) in vivo. miR‐7 expression was down‐regulated in lung tissue of PM group than control group, and NETs further decreased miR‐7 expression. TLR9 and Smad2 were up‐regulated in lung tissue of PM group than control group, and NETs further increased TLR9 and Smad2 expressions. In vitro experiments showed that PMA‐treated NETs accelerated the proliferation of LF and their differentiation into myofibroblast (MF), whereas DNase I decreased the promotion effect of NETs. Neutrophil extracellular trap components myeloperoxidase (MPO) and histone 3 also promoted the proliferation and differentiation of LF. In addition, we demonstrated that TLR9 involved in the regulation of NETs on LF proliferation and differentiation, and confirmed the interaction between miR‐7 and Smad2 in LF. Finally, miR‐7‐Smad2 pathway was confirmed to be involved in the regulation of TLR9 on LF proliferation and differentiation. Therefore, NETs promote PM‐related ILD, and TLR9‐miR‐7‐Smad2 signalling pathway is involved in the proliferation of LFs and their differentiation into MFs.     

lncRNA A1BG-AS1 suppresses proliferation and invasion of hepatocellular carcinoma cells by targeting miR-216a-5p

Extensive evidence indicate that long noncoding RNAs (lncRNAs) regulates the tumorigenesis and progression of hepatocellular carcinoma (HCC). However, the expression and biological function of lncRNA A1BG antisense RNA 1 (A1BG-AS1) were poorly known in HCC. Here, we found the underexpression of A1BG-AS1 in HCC via analysis of The Cancer Genome Atlas database. Further analyses confirmed that A1BG-AS1 expression in HCC was markedly lower than that in noncancerous tissues based on our HCC cohort. Clinical association analysis revealed that low A1BG-AS1 expression correlated with poor prognostic features, such as microvascular invasion, high tumor grade, and advanced tumor stage. Follow-up data indicated that low A1BG-AS1 level evidently correlated with poor clinical outcomes of HCC patients. Moreover, forced expression of A1BG-AS1 repressed proliferation, migration, and invasion of HCC cells in vitro. Conversely, A1BG-AS1 knockdown promoted these malignant behaviors in HepG2 cells. Mechanistically, A1BG-AS1 functioned as a competing endogenous RNA by directly sponging miR-216a-5p in HCC cells. Notably, miR-216a-5p restoration rescued A1BG-AS1 attenuated proliferation, migration and invasion of HCCLM3 cells. A1BG-AS1 positively regulated the levels of phosphatase and tensin homolog and SMAD family member 7, which were reduced by miR-216a-5p in HCC cells. Altogether, we conclude that A1BG-AS1 exerts a tumor suppressive role in HCC progression.     

Hepatitis B virus X protein acts as a tumor promoter in development of diethylnitrosamine-induced preneoplastic lesions

Chronic infection with hepatitis B virus (HBV) is one of the major etiological factors in the development of human hepatocellular carcinoma. Transgenic mice that express the HBV X protein (HBx) have previously been shown to be more sensitive to the effects of hepatocarcinogens. Although the mechanism for this cofactor role remains unknown, the ability of HBx to inhibit DNA repair and to influence cell cycle progression suggests two possible pathways. To investigate these possibilities in vivo, we treated double-transgenic mice that both express HBx (ATX mice) and possess a bacteriophage lambda transgene with the hepatocarcinogen diethylnitrosamine (DEN). Histological examination of liver tissue confirmed that DEN-treated ATX mice developed approximately twice as many focal lesions of basophilic hepatocytes as treated wild-type littermates. Treatment of mice with DEN resulted in a six- to eightfold increase in the mutation frequency (MF), as measured by a functional analysis of the lambda transgene. HBx expression was confirmed by immunoprecipitation and Western blotting and was associated with a modest 23% increase in the MF. Importantly, the extent of hepatocellular proliferation in 14-day-old mice, as measured by the detection of proliferating cell nuclear antigen and by the incorporation of 5-bromo-2'-deoxyuridine, was determined to be approximately twofold higher in ATX livers than in wild-type livers. These results are consistent with a model in which HBx expression contributes to the development of DEN-mediated carcinogenesis by promoting the proliferation of altered hepatocytes rather than by directly interfering with the repair of DNA lesions.     

Migration-stimulating factor (MSF) is over-expressed in non-small cell lung cancer and promotes cell migration and invasion in A549 cells over-expressing MSF

Migration-stimulating factor (MSF), an oncofetal truncated isoform of fibronectin, is a potent stimulator of cell invasion. However, its distribution and motogenic role in non-small cell lung cancer (NSCLC) have never been identified. In this study, real-time PCR and immunohistochemical staining (IHC) were performed to detect MSF mRNA and protein levels in tumor tissues and matched adjacent tumor-free tissues. Furthermore, to examine the effect of MSF on invasiveness, MSF was upregulated in A549 cells. The invasiveness and viability of A549 cells were then determined using a transwell migration assay and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assays, respectively. The expression level of MSF in NSCLC tissue was markedly higher than in matched adjacent tumor-free tissue. Additionally, the level of MSF protein expression in stage III and IV NSCLC samples was higher than in stage I and II NSCLC samples. More importantly, we also demonstrated that migration and invasion of A549 cells increased substantially after upregulating MSF, although proliferation remained unchanged. Meanwhile, we found no correlation between increasing motility and invasiveness of MSF-overexpressing cells and expression levels and activities of matrix metalloprotease MMP-2 and MMP-9. Our current study shows that MSF plays a role in migration and invasion of A549 cells and suggests that MSF may be a potential biomarker of NSCLC progression.     

Myostatin-null mice exhibit delayed skin wound healing through the blockade of transforming growth factor-β signaling by decorin

Myostatin (Mstn) is a secreted growth and differentiation factor that belongs to the transforming growth factor-β (TGF-β) superfamily. Mstn has been well characterized as a regulator of myogenesis and has been shown to play a critical role in postnatal muscle regeneration. Herein, we report for the first time that Mstn is expressed in both epidermis and dermis of murine and human skin and that Mstn-null mice exhibited delayed skin wound healing attributable to a combination of effects resulting from delayed epidermal reepithelialization and dermal contraction. In epidermis, reduced keratinocyte migration and protracted keratinocyte proliferation were observed, which subsequently led to delayed recovery of epidermal thickness and slower reepithelialization. Furthermore, primary keratinocytes derived from Mstn-null mice displayed reduced migration capacity and increased proliferation rate as assessed through in vitro migration and adhesion assays, as well as bromodeoxyuridine incorporation and Western blot analysis. Moreover, in dermis, both fibroblast-to-myofibroblast transformation and collagen deposition were concomitantly reduced, resulting in a delayed dermal wound contraction. These decreases are due to the inhibition of TGF-β signaling. In agreement, the expression of decorin, a naturally occurring TGF-β suppressor, was elevated in Mstn-null mice; moreover, topical treatment with TGF-β1 protein rescued the impaired skin wound healing observed in Mstn-null mice. These observations highlight the interplay between TGF-β and Mstn signaling pathways, specifically through Mstn regulation of decorin levels during the skin wound healing process. Thus we propose that Mstn agonists might be beneficial for skin wound repair.     

miR-101 Inhibiting Cell Proliferation,Migration and Invasion in Hepatocellular Carcinoma through Downregulating Girdin

miR-101 is considered to play an important role in hepato-cellular carcinoma (HCC), but the underlying molecular mechanism remains to be elucidated. Here, we aimed to confirm whether Girdin is a target gene of miR-101 and determine the tumor suppressor of miR-101 through Girdin pathway. In our previous studies, we firstly found Girdin protein was overexpressed in HCC tissues, and it closely correlated to tumor size, T stage, TNM stage and Edmondson-Steiner stage of HCC patients. After specific small interfering RNA of Girdin was transfected into HepG2 and Huh7.5.1 cells, the proliferation and invasion ability of tumor cells were significantly inhibited. In this study, we further explored the detailed molecular mechanism of Girdin in HCC. Interestingly, we found that miR-101 significantly low-expressed in HCC tissues compared with that in matched normal tissues while Girdin had a relative higher expression, and miR-101 was inversely correlated with Girdin expression. In addition, after miR-101 transfection, the proliferation, migration and invasion abilities of HepG2 cells were weakened. Furthermore, we confirmed that Girdin is a direct target gene of miR-101. Finally we confirmed Talen-mediated Girdin knockout markedly suppressed cell proliferation, migration and invasion in HCC while down-regulation of miR-101 significantly restored the inhibitory effect. Our findings suggested that miR-101/Girdin axis could be a potential application of HCC treatment.     

Actin microfilaments regulate vacuolar structures and dynamics: dual observation of actin microfilaments and vacuolar membrane in living tobacco BY-2 Cells

Actin microfilaments (MFs) participate in many fundamental processes in plant growth and development. Here, we report the co-localization of the actin MF and vacuolar membrane (VM), as visualized by vital VM staining with FM4-64 in living tobacco BY-2 cells stably expressing green fluorescent protein (GFP)-fimbrin (BY-GF11). The MFs were intensively localized on the VM surface and at the periphery of the cytoplasmic strands rather than at their center. The co-localization of MFs and VMs was confirmed by the observation made using transient expression of red fluorescent protein (RFP)-fimbrin in tobacco BY-2 cells stably expressing GFP-AtVam3p (BY-GV7) and BY-2 cells stably expressing gamma-tonoplast intrinsic protein (gamma-TIP)-GFP fusion protein (BY-GG). Time-lapse imaging revealed dynamic movement of MF structures which was parallel to that of cytoplasmic strands. Disruption of MF structures disorganized cytoplasmic strand structures and produced small spherical vacuoles in the VM-accumulating region. Three-dimensional reconstructions of the vacuolar structures revealed a disconnection of these small spherical vacuoles from the large vacuoles. Real-time observations and quantitative image analyses demonstrated rapid movements of MFs and VMs near the cell cortex, which were inhibited by the general myosin ATPase inhibitor, 2,3-butanedion monoxime (BDM). Moreover, both bistheonellide A (BA) and BDM treatment inhibited the reorganization of the cytoplasmic strands and the migration of daughter cell nuclei at early G1 phase, suggesting a requirement for the acto-myosin system for vacuolar morphogenesis during cell cycle progression. These results suggest that MFs support the vacuolar structures and that the acto-myosin system plays an essential role in vacuolar morphogenesis.     

HDAC6 interacts with PTPN1 to enhance melanoma cells progression

Histone deacetylase 6 (HDAC6) plays an important role in oncogenic transformation and cancer metastasis. Our previous study has demonstrated that HDAC6 was highly expressed in melanoma cells, and contributed to the proliferation and metastasis of melanoma cells. However, the underlying mechanism of HDAC6 in melanoma metastasis and progression remains largely unclear. In this study, we reported that HDAC6 directly interacted with Tyrosine-protein phosphatase non-receptor type 1 (PTPN1) by performing co-immunoprecipitation (Co-IP) combined with liquid chromatography tandem mass spectrometry (LC-MS/MS). HDAC6 increased the protein level of PTPN1 independent of histone modifying activity. In addition, PTPN1 promoted proliferation, colony formation and migration while decreased apoptosis of melanoma cells through activating extracellular signal-regulated kinase 1/2 (ERK1/2). Furthermore, we found that matrix metallopeptidase 9 (MMP9) was increased by HDAC6/PTPN1/ERK1/2 axis, which might serve as a mechanism for melanoma invasion and metastasis. In conclusion, HDAC6 might enhance aggressive melanoma cells progression via interacting with PTPN1, which was independent of its histone modifying activity.     

Constitutively active erythropoietin receptor expression in breast cancer cells promotes cellular proliferation and migration through a MAP-kinase dependent pathway

The role of erythropoietin receptor (EpoR) expression in tumor cells and the potential of EpoR-mediated signaling to contribute to cellular proliferation and invasiveness require further characterization. To determine whether EpoR expression and activation in tumor cells modulates intracellular signal transduction to promote cellular proliferation and migration, we employed a novel experimental model using human breast cancer cells engineered to stably express a constitutively active EpoR-R129C variant. EpoR-R129C expression resulted in increased cellular proliferation and migration of breast cancer cells and these effects were associated with significantly increased Epo-induced phosphorylation of ERK1/2, AKT and c-Jun-NH2-kinase (SAPK/JNK) proteins. Expression of the constitutively active EpoR-R129C receptor promoted the proliferation and migration of breast cancer cells via activation of ERK- and SAPK/JNK-dependent signaling pathways, respectively. These findings suggest that EpoR over-expression and activation in breast cancer cells has the potential to contribute to tumor progression by promoting the proliferation and invasiveness of the neoplastic cells.     

A role for parasite-induced PGE2 in IL-10-mediated host immunoregulation by skin stage schistosomula of Schistosoma mansoni

Significant quantities of PGE(2) were produced by cercariae of Schistosoma mansoni following incubation with linoleic acid, a free fatty acid found on the surface of the skin. Cyclooxygenase (COX) 2 inhibitors failed to block this PGE(2) production, suggesting that a different biochemical pathway may be involved in the production of PGE(2) by the parasite. In addition, the parasites were also able to induce PGE(2) and IL-10 from human and mouse keratinocytes. Analysis of mouse skin during skin migratory phases of infection confirmed these in vitro observations. COX2 inhibitors blocked the parasite-induced PGE(2) and IL-10 from keratinocytes. Further analysis of the parasite secretions showed that the PGE(2)/IL-10-inducing effect was associated with a fraction <30 kDa molecular size. Addition of this fraction or parasite-stimulated keratinocyte culture supernatant to Con A-stimulated spleen cells resulted in the suppression of cell proliferation. This effect could be blocked by anti-IL-10 treatment. In sharp contrast, attenuation of the parasites with gamma-irradiation significantly abrogated their ability to induce PGE(2) or IL-10 from skin cells. Significance of IL-10 in host immunoregulation by skin stage schistosomula of S. mansoni was further confirmed by using IL-10-deficient mice. In these mice the normal subdued cutaneous reaction to the parasite was absent. Instead, a prominent cellular reaction occurred around the parasite, and there was considerable delay in parasitic migration through the skin. Thus these results suggest a key role for parasite-induced PGE(2) in IL-10-dependent down-regulation of host immune responses in the skin.     

Inhibition of cell cycle progression and migration of vascular smooth muscle cells by prostaglandin D2 synthase: resistance in diabetic Goto-Kakizaki rats

The regulation of vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis plays a clear role in the atherosclerotic process. Recently, we reported on the inhibition of the exaggerated growth phenotype of VSMCs isolated from hypertensive rats by lipocalin-type prostaglandin D₂ synthase (L-PGDS). In the present study, we report the differential effects of L-PGDS on VSMC cell cycle progression, migration, and apoptosis in wild-type VSMCs vs. those from a type 2 diabetic model. In wild-type VSMCs, exogenously added L-PGDS delayed serum-induced cell cycle progression from the G₁ to S phase, as determined by gene array analysis and the decreased protein expressions of cyclin-dependent kinase-2, p21^Cip1, and cyclin D1. Cyclin D3 protein expression was unaffected by L-PGDS, although its gene expression was stimulated by L-PGDS in wild-type cells. In addition, platelet-derived growth factor-induced VSMC migration was inhibited by L-PGDS in wild-type cells. Type 2 diabetic VSMCs, however, were resistant to the L-PGDS effects on cell cycle progression and migration. L-PGDS did suppress the hyperproliferation of diabetic cells, albeit through a different mechanism, presumably involving the 2.5-fold increase in apoptosis and the concomitant 10-fold increase of L-PGDS uptake we observed in these cells. We propose that in wild-type VSMCs, L-PGDS retards cell cycle progression and migration, precluding hyperplasia of the tunica media, and that diabetic cells appear resistant to the inhibitory effects of L-PGDS, which consequently may help explain the increased atherosclerosis observed in diabetes. apoptosis; atherosclerosis; insulin resistance