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Abstract Background The death-domain-associated protein (DAXX) was originally identified as a protein that binds to the transmembrane death receptor FAS and enhances both FAS-induced and transforming growth factor-β-dependent apoptosis. In a previous study, we found that nude mice injected with DAXX-overexpressing cells (ES-2-DAXX) accumulated large concentrations of first-generation ascites cells (I ascites cells). The role of DAXX in the development of ascites is unknown. The aim of this study was to analyze the effect of DAXX on proliferation and migration of ascites cells in ovarian cancer in vitro and in vivo. Methods Nude mice were housed in cages with a 14:10 h light:dark cycle; water and food were provided ad libitum. ES-2-DAXX cells (1×106) were injected intraperitoneally into athymic nude mice (8-week-old female mice). After 4 weeks, I ascites cells were collected. The I ascites cells were injected intraperitoneally into athymic nude mice (8-week-old female mice). After 4 weeks, II ascites cells were collected and cultured. Ascites cell survival, migration, and colony formation were measured using colony formation and cell growth assays. Immunofluorescent staining revealed the co-localization of DAXX and promyelocytic leukemia protein (PML) in ascites cell nuclei. Western blotting and immunohistochemistry showed that extracellular signal-related kinase (p-ERK) 1/2 and CEBP-β were highly expressed in tumor tissues formed by II ascites cells. Through immunoprecipitation, we also found that DAXX can interact with CEBP-β. Results DAXX enhanced ascites cell survival, migration, and colony formation. DAXX and PML nuclear foci dramatically increased in a passage-dependent manner in ascites cells, DAXX promoted the tumor growth of ascites cells in vivo, increased ascites cell proliferation in vivo, and enhanced ascites cell survival and migration by activating the ERK signalling pathway and integrating with CEBP-β. Conclusions DAXX can interact with CEBP-β. DAXX can induce ovarian cancer ascites formation by activating the ERK signal pathway and binding to CEBP-β.

Colorectal cancer (CRC) is the most common malignancy worldwide, and its underlying molecular mechanisms remain largely unexplored. Accumulating evidences indicate cancer-associated fibroblasts (CAFs), abundant stromal cell population in the tumor microenvironment, play a key role in tumor development. Herein, we have successfully isolated CAFs and paired normal fibroblasts (NFs) from colorectal cancer tissues (n=10). By using a multiplex cytokine profiling assay, we have identified IL-6 as a major cytokine released by CAFs. Co-culturing of CAFs with CRC cell lines HCT116 or SW480 increases IL-6 release, and the secretion by CAFs can be further enhanced under hypoxia. By using the CCK-8 assay, we have found that HCT116 or SW480 cells treated with culture medium from CAFs, IL-6, or hypoxia showed a significant cell growth compared to control cells (p<0.01). Mechanistically, we have found that hypoxia could enhance the effect of the IL-6/STAT3 signaling on CRC cells, in part, through HIF-1α targeting PKM2. In conclusion, our data clearly proposes the interconnected mechanisms for constitutive activation of STAT3 signal by CAFs-derived IL-6 under hypoxia in colorectal cancer. The pharmacological inhibition of STAT3, PKM2, or HIF-1α can significantly reduce the oncogenic effect of IL-6, providing a potential therapeutic target for CRC patients. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the impact of overexpression of long noncoding RNA PEG10 (lncRNA PEG10) on the proliferation, invasion and metastasis of hypopharyngeal squamous cell carcinoma (HSCC). Quantitative reverse transcription polymerase chain reaction was used to quantify lncRNA PEG10 expression levels in HSCC tumor tissues samples, para-carcinoma tissue samples and the HSCC FaDu cell line. Cell proliferation assays, Transwell invasion assays and wound healing assays were used to evaluate the effects of lncRNA PEG10 on FaDu cells in vitro. In 56 eligible patients, lncRNA PEG10 was expressed at higher levels in HSCC tumor tissues compared with para-carcinoma tissues, and significant associations were observed between increased tumor expression of lncRNA PEG10 and primary tumor size, lymph node status and tumor node metastasis stage. In the in vitro experimental studies, enhanced expression of lncRNA PEG10 was significantly associated with increased proliferation, invasion and metastasis of FaDu cells. lncRNA PEG10 was upregulated in HSCC, and its overexpression in HSCC cells promoted an increase in the tumorigenic activities of proliferation, invasion and migration. The potential underlying mechanisms require investigation in future studies. [ABSTRACT FROM AUTHOR]

The aim of the present study was to elucidate the regulatory role of cell proliferation and apoptosis in testicular development of wild Daurian ground squirrels during the breeding season (April), the non-breeding season (June) and before hibernation (September). Gross mass and hormonal analysis showed that the testis : body mass ratio and plasma testosterone concentration fluctuated seasonally, with a peak in April and lowest values in June. Similarly, spermatogenesis was fully developed in April but suppressed in June and September. Testicular decellularisation and vacuolisation was seen during the transition from the breeding to the non-breeding season. Furthermore, testicular levels of proliferating cell nuclear antigen, cyclin D2 and caspase-3 protein were significantly increased in June and September. Intriguingly, positive terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling staining and nuclear translocation of caspase-3 in testicular germ cells appeared only during the prehibernation period, whereas accumulation of cyclin D2 in spermatocyte nuclei occurred in September. These findings demonstrate, for the first time, that both cell proliferation and apoptosis are stimulated during the prehibernation period, indicating that a hormonal-regulated balance of testicular germ cell proliferation and apoptosis may play a pivotal role in preparing for testicular recrudescence of wild Daurian ground squirrels. [ABSTRACT FROM AUTHOR]

The molecular mechanisms underlying protection and pathogenesis in spinal degenerative diseases remain unclear. Tumor necrosis factor-α (TNF-α) has been demonstrated to induce apoptosis of inte rvertebral disc (IVD) cells during IVD degeneration, and 17β-estradiol (17β-E2) has a protective effect against IVD cell apoptosis. However, the underlying molecular mechanism by which 17β-E2 protects nucleus pulposus (NP) cells remains to be investigated. The aim of the present study was to evaluate whether 17β-E2 modulates apoptosis of human NP cells induced by TNF-α. In addition, the concentration-response effect of 17β-E2 on human NP cells was investigated. Human NP cells were cultured in complete medium, which was replaced every three days until the culture was ~80% confluent. Cells were treated with 100 ng/ml TNF-α for 48 h, with or without pretreatment with various concentrations of 17β-E2, and ICI 182,780, for 30 min. Morphologic alterations characteristic of apoptosis were observed by inverted phase-contrast microscopy and Hoechst 33258 staining; the apoptosis rate was analyzed by flow cytometry. A Cell Counting kit-8 assay was used to assess cell proliferation. Furthermore, caspase-3 activity was determined and proteins associated with apoptosis were analyzed by western blotting. The level of apoptosis and caspase-3 activity in human NP cells increased, whereas proliferation and the expression of poly ADP-ribose polymerase decreased following TNF-α treatment. These effects of TNF-α were abolished by pretreatment with 17β-E2 in a concentration-dependent manner. The results of the present study indicated that 17β-E2 serves a critical role in the survival of degenerative human NP cells. [ABSTRACT FROM AUTHOR]

Objective: Although our previous studies have confirmed that 1, 3, 8-trihydroxy-6-methylant hraquinone (emodin) inhibits migration and invasion in epithelial ovarian cancer (EOC) cells, the underlying molecular mechanism remains unknown. Here, the aim was to investigate the effects of emodin on EOC cells and to study further the mechanism underlying this process, both in vitro and in vivo. Materials and methods: Cell proliferation was evaluated by the methylthiazolyl tetrazolium assay. Cell migration and invasion abilities were tested using the transwell assay. The expression of integrin-linked kinase (ILK) and epithelial-mesenchymal transition (EMT)-associated factors were measured with western blotting. Results: Exogenous ILK enhanced the proliferation, migration and invasion properties of A2780 and SK-OV-3 cells. After treatment with emodin, the survival rate of cells was gradually reduced, including those of SK-OV-3/pLVX-ILK and A2780/pLVX-ILK cells, with increasing emodin concentrations. The migration and invasion abilities of A2780 and SK-OV-3 cells were effectively increased by the transfection of pLVX-ILK, which could be abrogated by following this with 48 hours of emodin treatment. Treatment with emodin significantly downregulated the expression of ILK and EMT-related proteins. So, emodin suppressed proliferation, migration and invasion in ovarian cancer cells by downregulating ILK in vitro. SK-OV-3/pLVX-Con and SK-OV-3/pLVX-ILK cells were used to generate xenografts in nude mice. Tumors grew more rapidly in the SK-OV-3/pLVX-ILK group compared with the control group, and this could be significantly inhibited by emodin. Also, the expression of E-cadherin was downregulated, while the expression of Slug, MMP-9 and Vimentin were upregulated in the SK-OV-3/pLVX-ILK group, and this could be reversed by following treatment with emodin. Emodin did not demonstrate target toxicity on hepatocytes, nephrocytes and cardiomyocytes. Conclusion: Emodin suppresses proliferation, migration and invasion in ovarian cancer by targeting ILK. [ABSTRACT FROM AUTHOR]

The promyelocytic leukemia (PML) gene is a tumor suppressor gene. It was first identified in acute promyelocytic leukemia, in which it is fused to retinoic acid receptor a by the (15;17) chromosomal translocation. The function of the PML protein is frequently lost or aberrant in human solid tumors. In human ovarian carcinoma tissue, PML detected by immunohistochemistry was highly expressed. A PML-silencing vector, pSRG-shPml, was constructed and used to transfect human ovarian cancer cells. Cells were cultured and selected with puromycin for 10-15 days, and then the PML mRNA expression levels were detected by RT-qPCR and immunofluorescence. Proliferation and clone number of PML-depleted cells were detected using MTT assay and colony-forming assay. The protein expression associated with DNA damage and apoptosis was assessed in PML-depleted cells using western blot analysis and immunofluorescence. The results showed that PML was highly expressed in human ovarian tissue. The proliferation and colony formation of ovarian cancer cells were significantly inhibited after PML was depleted. Western blot analysis and immunofluorescence revealed that p-H2AX and cleaved caspase-3 expression significantly increased after PML silencing. PML was located in the nucleus, and it formed foci after X-ray irradiation. PML foci increased significantly with increasing irradiation doses. [ABSTRACT FROM AUTHOR]

Background: As a new anti-diabetic medicine, Liraglutide (LIRA), one of GLP-1 analogues, has been found to have an anti-atherosclerotic effect. Since vascular smooth muscle cells (VSMCs) play pivotal roles in the occurrence of diabetic atherosclerosis, it is important to investigate the role of LIRA in reducing the harmful effects of high-glucose (HG) treatment in cultured VSMCs, and identifying associated molecular mechanisms. Methods: Primary rat VSMCs were exposed to low or high glucose-containing medium with or without LIRA. They were challenged with HG in the presence of phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK)1/2, or glucagon-like peptide receptor (GLP-1R) inhibitors. Cell proliferation and viability was evaluated using a Cell Counting Kit-8. Cell migration was determined by Transwell migration and scratch wound assays. Flow cytometry and Western blotting were used to determine apoptosis and protein expression, respectively. Results: Under the HG treatment, VSMCs exhibited increased migration, proliferation, and phosphorylation of protein kinase B (Akt) and ERK1/2, along with reduced apoptosis (all p < 0.01 vs. control). These effects were significantly attenuated with LIRA co-treatment (all p < 0.05 vs. HG alone). Inhibition of PI3K kinase and ERK1/2 similarly attenuated the HG-induced effects (all p < 0.01 vs. HG alone). GLP-1R inhibitors effectively reversed the beneficial effects of LIRA on HG treatment (all p < 0.05). Conclusions: HG treatment may induce abnormal phenotypes in VSMCs via PI3K and ERK1/2 signaling pathways activated by GLP-1R, and LIRA may protect cells from HG damage by acting on these same pathways. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA) plays a key role in development and specific biological processes, such as cell proliferation, differentiation, and apoptosis. Extensive studies of mammary miRNAs have been performed in different species and tissues. However, little is known about porcine mammary gland miRNAs. In this study, we report the identification and characterization of miRNAs in the lactating mammary gland in two distinct pig breeds, Jinhua and Yorkshire. Many miRNAs were detected as significantly differentially expressed between the two libraries. Among the differentially expressed miRNAs, many are known to be related to mammary gland development and lactation by interacting with putative target genes in previous studies. These findings suggest that miRNA expression patterns may contribute significantly to target mRNA regulation and influence mammary gland development and peak lactation performance. The data we obtained provide useful information about the roles of miRNAs in the biological processes of lactation and the mechanisms of target gene expression and regulation. [ABSTRACT FROM AUTHOR]

Diabetes is a chronic disease that disrupts the balance between bone formation and bone desorption, which can lead to osteoporosis, increasing the risk of fracture. However, compared with osteoblasts, the biological effects of hyperglycemia on osteoclastogenesis remain to be elucidated. Therefore, we investigated the impact of glucose at different concentrations (5.5, 10.5, 15.5, 20.5, 25.5, and 30.5 mM) on osteoclastogenesis using RAW264.7 cells. Cell proliferation was measured with the cell counting kit-8 assay, and osteoclastogenesis was detected with tartrate-resistant acid phosphatase staining and bone resorption assays, as well as protein cathepsin K expression. Compound C, the AMP-activated protein kinase (AMPK) pathway inhibitor, was used to examine the relationship between the AMPK/mTOR/ULK1 signaling pathway and autophagy in osteoclasts. Autophagy was evaluated with transmission electron microscopy and immunofluorescence microscopy and associated proteins were detected with western blotting. The pharmacological autophagic reagents bafilomycin A1, 3-methyladenine, and rapamycin were used to determine the effect of autophagy on osteoclastogenesis. Our results showed that glucose negatively affected osteoclast formation and function but did not affect the proliferation of RAW264.7 cells. Suppression of the AMPK/mTOR/ULK1 signaling axis decreased autophagy in glucose-mediated osteoclast. Furthermore, High levels of glucose decreased autophagy level in osteoclasts. Additionally, interfering with autophagy affected osteoclast formation and function. These findings clarify the mechanisms underlying the effects of glucose-mediated osteoclastogenesis and will help identify novel therapeutic strategies for the protection of skeletal health in diabetic osteoporosis. [ABSTRACT FROM AUTHOR]

Abstract Purpose To explore the function and regulatory mechanism of IFITM3 in mouse neural retinal progenitor cells (mNRPCs), which was found to be very important not only in the development of the retina in embryos but also in NRPCs after birth. Methods Published single-cell sequencing data were used to analyze IFITM3 expression in mNRPCs. RNA interference was used to knock down the expression of IFITM3. CCK-8 assays were used to analyze cell viability. RNA-seq was used to assess mRNA expression, as confirmed by real-time quantitative PCR, and immunofluorescence assays and western blots were used to validate the levels of relative proteins, and autophagy flux assay. Lysosomal trackers were used to track the organelle changes. Results The results of single-cell sequencing data showed that IFITM3 is highly expressed in the embryo, and after birth, RNA-seq showed high IFITM3 expression in mNRPCs. Proliferation and cell viability were greatly reduced after IFITM3 was knocked down. The cell membrane system and lysosomes were dramatically changed, and lysosomes were activated and evidently agglomerated in RAMP-treated cells. The expression of LAMP1 was significantly increased with lysosome agglomeration after treatment with rapamycin (RAMP). Further detection showed that SQSTM1/P62, HSC70 and LAMP-2A were upregulated, while no significant difference in LC3A/B expression was observed; no autophagic flux was generated. Conclusion IFITM3 regulates mNRPC viability and proliferation mainly through chaperone-mediated autophagy (CMA) but not macroautophagy (MA). IFITM3 plays a significant role in maintaining the homeostasis of progenitor cell self-renewal by sustaining low-level activation of CMA to eliminate deleterious factors in cells.