Caveolin-1, an indispensable component of caveolae serving as a transformation suppressor protein, is highly expressed in poorly metastatic mouse osteosarcoma FBJ-S1 cells while highly metastatic FBJ-LL cells express low levels of caveolin-1. Calcium concentration is higher in FBJ-S1 cells than in FBJ-LL cells; therefore, we investigated the possibility that calcium signaling positively regulates caveolin-1 in mouse FBJ-S1 cells. When cells were treated with the calcium channel blocker nifedipine, cyclosporin A (a calcineurin inhibitor), or INCA-6 (a nuclear factor of activated T-cells [NFAT] inhibitor), caveolin-1 expression at the mRNA and protein levels decreased. RNA silencing of voltage-dependent L-type calcium channel subunit alpha-1C resulted in suppression of caveolin-1 expression. This novel caveolin-1 regulation pathway was also identified in mouse NIH 3T3 cells and Lewis lung carcinoma cells. These results indicate that caveolin-1 is positively regulated at the transcriptional level through a novel calcium signaling pathway mediated by L-type calcium channel/Ca(2+)/calcineurin/NFAT.

Circular RNAs (circRNAs) are a group of non-protein-coding RNAs generated from back splicing. Emerging evidence has demonstrated its vital regulation on angiogenesis. However, the underlying mechanism responsible for circRNAs effects on vascular endothelial cells is still unclear. In the present study, we screened the expression profiles and investigated the physiological role of circRNAs in hypoxia-induced human umbilical vein endothelial cells (HUVECs). Using circRNA microarray analysis, we identified 36 circRNAs that were significantly dysregulated including 14 down-regulated circRNAs and 22 up-regulated with 2-fold change (P < 0.05). From the over-expressed circRNAs, hsa_circ_0010729 was selected as candidate circRNA and which was validated to be significantly up-regulated using RT-PCR. In loss-of-function experiments of HUVECs, hsa_circ_0010729 knockdown suppressed the proliferation and migration ability and enhanced apoptosis. Bioinformatic prediction and luciferase assay revealed that hsa_circ_0010729 and hypoxia inducible factor 1 alpha (HIF-1α) were targeted by miR-186. Validation experiments verified that hsa_circ_0010729 was co-expressed with HIF-1α, being negatively correlated with miR-186. Moreover, rescue experiments demonstrated that miR-186 inhibitor could reverse the role of hsa_circ_0010729 knockdown on HUVECs progression. Overall, the present study identifies the crucial regulation of hsa_circ_0010729 on vascular endothelial cell proliferation and apoptosis via targeting miR-186/HIF-1α axis.

In March 2003, a novel coronavirus was isolated from patients exhibiting atypical pneumonia and subsequently proven to be the causative agent of the disease now referred to as severe acute respiratory syndrome (SARS). The complete genome of the SARS coronavirus (SARS-CoV) has since been sequenced. The SARS-CoV nucleocapsid (SARS-CoV N) shares little homology with other members of the coronavirus family. To determine if the N protein is involved in the regulation of cellular signal transduction, an ELISA-based assay on transcription factors was used. We found that the amount of transcription factors binding to promoter sequences of c-Fos, ATF2, CREB-1, and FosB was increased by the expression of SARS-CoV N. Since these factors are related to AP-1 signal transduction pathway, we investigated whether the AP-1 pathway was activated by SARS-CoV N protein using the PathDetect system. The results demonstrated that the expression of N protein, not the membrane protein (M), activated AP-1 pathway. We also found that SARS-CoV N protein does not activate NF-kappaB pathway, demonstrating that activation of important cellular pathways by SAS-CoV N protein is selective. Thus our data for the first time indicate that SARS-CoV has encoded a strategy to regulate cellular signaling process.

The ubiquitin-specific protease Usp7 plays roles in multiple cellular processes through deubiquitinating and stabilizing numerous substrates, including P53, Pten and Gli. Aberrant Usp7 activity has been implicated in many disorders and tumorigenesis, making it as a potential target for therapeutic intervention. Although it is clear that Usp7 is involved in many types of cancer, its role in regulating medulloblastoma (MB) is still unknown. In this study, we show that knockdown of Usp7 inhibits the proliferation and migration of MB cells, while Usp7 overexpression exerts an opposite effect. Furthermore, we establish Usp7 knockout MB cell line using the CRISPR/Cas9 system and further confirm that Usp7 knockout also blocks MB cell proliferation and metastasis. In addition, we reveal that knockdown of Usp7 compromises Shh pathway activity and decrease Gli protein levels, while P53 level and P53 target gene expression have no obvious changes. Finally, we find that Usp7 inhibitors apparently inhibit MB cell viability and migration. Taken together, our findings suggest that Usp7 is important for MB cell proliferation and metastasis by activating Shh pathway, and is a putative therapeutic target for MBs.

Previously, we confirmed the anti-tumor effects of sodium butyrate (NaBu) in nasopharyngeal carcinoma (NPC). However, its molecular mechanisms have not be fully elucidated. In this study, we studied the effects of NaBu on autophagy and explored the relation between NaBu associated autophagy and apoptosis in NPC cells. EGFP-LC3 plasmids were introduced into NPC cells to observed the effects of NaBu on autophagy flux with or without chloroquine (CQ) addition. Autophagy markers were also detected by Western blot. Under NaBu treatment, autophagy and apoptosis markers were detected simultaneously at different time. Then, to explore the roles of autophagy in NaBu induced apoptosis, the effects of autophagy inhibition, via specific inhibitor treatment or key gene knockdown, were analyzed. At last, the upstream signaling and its roles in NaBu induced autophagy and apoptosis were also analyzed. Increased LC3 dots and LC3-II accumulation indicated that NaBu can promote autophagy flux in NPC cells. LC3-II accumulation was earlier than cleaved PARP increment suggesting autophagy activation is prior to apoptosis activation, which was validated by flow cytometry mediated apoptosis analysis. Moreover, autophagy inhibition, achieved by 3-MA treatment or BECN1 knockdown, can antagonize NaBu induced apoptosis reflecting by re-deregulated cPARP and apoptotic rates. Furthermore, NaBu treatment inhibited the AKT/mTOR axis indicated by deregulated p-AKT(S473) and p-mTOR(S2448) and ectopic AKT expression both suppressed NaBu induced autophagy and apoptosis. At last, Western blot showed that HDAC6 dependent EGFR deregulation may account for the NaBu associated AKT/mTOR inhibition. NaBu can induce autophagic apoptosis via suppressing AKT/mTOR axis in NPC cells. Our results suggest that combination of autophagy inhibitors and deacetylase inhibitors may not be recommended in NPC clinical treatment.

Influenza A viruses are a major threat to human health and inflict a significant public health challenge worldwide. Hemagglutinin (HA) is the main contributor to the infectivity of the virus and is a potential target for the development of antiviral therapeutic agents. The stem region of HA, which has been shown to be conserved, is the primary target in the development of antibody-based therapeutic strategies and preventive tools. Here, we confirmed the neutralizing activity and prophylactic efficacy of murine monoclonal antibody PR8-25 as well as Western blot analysis of different influenza virus strains. Then, we identified the epitope of PR8-25, 328-LRMVTGLRNIPS-339, by phage-displayed 12-mer random peptide library. The identified epitope targeted in the stem region of HA, specifically at the C-terminal of the HA1 fragment. This result suggest that the identified epitope may be a potential basis for antiviral drugs and stem-based universal influenza vaccines.

The inflammation and immune hypothesis of major depressive disorder (MDD) explains the mechanism of neuroinflammatory response to promote depression-like behaviors and provides targets for immunotherapy. Previous studies revealed that the neuronal function of the entorhinal cortex (EC) was relative to the depression symptoms in MDD. However, it remains largely unknown what role of neuroinflammation plays in the EC. Hence, we used immunofluorescence to determine c-Fos expression in the EC of lipopolysaccharide (LPS)-treated mice. Mice model was constructed of 10-day LPS treatment, and depression-related behaviors were assessed. We used gene expression microarray to determine differentially expressed genes (DEGs) in the EC of LPS group comparing to control group, and molecular verification was performed by quantitative real-time PCR and Western blot. We found that c-Fos expression was significant reduced in the two layers (Lateral 3.25 mm and 3.00 mm) of the EC in LPS-treated mice compared to saline-treated mice. Mice in LPS group exhibited depression- and anxiety-like behaviors in chronic model. Gene expression analyses identified 339 DEGs in the EC between LPS and control group. The molecular verification showed activation of IL-1R1/NF-κB/CCL5 signaling and upregulation of markers of astrocyte (GFAP) and microglia (AIF1 and CD86) in the EC. Our results suggested that LPS-induced neuroinflammation inhibited neuronal activity in the EC of mice, and that activation of IL-1R1/NF-κB/CCL5 signaling could be involved in the neuroinflammation in the EC of LPS-treated depression model.

Neoadjuvant chemoradiation (nCRT) followed by radical surgery is the preferred option for locally advanced colorectal cancer (CRC) treatment. However, chemo/radio-resistance remains a main obstacle in CRC therapy. In the study, we analyzed the mRNA expression profiling of CRC patients and revealed that the aberrant expression of fibronectin type III domain containing 1 (FNDC1) was associated with disease progression and poor prognosis in CRC. FNDC1 expression was consistently increased in multiple independent cohorts of CRC. Upregulated FNDC1 in pretreated primary tumor tissues predicted a poor response to nCRT, recurrence, and poor disease-free survival in nCRT-treated CRC patients. FNDC1 overexpression accelerated CRC cell survival on 5-FU or radiation treatment both in vitro and in vivo, whereas FNDC1 inhibition sensitized CRC cells to chemoradiation. In addition, FNDC1 accelerated stem cell-like properties of CRC cells. Furthermore, tumor tissues from non-responders exhibited higher activation of PI3K/Akt signaling than those from responders. FNDC1 depletion repressed 5-FU or irradiation-induced activation of PI3K/AKT in CRC cells. More importantly, pharmacological inhibition of PI3K/Akt signaling effectively decreased the effect of FNDC1 on chemoradiation resistance. Taken together, our study reveals the potential function of FNDC1 as a biomarker to predict nCRT sensitivity in CRC and a therapeutic target in CRC treatment.

Glycosylation is one of the most common post-translational modifications which diversifies the structure and function of glycoproteins like immunoglobulin G (IgG). The effector function of IgG depends on N-glycan patterns located in the crystalline fragment (Fc). Fc gamma receptor (FcγR)-binding affinity is one of the most important effector functions in IgG, and it varies with different IgG isotypes. Murine IgG1 (mIgG1) triggers various immune effector functions via FcγRs, however, how N-glycans of mIgG1 impact interactions between mIgG1s and murine FcγRs remains largely unknown. Here, we generated mIgG1s with different N-glycan patterns by adding different types of N-glycan processing enzyme inhibitors to the hybridoma culture media, before comparing their FcγR-binding affinity using enzyme-linked immunosorbent assay (ELISA) analysis. We showed that N-glycans critically affect mIgG1 affinity to FcγRs. The removal of N-glycans nearly completely abolished mIgG1-FcγR binding. In comparison, when N-glycans are present, decreasing fucosylation levels enhanced the FcγR-binding affinity regardless of the types of N-glycans. Furthermore, high-mannose type and hybrid type N-glycans reduced FcγR-binding affinity, compared to complex type N-glycans. In conclusion, our findings clearly demonstrate that FcγR-binding affinity of mIgG1 is under the control of glycosylation. Importantly, we found that both the levels of specific glycosylation as well as the types of N-glycans affect FcγR-binding affinity. Together, these insights should greatly expand our understanding of N-glycans function in general, and assist in manipulating host immune responses by controlling antibody N-glycan patterns, which is important for designing therapeutic antibodies with improved characteristics.

To investigate the influence of miR-18a-3p and ADCY5 on OP and osteogenic differentiation of human Mesenchymal stem cell (hBMSCs) and its possible mechanism. Samples were collected from osteoporotic patients with or without vertebral compression fracture, and without OP volunteers. MiR-18a-3p and ADCY5 mRNA expression levels in the tissue samples and hBMSCs during osteogenic differentiation were detected。MiR-18a-3p mimic and OE-ADCY5 were introduced into hBMSCs to research the effects of miR-18a-3p and ADCY5 on osteogenesis differentiation of hBMSCs. Dual luciferase reporter system and RNA pull-down were applied to determine whether ADCY5 was a target gene of miR-18a-3p. Compared with the control group, ADCY5 expression level was down-regulated in patients with OP-no-Frx and OP-Frx, but that of miR-18a-3p was up-regulated. In addition, ADCY5 increased during osteogenesis differentiation of hBMSCs, whereas miR-18a-3p did not. OE-ADCY5 significantly facilitated calcium deposition, ALP activity, osteoblast protein expression (OSX, ALP and EUNX2), miR-18a-3p mimic inhibited osteogenic differentiation, and partially reversed the effect of OE-ADCY5 on osteogenic differentiation. In general, miR-18a-3p targets ADCY5 to promote OP and may be involved in spinal fracturs.

It is well-known that one of the most important features of intervertebral disc degeneration (IDD) is the extracellular matrix (ECM) degradation. Collagen and aggrecan are major components of ECM; the degradation of ECM in intervertebral discs (IVDs) is closely related to the activities of collagenase and aggrecanase. TIMP-3 is the most efficient inhibitor of aggrecanase in IVD. However, only few studies focus on the potential relationship between TIMP-3 and IDD. In our study, we found TIMP-3 gene expression was decreased after stimulating with LPS in rat nucleus pulposus (NP) cells. Then we used a lentivirus vector to reconstruct rat NP cells which high expressed TIMP-3 gene (LV-TIMP3). The upregulation of MMPs and ADAMTSs induced by LPS was significantly inhibited in LV-TIMP3 cells. After overexpression of TIMP-3, the aggrecan breakdown caused by LPS was also reduced in both monolayer culture and three-dimension culture model. To further study the relation between TIMP-3 and IDD, we collected human NP tissue samples of different degenerative degrees. Real-time PCR and immunohistochemical staining showed that the expression of TIMP-3 was negatively correlated with the degree of intervertebral disc degeneration, while MMP-1 and ADAMTS-4 were markedly increased in degenerative IVD. Taken together, our results suggest that the imbalance between aggrecanase and TIMP-3 may play an important role in the pathogenesis of IDD and therefore be a potential therapeutic target for treating IDD.

Upregulated gene 11 (URG11), recently identified as a new HBx-upregulated gene that may activate beta-catenin and Wnt signaling, was found to be upregulated in a human tubule cell line under low oxygen. Here, we investigated the potential role of URG11 in hypoxia-induced renal tubular epithelial-to-mesenchymal (EMT). Overexpression of URG11 in a human proximal tubule cell line (HK2) promoted a mesenchymal phenotype accompanied by reduced expression of the epithelial marker E-cadherin and increased expression of the mesenchymal markers vimentin and alpha-SMA, while URG11 knockdown by siRNA effectively reversed hypoxia-induced EMT. URG11 promoted the expression of beta-catenin and increased its nuclear accumulation under normoxic conditions through transactivation of the beta-catenin promoter. This in turn upregulated beta-catenin/T-cell factor (TCF) and its downstream effector genes, vimentin, and alpha-SMA. In vivo, strong expression of URG11 was observed in the tubular epithelia of 5/6-nephrectomized rats, and a Western blot analysis demonstrated a close correlation between HIF-1alpha and URG11 protein levels. Altogether, our results indicate that URG11 mediates hypoxia-induced EMT through the suppression of E-cadherin and the activation of the beta-catenin/TCF pathway.

The purpose of this study was to investigate the role of GnT-V on radiosensitivity in human nasopharyngeal carcinoma (NPC) both in vitro and in vivo, and the possible mechanism. The GnT-V stably suppressed cell line CNE-2 GnT-V/2224 was constructed from CNE-2 by transfection. The radiosensitivity of the cells was studied by CCK-8 assay, flow-cytometry, caspases-3 activity analysis and tumor xenografts model. The expression of Bcl-2, Bax and Bcl-xl was analyzed with or without radiation. The results showed that down-regulation of GnT-V enhanced CNE-2 radiosensitivity. The underlying mechanisms may be link to the cell cycle G2-M arrest and the reduction of Bcl-2/Bax ratio. The results suggest that GnT-V may be a potential target for predicting NPC response to radiotherapy.

The human adenovirus oncoprotein E4orf6 hijacks intracellular Cullin 5-based E3 ubiquitin ligases (CRL5s) to induce the degradation of host proteins, including p53, that impede efficient viral replication. The complex also relies on another viral protein, E1B55K, to recruit substrates for ubiquitination. However, the determinants of adenoviral E4orf6-CRL5 E3 ligase-mediated p53 degradation in the scaffolding protein Cullin5 remain rarely investigated. Here, we demonstrated that the viral protein E4orf6 triggered relocalization of the Cullin5 protein from the cytoplasm to the nucleus and induced activation of the CRL5 E3 ligase via facilitating neddylation. The expression of the deneddylase SENP8/Den1 was significantly downregulated by E4orf6. We then identified SENP8 as a natural restriction factor for E4orf6-induced p53 degradation. Furthermore, our results indicated that the NEDD8-conjugating E2 enzyme UBE2M was essential for E4orf6-mediated p53 degradation and that its dominant negative mutant UBE2M C111S dramatically blocked E4orf6 functions. The Nedd8-activating enzyme inhibitor MLN4924 decreased E4orf6-induced neddylation of the cullin5 protein and subsequently suppressed p53 degradation. Collectively, our findings illuminate the strategy by which this viral oncoprotein specifically utilizes the neddylation pathway to activate host CRL E3 ligases to degrade host restriction factors. Disrupting this post-translational modification is an attractive pharmacological intervention against human adenoviruses.

A number of homeobox genes are implicated in the malignancy of various cancers. Here, we investigated the role of the homeobox gene SIX4 in non-small-cell lung cancer (NSCLC). The sine oculis homeobox (SIX4) gene was found to be highly expressed at both mRNA and protein levels in NSCLC tumor tissues as compared with matching normal counterparts. In this study, the SIX4 gene of two human NSCLC cell lines (A549 and PC9) was overexpressed or silenced using the lentiviral system. We evaluated the malignancy-associated phenotype of transfected cells, which demonstrated that exogenous expression of the SIX4 gene greatly enhanced the proliferation, migration, and invasion of NSCLC cells. The opposite was true in the SIX4-silenced cells. Transcriptomic profiling analysis revealed that the SIX4 gene modulated the expression of hundreds of downstream target genes in a cell context-dependent manner. Most notably, the SIX4 gene controls the expression of crucial genes with evidently oncogenic function. We conclude that SIX4 plays an oncogenic role and may be potentially utilized as a diagnostic and therapeutic marker for NSCLC.

Diabetic retinopathy (DR), a major cause of blindness in working-age people, is attributed to the inflammatory response of retinal Müller cells (RMCs). The heparanase inhibitor PG545 plays proautophagic and anti-inflammatory roles. Intraperitoneal injection of PG545 at a dose of 20 mg/kg/d clearly reduced diabetes-induced body weight changes and fasting blood glucose levels in mice. PG545 also mitigated the reduction in retinal thickness and the formation of microaneurysms by promoting autophagy to inhibit the inflammatory response. In vitro, PG545 stimulated autophagy to downregulate the inflammatory response in high glucose-induced primary adult mouse RMCs. These data suggest that PG545 mitigates DR by promoting RMC autophagy to inhibit the inflammatory response.

Halogenated compounds are widely discovered in nature, and many of them exhibit biological activities, such as an important chlorinated natural product salinosporamide A serving as a potential anticancer agent. Compared with bromination, iodination and fluorination, chlorination is the mainly important modification. To shed light on the mechanism of SAM-dependent chlorinases, a recombinant chlorinase ClA1 was expressed in Escherichia coli and further purified for crystallization and X-ray diffraction experiments. The flake crystals of ClA1 were able to diffract to a resolution of 1.85 Å. The crystals belonged to space group R3, with unit-cell parameters α = β = 90.0°, γ = 120.0°. By determining the structure of ClA1, it is revealed that the side chain of Arg242 in ClA1 may have contacts with the L-Met. However, in SalL the equivalent Arg243's side chain is far from L-Met. Considering the ClA1 and SalL are from different environments and their enzyme kinetics are quite different, it is suggested that the side chain conformation differences of the conserved arginine are possibly related with the enzyme activity differences of the two chlorinases.

Y-box binding protein (YB-1), known as a multifunctional cellular protein in various biological processes, was recently reported to be associated with liver fibrosis. The critical role of TGF-β/Smad signaling pathway in stimulating the transcription of fibrotic genes in fibroblasts have already been identified, however, whether and how YB-1 modulated liver fibrosis via TGF-β/Smad signaling pathway remains largely unknown. In our previous study, we proved that ectopic TGF-β was associated with YB-1 expression. Herein, by combining in vitro experiments in LX2 human hepatic stellate cells and in vivo studies by building CCl4 based mice liver fibrosis model, we showed that YB-1 and p-YB-1 were upregulated in liver fibrosis tissue, and YB-1 promoted the deposition of excess extracellular matrix. Mechanistically, Smad2, a key member in TGF-β signaling pathway, acted as a transcription factor that triggered YB-1 promoter, while on the other hand, p-YB-1 stabilized Smad2 by attenuating its ubiquitination. Knockdown of Smad2 could reduce YB-1 expression, which in turn shorter the half time of Smad2. Furthermore, the serine102 residue of YB-1 both affected its binding and stabilizing activity to Smad2. These finding demonstrated that YB-1 and Smad2 played as a positive feedback loop in promoting liver fibrosis. In conclusion, TGF-β signaling pathway may influence liver fibrosis by incorporating with YB-1, indicating that YB-1 could be a potential target for therapies against liver fibrosis.

Cancer immunotherapy has many great achievements in recent years. One of the most promising cancer immunotherapies is PD-1/PD-L1 pathway blockade. miRNAs (MicroRNAs) belongs to small noncoding RNA and can regulate gene expression by binding to the 3'UTR. Many miRNAs can inhibit cancer growth by regulating the PD-L1 expression in cancer cells. Herein, we firstly found that PD-L1 could be the target of miR-142-5p by using bioinformatics methods, then we conduct luciferase activity assay, RT-PCR and western blot experiments to demonstrate that miR-142-5p can regulate PD-L1 expression by binding to its 3'UTR. And in vivo experiments certified that miR-142-5p overexpression can inhibit pancreatic cancer growth. Flow cytometry and RT-PCR experiment demonstrated that miR-142-5p overexpression on tumor cells inhibits the expression of PD-L1 on tumor cells which result in the increase of CD4+ T lymphocytes and CD8+ T lymphocytes, the decrease of PD-1+ T lymphocytes and increase of IFN-γ and TNF-α. So, miR-142-5p overexpression can enhance anti-tumor immunity by blocking PD-L1/PD-1 pathway. Our results identify a novel mechanism by which PD-L1 is regulated by miR-142-5p and overexpression of miR-142-5p could enhance the anti-tumor immunity.

DNA methyltransferase 3A (DNMT3A) catalyzes de novo DNA methylation and plays important roles in the pathogenesis of acute myeloid leukemia. However, the expression status of DNMT3A variants in acute myeloid leukemia remains obscure. This study aimed to assess the expression levels of alternative splicing of DNMT3A variants and explore their roles in acute myeloid leukemia (AML). DNMT3A variants gene expression were assessed, measuring their effects on cell proliferation. In addition, the expression of DNMT3A variants were evaluated in acute myeloid leukemia patients. Four DNMT3A variants were identified, with DNMT3A1 and DNMT3A2V found to be dominant in acute myeloid leukemia cell lines. Moreover, DNMT3A2V overexpression delayed cell proliferation; while, DNMT3A2V R882H mutation promoted cell proliferation. Further, DNMT3A1 and DNMT3A2V were detected in newly diagnosed acute myeloid leukemia (AML) patients and controls with non-malignant hematological disease, with DNMT3A2V significantly up-regulated in AML patients. The main transcript switched from DNMT3A1 to DNMT3A2V in some patients, especially the low risk group based on the NCCN 2016 guidelines. These findings suggest that DNMT3A1 and DNMT3A2V are the main variants in acute myeloid leukemia with different clinical association, and might play important roles in the pathophysiology of acute myeloid leukemia.