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.

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.

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.

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.

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.

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.

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.

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.

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.

Cerebral ischemia-reperfusion-induced microglial activation causes neuronal death through the release of inflammatory cytokines. Increasing evidence suggests that microRNAs (miRNAs) exert a neuroprotective effect by modulating the inflammatory process in cerebral ischemia-reperfusion injury. Furthermore, Toll-like receptor 4 (TLR4) is increasingly being considered to have a significant role in the regulation of inflammation. However, whether miRNAs mediate their neuroprotective effects by regulating TLR4-mediated inflammatory responses remains unknown. To explore this gap in the literature, we conducted both in vitro and in vivo experiments. In vitro: BV2 cells were activated by oxygen-glucose deprivation (OGD). TLR4 and inflammatory cytokine (TNF-a, IL-6, and IL-1β) transcription and translation expression levels were assessed using RT-PCR, ELISA, and western blot. BV2 cells were transfected with miR-182-5p mimics, inhibitors, siTLR4, or negative control (NC) using lipofectamine 2000 reagent. To confirm whether TLR4 is a direct target of miR-182-5p, we performed a luciferase reporter assay. In BV2 cells, we observed that OGD upregulated TLR4 expression, but downregulated miR-182-5p expression. We determined that miR-182-5p inhibited TLR4 by directly binding to its 3'-UTR. Furthermore, miR-182-5p suppressed the release of TNF-a, IL-6, and IL-1β. In vivo: A middle cerebral artery occlusion (MCAO) rat model was used to mimic cerebral ischemia-reperfusion. Iba1 and TLR4 double staining was used to demonstrate that the target of miR-182-5p in microglial cells, and the mediator of the anti-inflammatory effect, is TLR4. TTC staining was performed to evaluate the infarct volume. Compared to the animals treated with miR-182-5p NC and normal saline, rats treated with miR-182-5p mimics demonstrated significantly enhanced neurological functions. TTC staining results were consistent with neurological function test findings. In summary, our data suggested that miR-182-5p exhibits potential neuroprotective effects in the cerebral ischemia-reperfusion injury via the regulation of the TLR4-mediated inflammatory response.

Recent studies have shown that metformin, a widely used antidiabetic agent, may reduce the risk of cancer development. In this study, we investigated the antitumoral effect of metformin on both acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells. Metformin induced apoptosis with partial differentiation in an APL cell line, NB4, but only displayed a proapoptotic effect on several non-M3 AML cell lines. Further analysis revealed that a strong synergistic effect existed between metformin and all-trans retinoic acid (ATRA) during APL cell maturation and that metformin induced the hyperphosphorylation of extracellular signal-regulated kinase (ERK) in APL cells. U0126, a specific MEK/ERK activation inhibitor, abrogated metformin-induced differentiation. Finally, we found that metformin induced the degradation of the oncoproteins PML-RARα and c-Myc and activated caspase-3. In conclusion, these results suggest that metformin treatment may contribute to the enhancement of ATRA-induced differentiation in APL, which may deepen the understanding of APL maturation and thus provide insight for new therapy strategies.

HCA587, also known as MAGE-C2, belonging to the MAGE gene family which is characterized by a conserved MAGE Homology Domain, is active in various types of tumors and silent in normal tissues except in male germ-line cells. The biological function of HCA587 is largely unknown. To analyze it, we attempted to identify protein partners of HCA587. We immunopurified HCA587-containing complex from HEK293 cells and identified BS69, a potential tumor suppressor, as an associated protein by mass spectrometry, and the following Immunoprecipitation and GST pull-down assays confirmed HCA587 interaction with BS69. Interestingly, overexpression of HCA587 promoted ubiquitination and the proteasomal degradation of BS69 whereas knockdown of endogenous HCA587 increased the protein level of BS69. Consistent with a functional role for BS69 in negatively regulating LMP1-induced NF-κB activation, overexpression of HCA587 resulted in a significant enhancement of LMP1-induced IL-6 production. These data indicate that HCA587 is a new negative regulator of BS69.

Circular RNA (circRNA), a special class of non-coding RNA, is increasingly being realized as a critical regulator in human diseases, including carcinomas. However, its role in hepatocellular carcinoma (HCC) metastasis remains largely unknown. Herein, we enrolled three Gene Expression Omnibus (GEO) databases and screened and identified a novel circRNA, circ-ZNF652 (hsa_circ_0003258), which was significantly upregulated in HCC tissues and cell lines. Importantly, HCC patients with high circ-ZNF652 expression were more prone to vascular invasion, intrahepatic metastasis, distant metastasis, and poor outcome. Subsequent functional experiments showed that depletion of circ-ZNF652 dramatically suppressed the migratory and invasive capabilities of HCC cells in vitro as well as tumor metastasis in vivo by inhibiting the process of epithelial-mesenchymal transition (EMT). Mechanistically, circ-ZNF652 could physically interact with miR-203 and miR-502-5p to increase the expression of their common target gene Snail (a key transcription factor that triggers EMT), thereby promoting the metastasis of HCC. In turn, the upregulated Snail was capable of binding to the E-box motif (CAGGTG) on the promoter of circ-ZNF652 to elevate circ-ZNF652 expression. Collectively, our findings suggest that circ-ZNF652 is a novel driver of EMT and unveil the important regulatory role of circ-ZNF652/miR-203/miR-502-5p/Snail feedback loop in HCC metastasis.

Glycoprotein130 (gp130) is an important signal transducer in the interleukin-6 (IL-6) trans-signaling pathway, which plays a crucial role in chronic inflammation in atherosclerosis. Studies suggest that estrogen can inhibit IL-6/gp130 signaling and reduce the risk of coronary artery disease, but the precise mechanism is unclear. The aim of this study was to investigate whether and how estrogen regulates gp130 in human umbilical vein endothelial cells (HUVECs). HUVECs were first treated with IL-6 and soluble IL-6 receptor (sIL-6R) to induce inflammation, then treated with estradiol. We then measured the expression of gp130, a disintegrin and metalloproteinase 10 (ADAM10) and 17 (ADAM17) by RT-PCR and western blot. Levels of soluble gp130 (sgp130) in the culture supernatant were measured by ELISA. We found that IL-6 and sIL-6R increased expression of gp130 protein and decreased levels of sgp130 protein, without affecting gp130 mRNA levels. Estradiol treatment reversed these effects in a concentration- and time-dependent manner. These effects were regulated by ADAM10 and ADAM17 via an estrogen receptor α/β-dependent mechanism. These results shed further light on the mechanism underlying the clinical effects of estrogen therapy in atherosclerosis and coronary artery disease.

Uncontrollable vascular smooth cell proliferation is responsible for vascular remodeling during hypertension development. Glyoxalase 1 (GLO1), the major enzyme detoxifying methylglyoxal, has a critical role in regulating proliferation of several cell types. However, little is known whether GLO1 is involved in cerebrovascular remodeling and basilar smooth muscle cell (BASMC) proliferation during hypertension. Here we explored the role of GLO1 in angiotensin II (Ang II)-induced cerebrovascular remodeling and proliferation of BASMCs and the underlying mechanisms. The protein expression of GLO1 in basilar arteries from hypertensive mice was decreased, and GLO1 expression was negatively correlated with medial cross-sectional area and blood pressure in basilar arteries during hypertension. Knockdown of GLO1 promoted while overexpression of GLO1 prevented Ang II-induced cell proliferation and cell cycle transition in BASMCs. These results were related to the inhibitory effects of GLO1 on PI3K/AKT/CDK2 cascade activation upon Ang II treatment. In addition, in vivo study, GLO1 overexpression with adeno-associated virus harboring GLO1 cDNA improved cerebrovascular remodeling in basilar artery tissue during Ang II-induced hypertension development. These data indicate that GLO1 reduction mediates cerebrovascular modeling via PI3K/AKT/CDK2 cascade-dependent BASMC proliferation. GLO1 acts as a negative regulator of hypertension-induced cerebrovascular remodeling and targeting GLO1 may be a novel therapeutic strategy to prevent hypertension-associated cardiovascular complications such as stroke.

Although major depressive disorder (MDD) has caused severe mental harm to overwhelming amounts of patients, the pathogenesis of MDD remains to be studied. Due to the in-depth discussion of the mechanism of new antidepressants like ketamine, the habenula (Hb) was reported to be significant in the onset of MDD and the antidepressant mechanism. In the Hb of depressive-like rodents, various molecular mechanisms and neuronal electrical activities have been reported, but neurotransmitters disorder in response to stress are still unclear. Thus, we divided stress-susceptible and stress-resilient rats after exposure to chronic unpredictable mild stress (CUMS). Multiple metabolites in the Hb were determined by liquid chromatography-tandem mass spectrometry. Based on this approach, we found that glutamate was significantly increased in susceptible group and resilient group, while dopamine was significant decreased in two groups. Gamma-aminobutyric acid was significantly upregulated in susceptible group but downregulated in resilient group. Our study firstly provides quantitative evidence regarding alterations of main neurotransmitters in the Hb of CUMS rats, showing the different role of neurotransmitters in stress susceptibility and stress resilience.