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Mounting studies have demonstrated that RAB3GAP1 expression is modified in brain diseases with multiple neurobiological functions and processes and acts as a potentially significant target. However, the cellular and molecular events arising from RAB3GAP1 dysexpression are still incompletely understood. In this work, underexpression and overexpression of RAB3GAP1 were first induced into cultured mouse cortical neurons by transfection with lentivirus plasmids. Then we globally explored the effects of RAB3GAP1 dysexpression on the proteome of the neurons through the use of isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics with bioinformatics. A total of 364 proteins in the RAB3GAP1-underexpression group and 314 proteins in the RAB3GAP1-overexpression group were identified to be differentially expressed. Subsequent bioinformatics analysis indicated that the proteome functional expression profiles induced by RAB3GAP1 underexpression and overexpression were different, suggesting the potential differences in biological processes and cellular effects. Subsequent intergroup cross-comparison revealed some candidate target proteins regulated directly by RAB3GAP1. Further parallel reaction monitoring (PRM) analysis illustrated that Sub1, Ssrp1, and Top1 proteins might serve as new potentially important linkers in the RAB3GAP1-mediated autophagy pathway in the cortical neurons. Collectively, the current proteomics data furnished new valuable insights to better understand the regulatory molecular mechanism of neuronal RAB3GAP1.

Recent studies have demonstrated that Camk2b expression is modified in neuropsychiatric illnesses and potentially affects synaptic plasticity. However, the molecular events arising from Camk2b dysregulation are not fully elucidated and need to be comprehensively explored. In the present study, we first induced over-expression and under-expression of Camk2b in cultured rat hippocampal neurons through transfection with lentivirus plasmids. Then isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics followed by bioinformatics analyses were carried out to explore the impacts of Camk2b dysexpression on the proteome of the neurons. Compared with the respective controls, a total of 270 proteins in the Camk2b-overexpression group and 209 proteins in the Camk2b-underexpression group were experienced a divergence in expression. Gene ontology and pathway analyses indicated that Camk2b overexpression and under-expression respectively induced two different change profiles of protein expressions and functions, reflecting the potential differences in cellular processes and biological events. Through cross comparison, several candidate target proteins regulated directly by Camk2b were revealed. Further network and immunoblot analyses demonstrated that Mapk3 could be an important linker and Camk2b-Mapk3 might serve as a new potential pathway affecting the expression of synaptic proteins in hippocampal neurons. Collectively, the present results offer a new comprehension of the regulatory molecular mechanism of Camk2b and thereby increase our understanding of Camk2b-mediated synaptogenesis in synaptic plasticity.

Comprehensive knowledge of the intracellular protein interactions of cell-surface receptors will greatly advance our comprehension of the underlying trafficking mechanisms. Hence, development of effective and high-throughput approaches is highly desired. In this work, we presented a strategy aiming to tailor toward the analysis of intracellular protein interactome of cell-surface receptors. We used α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors subunit GluA1 as an example to illustrate the methodological application. To capture intracellular proteins that interact with GluA1, after surface biotinylation of the prepared hippocampal neurons and slices, the non-biotinylated protein components as intracellular protein-enriched fraction were unconventionally applied for the following co-immunoprecipitation. The co-immuno-precipitated proteins were then analyzed through mass spectrometry-based proteomics and bioinformatics platforms. The detailed localizations indicated that intracellular proteins accounted for up to 93.7 and 90.3% of the analyzed proteins in the neurons and slices, respectively, suggesting that our protein preparation was highly effective to characterize intracellular interactome of GluA1. Further, we systematically revealed the protein functional profile of GluA1 intracellular interactome, thereby providing complete overview and better comprehension of diverse intracellular biological processes correlated with the complex GluA1 trafficking. All experimental results demonstrated that our methodology would be applicable and useful for intracellular interaction proteomics of general cell-surface receptors.

Chronic stress causes the abnormality of olfactory bulb (OB) in both anxiety and depression, however, the unique and common neurobiological underpinnings are still poorly understood. Previously, we built the three groups by chronic mild stress (CMS), depression-susceptible (Dep-Sus): with depression-like behavior, anxiety-susceptible (Anx-Sus): with anxiety-like behavior and insusceptible (Insus): without depression- and anxiety-like behaviors. To continuously explore the protein expression changes in these three groups, comparative quantitative proteomics analysis was conducted on the rat OB as crucial part of the olfactory system. Next, bioinformatics analyses were implemented whereas protein expressions were independently analyzed by parallel reaction monitoring (PRM) or Western blot (WB). The OB-proteome analysis identified totally 133 differentially expressed proteins as a CMS response. These deregulated proteins were involved in multiple functions and significant pathways potentially correlated with phenotypes of maladaptive behavior of depression or anxiety as well as adaptive behavior, and hence might act as potential candidate protein targets. The subsequent PRM-based or WB-based analyses showed that changes in Nefl, Mtmr7 and Tk2; Prkaca, Coa3, Cox6c2, Lamc1 and Tubal3; and Pabpn1, Nme3, Sos1 and Lum were uniquely associated with Dep-Sus, Anx-Sus, and Insus groups, respectively. These phenotype-specific deregulated proteins were primarily involved in multiple metabolic and signaling pathways, suggesting that the identical CMS differently impacted the olfactory protein regulation system and biological processes. To sum up, our present data as a useful proteomics underpinning provided the common and distinct molecular insights into the biochemical understanding of OB dysfunction underlying susceptibility and resiliency to chronic-stress-induced anxiety or depression.

Chronic stress is a significant risk factor for depression as well as anxiety disorders. Yet, the stress-induced specific and common molecular dysregulations of these disorders have not been fully understood. Previously, we constructed a chronic mild stress (CMS) rat model to separate and obtain depression-susceptible, anxiety-susceptible, and insusceptible groups. In this study, the prefrontal cortical proteomes of the three stressed groups were comparatively profiled utilizing isobaric tags for relative and absolute quantitation (iTRAQ)-coupled tandem mass spectrometry approach. A total of 212 protein dysregulations were identified, potentially correlating to susceptibility or resilience to CMS-induced depression or anxiety, and thus might serve as potential protein targets for further investigation. In addition, independent analysis by parallel reaction monitoring identified changes in Gfap, Rhog, Gnai2, Ppp1r1b, and Uqcrh; Tubb6, Urod, Cul1, Spred1, and Gpcpd1; Acadl, Ppp1r1a, Grm2, Mtor, Lsm8, Cplx2, and Tsta3 that were distinctly correlated to depression-susceptible, anxiety-susceptible, or insusceptible groups, respectively. This suggested that identical CMS had different effects on the protein regulation system of the rat prefrontal cortex. Collectively, the present proteomics study of the prefrontal cortex established a significant molecular basis and offered new insights into the specificity and commonality of pathophysiologic mechanisms underlying susceptibility and resiliency to stress-induced depression or anxiety.

Despite studies providing insight into the neurobiology of chronic stress, depression and anxiety, long noncoding RNA (lncRNA)-mediated mechanisms underlying the common and distinct pathophysiology of these stress-induced disorders remain nonconclusive. In a previous study, we used the chronic mild stress paradigm to separate depression-susceptible, anxiety-susceptible and insusceptible rat subpopulations. In the current study, lncRNA and messenger RNA (mRNA) expression was comparatively profiled in the hippocampus of the three stress groups using microarray technology. Groupwise comparisons identified distinct sets of lncRNAs and mRNAs associated with the three different behavioral phenotypes of the stressed rats. To investigate the regulatory roles of the dysregulated lncRNAs upon mRNA expression, correlations between the differential lncRNAs and mRNAs were first analyzed by combined use of weighted gene coexpression network analysis and ceRNA theory-based methods. Subsequent functional analysis of strongly correlated mRNAs indicated that the dysregulated lncRNAs were involved in various biological pathways and processes to specifically induce rat susceptibility or resiliency to depression or anxiety. Further intersectional analysis of phenotype-associated and drug-associated lncRNA-mRNA networks and subnetworks assisted in identifying 16 hub lncRNAs as potential targets of anti-depression/anxiety drugs. Collectively, our study established the molecular basis for understanding the similarities and differences in pathophysiological mechanisms underlying stress-induced depression or anxiety and stress resiliency, revealing several important lncRNAs that represent potentially new therapeutic drug targets for depression and anxiety disorders., Highlights • LncRNAs/mRNAs and pathways related to three CMS-induced phenotypes were identified. • Specific and common molecular basis of three CMS-induced phenotypes was provided. • Phenotype- and drug-associated lncRNA-mRNA networks were intersectionally applied. • 16 hub lncRNAs as potential targets of anti-depression/anxiety drugs were revealed.

Platelet‐derived growth factor receptor beta belongs to the tyrosine kinase family. Platelet‐derived growth factor receptor beta binds to its specific ligand and induces autophosphorylation to activate the downstream phosphatidylinositol 3‐kinase/AKT/mammalian target of rapamycin pathway. The activated pathway molecules then influence the epithelial–mesenchymal transition process by regulating the expression of epithelial–mesenchymal transition‐related proteins, and ultimately regulating tumor metastasis., Osteosarcoma (OS) is the most common malignant bone tumor primarily influencing children and adults. Approximately one‐fifth of patients have micrometastasis in the lungs when OS is diagnosed. Platelet‐derived growth factor receptor (PDGFR) beta (PDGFRβ) is a subtype of PDGFR. PDGFRβ has been noted to be highly expressed in OS cell lines and patient specimens, and is associated with metastasis and poor prognosis of OS. However, mechanistic insights into the exact role of PDGFRβ in OS pathogenesis and development are still lacking. Here we assessed the effects of PDGFRβ on invasive and migratory abilities, such as the epithelial–mesenchymal transition and phosphatidylinositol 3‐kinase (PI3K), Akt and mammalian target of rapamycin (mTOR) pathways in HOS cells. Depleting PDGFRβ resulted in reduced migration of HOS cells in the small interfering RNA duplexes specific for the PDGFRβ group compared with the mock and scramble‐treated groups in Transwell invasion assays. Using wound‐healing assays, we demonstrate the rate of wound healing in the PDGF‐BB‐stimulated group was higher compared with the mock‐treated group. Western blot showed that down‐regulation of PDGFRβ decreased the expression of stromal phenotype markers and phosphorylation pathway proteins (PI3K, AKT and mTOR), but the epithelial phenotype marker was increased in HOS cells. Treating HOS cells with PDGF‐BB revealed a treatment time‐dependent increase of phosphorylated, but not total, PI3K, AKT and mTOR. Taken together, we suggest that PDGFRβ plays an important role in OS invasion, migration and epithelial–mesenchymal transition by influencing the PI3K, Akt and mTOR pathways, hence highlighting PDGFRβ as a potential therapeutic target for OS.

Objective To study the effect of inhibiting the expression of glucose transporter 1 (GLUT1) in neuroblastoma cells on the proliferation, invasion and migration of tumor cells. Methods The specific small molecule inhibitor WZB117 was used to inhibit the expression of GLUT1 in neuroblastoma cells. The expression of GLUT1 mRNA was detected by real-time quantitative PCR; the expression of GLUT1 protein was detected by Western blotting; the ability of cell proliferation was detected by CCK-8 assay; the ability of cell invasion and migration were detected by Transwell

MCPH1, initially identified as an hTERT repressor, has recently been implicated in mediating DNA damage response and maintaining chromosome integrity. This study is to investigate its potential role in the onset of cervical cancer. In the study, decreased expression of MCPH1 was observed in 19 of 31 cases (61.3 %) at mRNA level and 44 of 63 cases (69.8 %) at protein level of cervical tumor tissues compared with the paired nontumor tissues. Reduced MCPH1 protein expression was significantly associated with high-tumor grade (1 vs. 3 P = 0.013; 2 vs. 3 P = 0.047). In addition to inhibit SiHa cell migration and invasion, the overexpression of MCPH1 inhibited cervical cancer cells growth through inducing S phase arrest and mitochondrial apoptosis. Further analysis demonstrated cyclinA2/CDK2, CDC25C-cyclinB/CDC2, and p53/p21 pathways were involved in the MCPH1 overexpression-induced S phase arrest. Moreover, the overexpression of MCPH1 activated mitochondrial apoptosis through regulating several apoptosis-related proteins such as p53, Bcl-2, Bax, cytochrome c, caspase-3, and PARP-1. Our findings indicate that downregulated MCPH1 correlates with tumor progression in cervical cancer, and MCPH1 has an important role in regulating cell growth through regulating the cell cycle and apoptosis. Thus, it may be a crucial tumor suppressor gene and a novel candidate therapeutic target for cervical cancer.

Mouse ovarian surface epithelium (OSE) is a single layer of cubodial epithelial cells that covers the ovary surface and is involved in regulating the secretion and transport of 17β-hydroxysteroid dehydrogenase. Recently, OSE cells have attracted particular interest as a major source of ovarian cancer. Death-associated protein DAXX along with PML (promyelocytic leukemia protein) nuclear bodies (PML-NBs) reportedly play roles in transcriptional regulation and apoptosis. However, little is known regarding a role for DAXX in mOSE cells. In this study, we both over-expressed DAXX and depleted DAXX in primary mOSE cells. We found that Daxx deletion accelerated senescence in a p53/p21-dependent manner and promoted DNA damage by interacting with PML bodies without affecting cell cycle progression. These results suggest that DAXX may transform mOSE cells to an ovarian oncogenic phenotype and may be an anti-cancer target.

This study was undertaken to determine the effects of Pyracantha fortuneana (Maxim.) Li polysaccharides (PFP) on antioxidant and immune functions in mice. Results from this study showed that PFP administration significantly increased thymus and spleen indices, promoted splenocyte proliferation and natural killer (NK) cell activity, and elevated CD4 T cell numbers as well as CD4(+)/CD8(+) ratios. PFP also increased interleukin-2 (IL-2) levels, and decreased the levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) in splenocytes. In addition, PFP treatment led to remarkable increases in glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) activities, and dramatic decreases in malondialdehyde (MDA) levels in splenocytes. Moreover, PFP increased mRNA and protein expression of nuclear factor erythroid 2-related factor (Nrf2) in splenocytes. Taken together, these results suggest that PFP treatment enhances the immune function and decreases the oxidative stress in mice.

The oncogene Bmi-1 is a member of the Polycomb group gene family. Its expression is found to be greatly increased in a number of malignant tumors including breast cancer. This could suggest Bmi-1 as a potent therapeutic target. In this study, RNAi was introduced to down-regulate the expression of Bmi-1 in a highly malignant breast adenocarcinoma cell line, MCF-7. A thorough study of the biological behavior and chemosensitivity changes of the MCF-7 cells was carried out in context to the therapeutic potential of Bmi-1. The results obtained indicated that siRNA targeting of Bmi-1 could lead to an efficient and specific inhibition of endogenous Bmi-1 activity. The mRNA and protein expression of Bmi-1 were determined by RT-PCR and Western blot, respectively. Furthermore, silencing of Bmi-1 resulted in a drastic inhibition of the growth of MCF-7 cells as well as G(1) /S phase transition. The number of target cells was found to increase in phase G (0) /G (1) and decrease in the S phase, but no increase in the basal level of apoptosis was noticed. On the other hand, a reduction in the expression of cyclin D1 and an increase in the expression of p21 were also noticed. Silencing of Bmi-1 made the MCF-7 cells more sensitive to the chemotherapeutic agent doxorubicin and induced a significantly higher percentage of apoptotic cells. Here, we report on a study regarding the RNAi-mediated silencing of the Bmi-1 gene in breast cancer.

To investigate the influence of melanin-concentrating hormone receptor 2 (MCHR2) on preadipocyte proliferation and differentiation, a 3T3-L1-MCHR2 cell line stably expressing human MCHR2 was established. The expression level and biological activity of MCHR2 were evaluated by immunofluorescence and radioligand binding assays. A 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell cycle analysis showed that MCH did not affect 3T3-L1-MCHR2 and 3T3-L1-mock cell proliferation. MCH accelerated preadipocyte differentiation of the two cell lines when they were administered various doses of standard adipogenic inducers. The rate and degree of differentiation of the 3T3-L1-MCHR2 cells were higher than those of the 3T3-L1-mock cells. The expression levels of adipocyte-specific marker genes, including peroxisome proliferator-activated receptorgamma2 (PPARgamma2), CCAAT enhancer-binding proteinalpha (C/EBPalpha), adipocyte-specific fatty acid-binding protein (aP(2)) and leptin were significantly up-regulated, especially in 3T3-L1-MCHR2 cells. These findings suggest that MCHR2 positively mediates the regulation of MCH during preadipocyte differentiation and is involved in energy balance regulation without affecting preadipocyte proliferation.

The potential protective effects of Phellinus ribis glucan (PRG) administration against immune injury due to free radical formation were evaluated in mice. The results showed that glucan administration significantly increased thymus and spleen indices, spleen lymphocyte proliferation and NK cells activity, as well as CD8 T cell numbers, and decreased CD4+/CD8+. In accordance with a previous study, glucan administration significantly enhanced plasma glutathione peroxidase, superoxide dismutases activity and reduced plasma thiobarbituric acid reactive species level. In conclusion, our results indicate that glucan administration improve immune function by its free radicals scavenging activity and reducing oxidative stress in mice.

Ad.mda-7 inhibited growth and decreased survival in a broad array of human tumor cells, without eliciting detrimental effects in normal cells. This study demonstrates that Ad.mda-7 can effectively impede the proliferation and induce apoptosis of human cervical carcinoma cells, but the underlying mechanisms inducing cell death at protein level are unknown. Using proteome analysis, an investigation aimed at a better understanding of the antiproliferative mechanisms by Ad.mda-7 was carried out in CaSki cervical cancer cells. A total of 43 differentially expressed proteins were visualized by 2-DE and silver stain., 29 proteins of which were identified via matrix-assisted laser desorption/ionization-time of flight mass spectrometry(MALDI-TOF-MS) analysis, 15 were upregulated (eg., Tumor suppressor p53, Apoptosis regulator BAX, Adenylate kinase isoenzyme 1(AK1), Growth arrest and DNA-damage-inducible protein GADD45 gamma (GADD45gamma)) and 14 were downregulated (e.g., Eukaryotic translation initiation factor 5A(eIF-5A), Protein DJ-1, Annexin V, Transcription elongation factor B polypeptide 2 (TCEB2), TRAF family member-associated NFkappaB activator (TRAF2),c-Myc-responsive protein Rcl (RCL)). Among the identified proteins, the protein and mRNA alterations of six proteins were further confirmed by Western blot and semi-quantitative RT-PCR. Together, at both the mRNA and protein levels, p53, BAX, AK1, GADD45gamma and BCCIP were upregulated, while eIF-5A was downregulated following Ad.mda-7 treatment. Our findings may offer new insights into the antiproliferative mechanisms by Ad.mda-7 and its mode of action in cervical carcinoma cells.

A large nuclear protein of 2089 amino acids, NFBD1/MDC1 has recently been implicated in tumorigenesis and tumor growth. In this study, we investigated its expression in cervical cancers and explored its function using gene knockdown approaches. We report here that NFBD1 expression is substantial increased in 24 of 39 cases (61.5%) of cervical cancer tissues at the mRNA level and in 35 of 60 cases (58.3%) at the protein level compared with the case matched normal tissues. Tumors with higher grade of malignancy tend to have higher levels of NFBD1 expression. By infecting cells with retroviruses expressing NFBD1 shRNA, we successfully knocked down NFBD1 expression in cervical cancer cell lines HeLa, SiHa, and CaSki. NFBD1 knockdown cells display significant growth inhibition, cell cycle arrest, higher apoptotic rate, and enhanced sensitivity to adriamycin. Furthermore, NFBD1 knockdown also inhibits the growth of HeLa cells in nude mice. Western blot analyses further revealed that NFBD1 knockdown induced Bax, Puma, and Noxa while down-regulating Bcl-2; it also up-regulated cytochrome C and activated caspases 3 and 9. Therefore, the function of NFBD1 may be involved in the CDC25C–CyclinB1/CDC2 pathway at the G2/M checkpoint, and the cytochrome C/caspase 3 apoptotic pathway. Since expression of NFBD1 seems to be related to the oncogenic potential of cervical cancer, and suppression of its expression can inhibit cancer cell growth both in vitro and in vivo, NFBD1 may be a potential therapeutic target in human cervical cancer.

The oncogene Bmi-1 is highly up-regulated in breast carcinoma and is found to be efficient in preventing apoptosis of the cancer cells. Doxorubicin is an important chemotherapeutic agent against breast carcinoma. However, the effective therapeutic response to doxorubicin is often associated with severe toxicity. The present study is targetted at developing a strategy to increase doxorubicin sensitivity to lower doses without compromising its efficacy. A stable cell line with a persistent silencing of Bmi-1 was established. MTT assay was performed to evaluate 50% inhibitory concentration (IC50) values of doxorubicin. Apoptosis was detected by FCM and the expression of related genes [phosphor-Akt (pAkt), totle-Akt (tAkt), Bcl-2 and Bax] was studied by Western blot. In vivo, the sensitivity of the tumor tissues against doxorubicin was evaluated by transplanted MCF-7 nude mice model and the apoptosis of tissue cells was detected by TUNEL assay. The expression of pAkt and Bcl-2 was down-regulated, whereas Bax was up-regulated in Bmi-1 silencing cells. The results obtained indicated that silencing of Bmi-1 can render MCF-7 cells more sensitive to doxorubicin which induced a significantly higher percentage of apoptosis cells in vitro and in vivo. All together these results clearly demonstrate that Bmi-1 siliencing combined treatment of doxorubicin might be a new strategy for biological treatment on breast cancer.

The whole length of MCHR2 gene cDNA fragment was amplified by PCR using human fetal brain cDNA library as template. The pcDNA3.1 (+)/MCHR2 eukaryotic expression vector was constructed successfully. The recombinant pcDNA3.1 (+)/MCHR2 plasmid was transfected into Chinese hamster ovary (CHO) cell by lipofectamine 2000, after G418 selection and then the CHO cell line expressing MCHR2 gene was established. The MCHR2 gene expression was tested by RT-PCR, western blotting and immunofluorescence. The maximum binding (B(max)) of CHO cell line was 309.97 +/-1.14 fM x mg(-1) protein and the dissociation constant (K(d) value) was 0.170 +/- 0.0006 nM. MCH could stimulate Ca2+ release, its 50% effective concentration (EC50) was 2.32 +/- 0.01 nM. The construction of the CHO cell line and the research of MCHR2 molecular characteristics have established a good experimental basis for the further research about the function of MCHR2 gene.

The sericulture industry plays a very important role in our national economy. Silkworm (Bombyx mori) is always regarded as a model animal and biological reactor. There have been detailed studies on the structure, expression and control and molecular evolution of silk genes. However, few, if any, reports are available on the localization of structural genes in silkworm by molecular cytogenetics. The present experiment has tentatively localized the Fib-H gene at the distal end of the 25th linkage group, namely at the 25-0.0 position, and verified that Fib-H has only one locus, thus providing a temporary solution to the problem about its localization.