The Identification of Potential Anti-Depression/Anxiety Drug Targets by Stress-Induced Rat Brain Regional Proteome and Network Analyses.

Depression and anxiety disorders are prevalent stress-related neuropsychiatric disorders and involve multiple molecular changes and dysfunctions across various brain regions. However, the specific and shared pathophysiological mechanisms occurring in these regions remain unclear. Previous research used a rat model of chronic mild stress (CMS) to segregate and identify depression-susceptible, anxiety-susceptible, and insusceptible groups; then the proteomes of six distinct brain regions (the hippocampus, prefrontal cortex, hypothalamus, pituitary, olfactory bulb, and striatum) were separately and quantitatively analyzed. To gain a comprehensive and systematic understanding of the molecular abnormalities, this study aimed to investigate and compare differential proteomics data from the six regions. Differentially expressed proteins (DEPs) were identified in between specific regions and across all regions and subjected to a series of bioinformatics analyses. Regional comparisons showed that stress-induced proteomic changes and corresponding gene ontology and pathway enrichments were largely distinct, attributable to differences in cell populations, protein compositions, and brain functions of these areas. Additionally, a notable degree of overlap in the significantly enriched terms was identified, potentially suggesting strong connections in the enrichment across different regions. Furthermore, intra-regional and inter-regional protein-protein interaction networks and drug-target-DEP networks were constructed. Integrated analysis of the three association networks in the six regions, along with the DisGeNET database, identified ten DEPs as potential targets for anti-depression/anxiety drugs. Collectively, these findings revealed commonalities and differences across different brain regions at the protein level induced by CMS, and identified several novel protein targets for the development of new therapeutics for depression and anxiety.

A strategy can be used to analyze intracellular interaction proteomics of cell-surface receptors.

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.

PDGF-BB/PDGFRß promotes epithelial-mesenchymal transition by affecting PI3K/AKT/mTOR-driven aerobic glycolysis in Wilms' tumor G401 cells.

Wilms' tumor (WT) is the most common pediatric renal malignancy. PDGFRß belongs to the type III receptor tyrosine kinase family and is known to be involved in tumor metastasis and angiogenesis. Here, we studied the effect and underlying mechanism of PDGFRß on WT G401 cells. Transwell assay and wound-healing assay were used to detect the effect of PDGFRß on G401 cells invasion and migration. Western blot and immunofluorescence were used to detect the expression of EMT-related genes. The expression of PI3K/AKT/mTOR pathway proteins was detected by Western blot. The relationship between PDGFRß and aerobic glycolysis was studied by assessing the expression of glycolysis-related enzymes detected by qRT-PCR and Western blot. The activity of HK, PK, and LDH was detected by corresponding enzyme activity kits. The concentration of lactic acid and glucose was detected by Lactic Acid Assay Kit and Glucose Assay Kit-glucose oxidase method separately. To investigate the mechanism of PDGFRß in the development of WT, the changes of glucose and lactic acid were analyzed after blocking PI3K pathway, aerobic glycolysis, or PDGFRß. The key enzyme was screened by Western blot and glucose metabolism experiment after HK2, PKM2, and PDK1 were inhibited. The results showed that PDGFRß promoted the EMT process by modulating aerobic glycolysis through PI3K/AKT/mTOR pathway in which PKM2 plays a key role. Therefore, our study of the mechanism of PDGFRß in G401 cells provides a new target for the treatment of WT.

Global effects of RAB3GAP1 dysexpression on the proteome of mouse cortical neurons.

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.

The overexpression of MCPH1 inhibits cell growth through regulating cell cycle-related proteins and activating cytochrome c-caspase 3 signaling in cervical cancer.

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.

IRTKS contributes to the malignant progression of cervical cancer cells.

Cervical cancer (CC), one of the most aggressive tumors in women, has high risk rates of recurrence and metastasis. It is essential to study the key genes and proteins involved in CC development. IRTKS, a member of the IRSp53 family, has been reported as a tumor promoter in gastric and breast cancers. However, the biological role of IRTKS in CC is still unclear. The purpose of this study was to explore the biological function of IRTKS in CC cells in vitro and the effect of IRTKS on tumorigenesis in vivo. Siha and Hela cells were treated with si-RNA and plasmids. Cell proliferation and growth were detected by CCK8, colony formation assay and nude mouse tumorigenicity assay, respectively. Transwell assay was used to analyze cell migration and invasion. The expression of epithelial-mesenchymal transition (EMT)-related proteins was determined by western blot. IRTKS was highly expressed in CC. IRTKS contributed to the proliferation of CC cells in vitro and in vivo. Furthermore, IRTKS facilitated the migration and invasion of CC cells and modulated EMT. IRTKS plays a crucial role in CC tumorigenesis, suggesting it may be a potential key gene for new therapeutic strategies in CC.

NFBD1/MDC1 is a protein of oncogenic potential in human cervical cancer.

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.

Influence of Dichloroacetate on Wilms'Tumor in vitro.

OBJECTIVE: To investigate the effect of dichloroacetate (DCA) on Wilms' tumor (WT) G401 cells. METHODS: CCK-8 assay was used to detect the influence of DCA on G401 cells viability and 10 mmol/L DCA was selected for subsequent experiments. The expression of glycolysis-related enzymes, such as hexokinase 2 (HK2), pyruvate kinase M2 (PKM2), lactic acid dehydrogenase A (LDHA), pyruvate dehydrogenase kinase 1 (PDK1), and pyruvate dehydrogenase (PDH), were detected by qRT-PCR and western blot. The extracellular lactic acid and glucose concentrations were measured by the lactic acid assay kit and glucose oxidase method kit respectively. Flow cytometry was used to detect the effect of DCA on G401 cells apoptosis. The invasion and migration ability of G401 cells were detected by Transwell assay and wound-healing assay. RESULTS: The results showed that DCA reduced glycolysis-related enzymes expression, inhibited lactic acid production, and glucose consumption. DCA also suppressed cells growth, induced cells apoptosis and inhibited cells invasion and migration. CONCLUSION: Inhibition of aerobic glycolysis by DCA can reduce the viability of G401 cells, promote cells apoptosis and inhibit cells invasion and migration. Therefore, aerobic glycolysis may be a potential therapeutic target for Wilms' tumor.

Effects of Camk2b Overexpression and Underexpression on the Proteome of Rat Hippocampal Neurons.

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.

Silencing of Bmi-1 gene by RNA interference enhances sensitivity to doxorubicin in breast cancer cells.

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.

Proteomic Insights Into Susceptibility and Resistance to Chronic-Stress-Induced Depression or Anxiety in the Rat Striatum.

Chronic stress is a key factor for the onset of anxiety and depression disorders. However, the stress-induced common and unique molecular basis of the two psychiatric disorders is not fully known and still needs to be explored. Previously, we employed a chronic mild stress (CMS) procedure to induce a rat model including depression-susceptible (Dep-Sus), anxiety-susceptible (Anx-Sus), and insusceptible (Insus) cohorts. In this work, we continuously analyze the striatal proteomes of the three stressed cohorts by the use of comparative proteomics and bioinformatics approaches. Through isobaric tags for relative and absolute quantitation (iTRAQ)-based analysis, 386 abnormally expressed proteins in total were identified. These deregulated proteins are involved in various biological functions and significant pathways that are potentially connected with resistance and susceptibility to CMS-caused anxious- or depressive-like behaviors and, hence, could act as suggestive protein targets. A further parallel reaction monitoring-based independent investigation shows that alterations in Pak5, Dgkg, Scn4b, Rb1cc1, and Acin1; Ggps1, Fntb, Nudt19, Ufd1, and Ndufab1; and Dnajb12, Hbb2, Ap2s1, Ip6k1, and Stk4 were specifically connected with Dep-Sus, Anx-Sus, or Insus groups, respectively, potentially indicating that identical CMS treatment results in the different changes in the striatal protein regulations. Overall, our current proteomics study of the striatum provides an important molecular foundation and comprehensive insights into common and specific deregulations correlated with pathophysiological mechanisms that underlie resistance and susceptibility to chronic stress-induced anxiety or depression.

Intersectional analysis of chronic mild stress-induced lncRNA-mRNA interaction networks in rat hippocampus reveals potential anti-depression/anxiety drug targets.

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.

Proteomic Response of Rat Pituitary Under Chronic Mild Stress Reveals Insights Into Vulnerability and Resistance to Anxiety or Depression.

Chronic stress as one of the most significant risk factor can trigger overactivity of hypothalamic-pituitary-adrenal (HPA) axis in depression as well as anxiety. Yet, the shared and unique neurobiological underpinnings underlying the pituitary abnormality in these two disorders have not been made clear. We previously have established depression-susceptible, anxiety-susceptible and insusceptible groups using a valid chronic mild stress (CMS) model. In this work, the possible protein expression changes in the rat pituitary of these three groups were continuously investigated through the use of the comparative quantitative proteomics and bioinformatics approaches. The pituitary-proteome analysis identified totally 197 differential proteins as a CMS response. These deregulated proteins were involved in diverse biological functions and significant pathways potentially connected with the three different behavioral phenotypes, likely serving as new investigative protein targets. Afterwards, parallel reaction monitoring-based independent analysis found out that expression alterations in Oxct1, Sec24c, Ppp1cb, Dock1, and Coq3; Lama1, Glb1, Gapdh, Sccpdh, and Renbp; Sephs1, Nup188, Spp1, Prodh1, and Srm were specifically linked to depression-susceptible, anxiety-susceptible and insusceptible groups, respectively, suggesting that the same CMS had different impacts on the pituitary protein regulatory system. Collectively, the current proteomics research elucidated an important molecular basis and furnished new valuable insights into neurochemical commonalities and specificities of the pituitary dysfunctional mechanisms in HPA axis underlying vulnerability and resistance to stress-induced anxiety or depression.

Comparative Proteomics of Rat Olfactory Bulb Reveal Insights into Susceptibility and Resiliency to Chronic-stress-induced Depression or Anxiety.

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 mild stress-induced protein dysregulations correlated with susceptibility and resiliency to depression or anxiety revealed by quantitative proteomics of the rat prefrontal cortex.

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.

Analysis of Chronic Mild Stress-Induced Hypothalamic Proteome: Identification of Protein Dysregulations Associated With Vulnerability and Resiliency to Depression or Anxiety.

Chronic stress as a known risk factor leads to hyperactivity of the hypothalamus-pituitary-adrenal (HPA) axis in both depression and anxiety. However, the stress-induced dysfunction of the HPA axis in these disorders especially the common and unique molecular dysregulations have not been well-explored. Previously, we utilized a chronic mild stress (CMS) paradigm to segregate and gain depression-susceptible, anxiety-susceptible, and insusceptible groups. In this study, we continue to examine the possible protein expression alterations of the hypothalamus as the center of the HPA axis in these three groups by using a proteomic approach. Though isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative analysis, a total of 593 dysregulated proteins were identified. These were potentially associated with vulnerability and adaptability of CMS-caused depression or anxiety and therefore might become novel investigative protein targets. Further independent analysis using parallel reaction monitoring (PRM) indicated that 5, 7, and 21 dysregulated proteins were specifically associated with depression-susceptible, anxiety-susceptible, and insusceptible groups, respectively, suggesting that the same CMS differently affected the regulation system of the rat hypothalamic proteome. In summary, the current proteomic research on the hypothalamus provided insights into the specific and common molecular basis for the HPA dysfunction mechanisms that underlie resiliency and vulnerability to stress-induced depression or anxiety.

Down-regulation of PDGFRß suppresses invasion and migration in osteosarcoma cells by influencing epithelial-mesenchymal transition.

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 (PDGFRß) is a subtype of PDGFR. PDGFRß 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 PDGFRß in OS pathogenesis and development are still lacking. Here we assessed the effects of PDGFRß 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 PDGFRß resulted in reduced migration of HOS cells in the small interfering RNA duplexes specific for the PDGFRß 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 PDGFRß 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 PDGFRß plays an important role in OS invasion, migration and epithelial-mesenchymal transition by influencing the PI3K, Akt and mTOR pathways, hence highlighting PDGFRß as a potential therapeutic target for OS.

Establishment of CHO cell line expressing human MCHR2 gene and research of its molecular characteristics.

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.

Influence of glucose transporter 1 activity inhibition on neuroblastoma in vitro.

Most cancer cells predominantly produce their energy through a high rate of glycolysis in the presence of abundant oxygen. Glycolysis has become a target of anticancer strategies. Previous researches showed that glucose transporter 1 (GLUT1) inhibitor is effective as anticancer agents. This study assessed the effects of the selective GLUT1 inhibitor WZB117 on regulation of neuroblastoma (NB) cell line SH-SY5Y viability, cell cycle and glycolysis in vitro. SH-SY5Y cells were grown and treated with WZB117 for up to 72 h and then subjected to cell viability, qRT-PCR, Western blot and flow cytometry analysis. Level of ATP and LDH was also analyzed. The result showed that WZB117 treatment reduced tumor cells viability, downregulated level of GLUT1 protein. Moreover, WZB117 treatment arrested tumor cells at the G0-G1 phase of the cell cycle, induced tumor cells to undergo necrosis instead of apoptosis. In addition, WZB117 treatment downregulated the levels of intracellular ATP, LDH and glycolytic enzymes. Thus, WZB117-induced GLUT1 inhibition suppressed tumor cell growth, induced cell cycle arrest and reduced glycolysis metabolites in NB cells in vitro. This study suggested that GLUT1 can be used as a potential therapeutic target for NB.

Chromosomal localization of silkworm (Bombyx mori) sericin gene 1 and chymotrypsin inhibitor 13 using fluorescence in situ hybridization.

The chromosomal locations of two single-copy genes, Ser-1 and CI-13, in silkworm (Bombyx mori) were detected at the molecular cytogenetics level by fluorescence in situ hybridization in the study. The results showed that Ser-1 is located near the distal end of the 11th linkage group, relatively at the 12.5+/-1.4 position in pachytene; and that CI-13 has been mapped near the distal end of the 2nd linkage group, relatively at the 8.2+/-1.2 position in pachytene. Furthermore, their location model map-FISH map on silkworm chromosome was drawn. The FISH technique and its application to silkworm are also discussed in this paper.