Pingzhang Wang

VSTM1-v2, a novel soluble glycoprotein, promotes the differentiation and activation of Th17 cells

Cytokines are soluble proteins that mediate immune reactions and are responsible for communication among immune cells. CD4(+) T cells are the principle sources of cytokines of adaptive immunity. Cytokines play critical roles in the differentiation and effector function of CD4(+) T cells. They also play key roles in diseases, and some of them have been developed into drugs in the forms of recombinant cytokines, soluble receptors and neutralizing antibodies. Therefore, identifying novel potential cytokines is necessary and beneficial for better understanding immunology and enhancing human health. To find novel potential cytokines, we carried out an integrated bioinformatics analysis on the whole human genome. Cytokine candidates were selected for cDNA cloning, sub-cloning, secretion verification, expression profile analysis and functional study. Here, we report a novel soluble protein, VSTM1-v2, which is a classical secretory glycoprotein mainly expressed in immune tissues, and can promote the differentiation and activation of Th17 cells.

CSBF/C10orf99, a novel potential cytokine, inhibits colon cancer cell growth through inducing G1 arrest

Cytokines are soluble proteins that exert their functions by binding specific receptors. Many cytokines play essential roles in carcinogenesis and have been developed for the treatment of cancer. In this study, we identified a novel potential cytokine using immunogenomics designated colon-derived SUSD2 binding factor (CSBF), also known as chromosome 10 open reading frame 99 (C10orf99). CSBF/C10orf99 is a classical secreted protein with predicted molecular mass of 6.5 kDa, and a functional ligand of Sushi Domain Containing 2 (SUSD2). CSBF/C10orf99 has the highest expression level in colon tissue. Both CSBF/C10orf99 and SUSD2 are down-regulated in colon cancer tissues and cell lines with different regulation mechanisms. CSBF/C10orf99 interacts with SUSD2 to inhibit colon cancer cell growth and induce G1 cell cycle arrest by down-regulating cyclin D and cyclin-dependent kinase 6 (CDK6). CSBF/C10orf99 displays a bell-shaped activity curve with the optimal effect at ~10 ng/ml. Its growth inhibitory effects can be blocked by sSUSD2-Fc soluble protein. Our results suggest that CSBF/C10orf99 is a novel potential cytokine with tumor suppressor functions.

ImmuCo: a database of gene co-expression in immune cells

Current gene co-expression databases and correlation networks do not support cell-specific analysis. Gene co-expression and expression correlation are subtly different phenomena, although both are likely to be functionally significant. Here, we report a new database, ImmuCo (http://immuco.bjmu.edu.cn), which is a cell-specific database that contains information about gene co-expression in immune cells, identifying co-expression and correlation between any two genes. The strength of co-expression of queried genes is indicated by signal values and detection calls, whereas expression correlation and strength are reflected by Pearson correlation coefficients. A scatter plot of the signal values is provided to directly illustrate the extent of co-expression and correlation. In addition, the database allows the analysis of cell-specific gene expression profile across multiple experimental conditions and can generate a list of genes that are highly correlated with the queried genes. Currently, the database covers 18 human cell groups and 10 mouse cell groups, including 20 283 human genes and 20 963 mouse genes. More than 8.6 × 108 and 7.4 × 108 probe set combinations are provided for querying each human and mouse cell group, respectively. Sample applications support the distinctive advantages of the database.

ImmuSort, a database on gene plasticity and electronic sorting for immune cells.

Gene expression is highly dynamic and plastic. We present a new immunological database, ImmuSort. Unlike other gene expression databases, ImmuSort provides a convenient way to view global differential gene expression data across thousands of experimental conditions in immune cells. It enables electronic sorting, which is a bioinformatics process to retrieve cell states associated with specific experimental conditions that are mainly based on gene expression intensity. A comparison of gene expression profiles reveals other applications, such as the evaluation of immune cell biomarkers and cell subsets, identification of cell specific and/or disease-associated genes or transcripts, comparison of gene expression in different transcript variants and probe set quality evaluation. A plasticity score is introduced to measure gene plasticity. Average rank and marker evaluation scores are used to evaluate biomarkers. The current version includes 31 human and 17 mouse immune cell groups, comprising 10,422 and 3,929 microarrays derived from public databases, respectively. A total of 20,283 human and 20,963 mouse genes are available to query in the database. Examples show the distinct advantages of the database. The database URL is http://202.85.212.211/Account/ImmuSort.html.

Novel transcript variants of TRAIL show different activities in activation of NF-κB and apoptosis.

AIMS Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has many transcript variants, but whether they possess distinct function is not completely known. In the present study, we compared the function of these TRAIL variants. MAIN METHODS A bioinformatics analysis was performed to examine potential TRAIL variants. For the functional study, over-expression of TRAIL isoforms was used to examine their NF-κB inducing and apoptotic activities in both cancer and normal cells. Moreover, soluble TRAIL E4 variant protein was expressed and purified in prokaryotic cells, and was used for apoptotic assay. KEY FINDINGS We cloned seven truncated TRAIL variants, designated as AK, E2, E3, E4, DA, BX424, and BX439. In comparison with the wild type TRAIL protein expressed from full-length RefSeq, over-expression of all these TRAIL variants activated NF-κB and its targeting genes in human cells at varying degrees. Some isoforms including BX424, DA and E4 even showed NF-κB, IL8, CCL4 and CCL20 promoter activating activity stronger than the wild type protein. All truncated variant proteins had no toxicity to normal human cells, similar to the wild type protein; however, they all failed to induce apoptosis in cancer cells that are sensitive to TRAIL. Recombinant soluble TRAIL E4 protein also failed to antagonize TRAIL-induced apoptosis in cancer cells. SIGNIFICANCE Truncated TRAIL variant proteins lost apoptotic activity but retained or even enhanced the NF-κB activating potentials, these results suggest that TRAIL variants may play roles in non-apoptotic cellular processes that are more important than we previously thought.

PC3-Secreted Microprotein Is a Novel Chemoattractant Protein and Functions as a High-Affinity Ligand for CC Chemokine Receptor 2

PC3-secreted microprotein (PSMP) or microseminoprotein is a newly discovered secreted protein whose function is currently unknown. In this study, PSMP was found to possess chemotactic ability toward monocytes and lymphocytes, and its functional receptor was identified as CCR2B. PSMP was identified as a chemoattractant protein from a PBMC chemoattractant platform screen that we established. The mature secreted PSMP was able to chemoattract human peripheral blood monocytes, PBLs, and CCR2B-expressing THP-1 cells, but not peripheral blood neutrophils, even though it does not contain the classical structure of chemokines. CCR2B was identified as one receptor for PSMP-mediated chemotaxis by screening HEK293 cells that transiently expressed classical chemokine receptors; results obtained from the chemotaxis, calcium flux, receptor internalization, and radioligand-binding assays all confirmed this finding. To further identify the major function of PSMP, we analyzed its expression profile in tissues. PSMP is highly expressed in benign prostatic hyperplasia and in some prostate cancers, and can also be detected in breast tumor tissue. In response to PSMP stimulation, phosphorylated ERK levels downstream of CCR2B signaling were upregulated in the PC3 cell line. Taken together, our data collectively suggest that PSMP is a chemoattractant protein acting as a novel CCR2 ligand that may influence inflammation and cancer development.

Discovery of novel human transcript variants by analysis of intronic single-block EST with polyadenylation site.

BackgroundAlternative polyadenylation sites within a gene can lead to alternative transcript variants. Although bioinformatic analysis has been conducted to detect polyadenylation sites using nucleic acid sequences (EST/mRNA) in the public databases, one special type, single-block EST is much less emphasized. This bias leaves a large space to discover novel transcript variants.ResultsIn the present study, we identified novel transcript variants in the human genome by detecting intronic polyadenylation sites. Poly(A/T)-tailed ESTs were obtained from single-block ESTs and clustered into 10,844 groups standing for 5,670 genes. Most sites were not found in other alternative splicing databases. To verify that these sites are from expressed transcripts, we analyzed the supporting EST number of each site, blasted representative ESTs against known mRNA sequences, traced terminal sequences from cDNA clones, and compared with the data of Affymetrix tiling array. These analyses confirmed about 84% (9,118/10,844) of the novel alternative transcripts, especially, 33% (3,575/10,844) of the transcripts from 2,704 genes were taken as high-reliability. Additionally, RT-PCR confirmed 38% (10/26) of predicted novel transcript variants.ConclusionOur results provide evidence for novel transcript variants with intronic poly(A) sites. The expression of these novel variants was confirmed with computational and experimental tools. Our data provide a genome-wide resource for identification of novel human transcript variants with intronic polyadenylation sites, and offer a new view into the mystery of the human transcriptome.

SUSD2 is frequently downregulated and functions as a tumor suppressor in RCC and lung cancer.

Sushi domain containing 2 (SUSD2) is type I membrane protein containing domains inherent to adhesion molecules. There have been few reported studies on SUSD2, and they have mainly focused on breast cancer, colon cancer, and HeLa cells. However, the expression and function of SUSD2 in other cancers remain unclear. In the present study, we conducted an integrated bioinformatics analysis based on the array data from the GEO database and found a significant downregulation of SUSD2 in renal cell carcinoma (RCC) and lung cancer. Western blotting and quantitative RT-PCR (qRT-PCR) confirmed that SUSD2 was frequently decreased in RCC and lung cancer tissues compared with the corresponding levels in normal adjacent tissues. The restoration of SUSD2 expression inhibited the proliferation and clonogenicity of RCC and lung cancer cells, whereas the knockdown of SUSD2 promoted A549 cell growth. Our findings suggested that SUSD2 functions as a tumor suppressor gene (TSG) in RCC and lung cancer.

Cytokine-like 1 Chemoattracts Monocytes/Macrophages via CCR2.

Cytokine-like 1 (CYTL1) is a novel potential cytokine that was first identified in CD34+ cells derived from bone marrow and cord blood, and it was also found using our immunogenomics strategy. The immunobiological functions of CYTL1 remain largely unknown, and its potential receptor(s) has not been identified. A previous proposed hypothesis suggested that CYTL1 had structural similarities with CCL2 and that CCR2 was a potential receptor of CYTL1. In this study, we verify that CYTL1 possesses chemotactic activity and demonstrate that its functional receptor is CCR2B using a series of experiments performed in HEK293 cells expressing CCR2B or CCR2B-EGFP, including chemotaxis, receptor internalization, and radioactive binding assays. CYTL1 chemoattracts human monocytes but not PBLs, and its chemotactic activity toward monocytes is dependent on the CCR2B-ERK pathway. Furthermore, both human and mouse recombinant CYTL1 protein have chemotactic effects on macrophages from wild-type mice but not from Ccr2−/− mice. Furthermore, the chemotactic activity of CYTL1 is sensitive to pertussis toxin. All of the above data confirm that CCR2B is a functional receptor of CYTL1.

CMTM4 is frequently downregulated and functions as a tumour suppressor in clear cell renal cell carcinoma

BackgroundChemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing family (CMTM) is a gene family involved in multiple malignancies. CMTM4 is a member of this family and is located at chromosome 16q22.1, a locus that harbours a number of tumour suppressor genes. It has been defined as a regulator of cell cycle and division in HeLa cells; however, its roles in tumourigenesis remain poorly studied.MethodsAn integrated bioinformatics analysis based on the array data from the GEO database was conducted to view the differential expression of CMTM4 across multiple cancers and their corresponding control tissues. Primary clear cell renal cell carcinoma (ccRCC) and the paired adjacent non-tumour tissues were then collected to examine the expression of CMTM4 by western blotting, immunohistochemistry, and quantitative RT-PCR. The ccRCC cell lines A498 and 786-O and the normal renal tubular epithelial cell line HK-2 were also tested for CMTM4 expression by western blotting. Cell Counting Kit-8 (CCK-8) and viable cell counting assays were used to delineate the growth curves of 786-O cells after CMTM4 overexpression or knockdown. Wound healing and transwell assays were performed to assess the cells’ ability to migrate. The effects of CMTM4 on cellular apoptosis and cell cycle progression were analysed by flow cytometry, and cell cycle hallmarks were detected by western blotting and RT-PCR. The xenograft model in nude mice was used to elucidate the function of CMTM4 in tumourigenesis ex vivo.ResultsBy omic data analysis, we found a substantial downregulation of CMTM4 in ccRCC. Western blotting then confirmed that CMTM4 was dramatically reduced in 86.9 % (53/61) of ccRCC tissues compared with the paired adjacent non-tumour tissues, as well as in the 786-O and A498 ccRCC cell lines. Restoration of CMTM4 significantly suppressed 786-O cell growth by inducing G2/M cell cycle arrest and p21 upregulation, and cell migration was also inhibited. However, knockdown of CMTM4 led to a completely opposite effect on these cell behaviours. Overexpression of CMTM4 also markedly inhibited the tumour xenograft growth in nude mice.ConclusionsCMTM4 is downregulated and exhibits tumour-suppressor activities in ccRCC, and could be exploited as a target for ccRCC treatment.