Adult ; Cell Adhesion Molecules ; genetics ; Genetic Predisposition to Disease ; Genotype ; HIV Infections ; etiology ; genetics ; Humans ; Lectins, C-Type ; genetics ; Middle Aged ; Receptors, Cell Surface ; genetics

OBJECTIVE: To evaluate the effects of CLEC5A expression level on cell proliferation, migration and invasion and epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) and explore the role of CLEC5A in the tumorigenesis and progression of HCC. METHODS: The expression level of CLEC5A was detected in 50 pairs of HCC and adjacent tissues using immunohistochemical staining, and its association with clinicopathological parameters of HCC patients was analyzed. Cultured HCC cell line SK-HEP-1 was transfected with a lentiviral vector overexpressing CLEC5A, and the transfection efficiency was verified using real-time fluorescence quantitative PCR and Western blotting. The changes in proliferation, migration and invasion abilities of the transfected cells were analyzed using CCK-8, 5-ethynyl-29-deoxyuridine (EdU) and Transwell assays, and EMT of the cells was determined using Western blotting. RESULTS: The protein expression level of CLEC5A was significantly lower in HCC tissues than in the adjacent tissues (P < 0.001). The expression level of CLEC5A was significantly correlated with tumor size (P=0.008), tumor number (P=0.010), histological differentiation (P=0.016), microvascular invasion (P=0.024) and BCLC stage (P=0.040). In SK-HEP-1 cells, overexpression of CLEC5A obviously inhibited the cell proliferation, migration and invasion and reversed EMT phenotype of the cells. CONCLUSION CLEC5A is a potential HCC suppressor gene and may serve as a promising therapeutic target for HCC.
Humans ; Carcinoma, Hepatocellular/genetics* ; Epithelial-Mesenchymal Transition ; Liver Neoplasms/genetics* ; Cell Proliferation ; Cell Differentiation ; Receptors, Cell Surface/genetics* ; Lectins, C-Type/genetics*

OBJECTIVETo discover and identify differentially expressed genes associated with colorectal adenoma formation and the role of RegIV in colorectal adenoma differentiation.

METHODSA subtracted cDNA library was constructed with cDNAs that were isolated from either the normal mucosa or adenoma tissue of a single patient. Suppressive subtractive hybridization (SSH) combined with virtual northern blotting was used to characterize differentially expressed genes and contigs were assembled by electronic cloning (in silico cloning) with the EST database. Semi-quantitative RT-PCR was performed in 9 colorectal adenomas.

RESULTSThe amino acid sequence was determined with open reading frame (ORF) prediction software and was found to be 100% homologous to the protein product of RegIV (a novel gene isolated from a large inflammatory bowel disease library). RegIV was found to be highly expressed in all of the adenoma samples (9/9) compared with the normal mucosa samples, while 5/6 cases showed RegIV to be more strongly expressed in adenocarcinoma.

CONCLUSIONRegIV may play an important role in the initiation of colorectal adenoma differentiation, and its detection may be useful in the early diagnosis of colorectal adenoma formation.

Adenoma ; genetics ; metabolism ; Blotting, Northern ; Colorectal Neoplasms ; genetics ; metabolism ; Humans ; Lectins, C-Type ; biosynthesis ; genetics ; Nucleic Acid Hybridization ; Pancreatitis-Associated Proteins ; Prognosis ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUNDTrichophyton rubrum (T. rubrum) represents the most important agent of dermatophytosis in humans. T. rubrum infection causes slight inflammation, and tends to be chronic and recurrent. It is suggested that it may result from the failure of epithelial cells to recognize T. rubrum effectively and initiate effective immune responses. The C-type lectin receptors (CLR) and toll-like receptors (TLR) are the two major pattern recognition receptors (PRRs) that recognize fungal components. Therefore, the purpose of the study was to analyze the expression of those PRRs and the cytokines in HaCaT cells stimulated with heat-inactivated T. rubrum conidia and hyphae, respectively.

METHODSHaCaT cells were unstimulated or stimulated with heat-inactivated T. rubrum conidia and hyphae (1×10(6) and 1.5×10(5) colony-forming unit (CFU) in 2 ml medium, respectively) for 6, 12 and 24 hours. The mRNA expression of PRRs involved in recognizing fungal pathogen-associated molecular patterns (PAMPs) and signaling molecules were measured by quantitative reverse transcription polymerase chain reaction (RT-PCR). Meanwhile, surface toll-like receptor (TLR) 2, TLR4 and Dectin-1 were analyzed by fluorescence-activated cell sorter (FACS) 24 hours after treatment. The cytokines were detected in cell culture supernatants of HaCaT cells in 12 and 24 hours after treatment.

RESULTSHaCaT cells constitutively expressed mRNA of membrane-bound TLR1, 2, 4 and 6, Dectin1 and DC-SIGN, but not Dectin-2 or Mincle. Heat-killed T. rubrum did not significantly upregulate gene transcriptions of the PRRs of HaCaT cells. Heat-inactivated T. rubrum conidia significantly reduced the surface expression of TLR2 and Dectin-1, and suppressed the secretions of interferon-inducible protein-10 (IP-10) and monocyte chemotactic protein-1 (MCP-1) of HaCaT cells, while heat-killed T. rubrum hyphae significantly induced the secretions of IP-10 and MCP-1.

CONCLUSIONThe cell-wall antigens of T. rubrum fail to activate transcriptional expression of PRRs and induce a lower immune response of HaCaT cells by limited cytokines secretion.

Cells, Cultured ; Cytokines ; biosynthesis ; Humans ; Keratinocytes ; immunology ; Lectins, C-Type ; genetics ; physiology ; RNA, Messenger ; analysis ; Receptors, Pattern Recognition ; genetics ; physiology ; Toll-Like Receptor 2 ; physiology ; Trichophyton ; immunology

OBJECTIVETo study the distribution of DC-SIGN/DC-SIGNR alleles among drug user (DUs) populations with or without HIV/HCV infection in Shenzhen, and to evaluate the role of these alleles in the construction of genetic resistance to HIV or HCV and screen out the anti-HIV/HCV gene in Shenzhen.

METHODSAll 500 DU blood samples were collected from Shenzhen Detoxification Center, including 313 from injected drug users (IDUs). All samples were screened for HIV and HCV antibody by means of ELISA. The genomic DNA were extracted and amplified by PCR. The neck domain repeat regions of DC-SIGN/DC-SIGNR were sequenced directly from the PCR products to confirm the amplification for some samples and all positive PCR products were analyzed by agarose gel electrophoresis.

RESULTSOf 500 samples, 97 were found HIV positive, all of which were IDUs and HCV positive. The total positive rate of HCV among all HIV negative DU was 57.57% (232/403), and it was 63.89% (138/216) among IDUs; in comparing to the 50.26% (94/187) of DUs with other manners there showed significant difference (chi(2) = 7.61, P = 0.0058). Among HIV + DUs, there was a higher proportion of patient with the DC-SIGNR 5/6 and 5/8 (Fisher's exact, P = 0.043 and P = 0.034) with statistical significance; there was no statistically significant difference between HCV + and HCV-DUs and no significant difference between IDUs and other DUs for the DC-SIGNR polymorphism.

CONCLUSIONThe results might indicate that DC-SIGN/DC-SIGNR polymorphism might not influence the susceptibility to HCV. Genotype 5/6 might probably have a relation with HIV infection, but still need further investigation for the low frequency.

Adolescent ; Adult ; Alleles ; Cell Adhesion Molecules ; genetics ; Drug Users ; Female ; Gene Frequency ; Genotype ; HIV Infections ; genetics ; HIV-1 ; Hepacivirus ; Hepatitis C ; genetics ; Humans ; Lectins, C-Type ; genetics ; Male ; Middle Aged ; Polymorphism, Genetic ; Receptors, Cell Surface ; genetics ; Young Adult

Coronaviruses (CoVs) are single-stranded RNA viruses which contain the largest RNA genomes, and severe acute respiratory syndrome coronavirus (SARS-CoV), a newly found group 2 CoV, emerged as infectious disease with high mortality rate. In this study, we compared the synonymous codon usage patterns between the nucleocapsid and spike genes of CoVs, and C-type lectin domain (CTLD) genes of human and mouse on the codon basis. Findings indicate that the nucleocapsid genes of CoVs were affected from the synonymous codon usage bias than spike genes, and the CTLDs of human and mouse partially overlapped with the nucleocapsid genes of CoVs. In addition, we observed that CTLDs which showed the similar relative synonymous codon usage (RSCU) patterns with CoVs were commonly derived from the human chromosome 12, and mouse chromosome 6 and 12, suggesting that there might be a specific genomic region or chromosomes which show a more similar synonymous codon usage pattern with viral genes. Our findings contribute to developing the codon-optimization method in DNA vaccines, and further study is needed to determine a specific correlation between the codon usage patterns and the chromosomal locations in higher organisms.
Animals ; Codon/*genetics ; Humans ; Lectins, C-Type/*genetics ; Membrane Glycoproteins/*genetics ; Mice ; Nucleocapsid/*genetics ; Phylogeny ; SARS Virus/*genetics/pathogenicity ; Severe Acute Respiratory Syndrome/prevention & control ; Species Specificity ; Vaccines, DNA ; Viral Envelope Proteins/*genetics ; Virus Attachment

OBJECTIVETo understand the genetic polymorphism of DC-SIGN's and DC-SIGNR's neck regions in normal Chinese Han population, and to obtain the genetic data of the two loci in Chinese Han population.

METHODSThe genotypes and alleles of repeat sequences of DC-SIGN and DC-SIGNR neck region were typed by PCR, agarose gel electrophoresis and sequencing. Polymorphism information content (PIC) of DC-SIGNR was calculated.

RESULTSDC-SIGN genetic polymorphism was rare. Allele 7 was most and its frequency was 0.9808. 4-, 5-, 6- and 8- alleles were also found, although their frequencies were very low. Caucasians had only 6- and 8- allele mutants; DC-SIGNR genetic polymorphism was high, its PIC was 0.5312, 4-,5-,6-,7-,8-,9- alleles and 16 genotypes were found in normal Chinese Han population. The differences of 6/5,7/4,7/5,7/6,7/7,9/5,9/7,9/9 genotypes distribution and 5-,6-,7-,9- alleles frequency between normal Chinese Han population and Caucasian population were all extremely distinct (P<0.01). The inserted mutation seemed more in Chinese Hans than Caucasian population.

CONCLUSIONDC-SIGN and DC-SIGNR genotypes and alleles distribution in Chinese Han population are significantly different from Caucasian population and with Chinese own population genetic characteristics, compared with Caucasians.

Adolescent ; Adult ; Alleles ; Asian Continental Ancestry Group ; genetics ; Cell Adhesion Molecules ; genetics ; China ; Female ; Gene Frequency ; Genotype ; Humans ; Lectins, C-Type ; genetics ; Male ; Polymerase Chain Reaction ; Polymorphism, Genetic ; genetics ; Receptors, Cell Surface ; genetics ; Young Adult

OBJECTIVETo construct the expression vector pLCK-CD69-IRES-EGFP that contains mouse cell surface activation protein CD69 and enhanced green fluorescent protein(EGFP),and to generate CD69 transgenic mice based on this vector.

METHODSFirst, RNA was extracted from mouse lung tissue and cDNA was synthesized via reverse transcription. PCR primer was designed through the PubMed searching, then mouse CD69 DNA fragment was amplified with PCR. Second, this DNA fragment was subcloned to the pInsulater-LCK-IRES-EGFP plasmid and constructed the transgenic vector after the verification of nucleotide sequence. Third, the expression vector was then transfected into 293 T cells and its expression in 293 T cells was observed under fluorescence microscope. Last, microinjection was performed to transfer the expression vector pLCK-CD69-IRES-EGFP into fertilized eggs, which were implanted into pseudo-pregnant recipient mice. After birth the tail samples of the pups were obtained for the purpose of genotyping to determine the transgenic founders. Fluorescence microscope and flow cytometer were used to measure the expression of CD69 on cells.

RESULTSThe construction of the expression vector pLCK-CD69-IRES-EGFP was verified by enzyme digestion and DNA sequencing. The transfected 293 T cell showed expression of the protein under fluorescence microscope. Identification of PCR for the tail tissue of the pups confirmed the present of CD69 transgene and resting lymphocytes demonstrated the expression of CD69.

CONCLUSIONThe construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice have been successfully processed, which lays a foundation of the solid pattern studies in inflammatory diseases.

Animals ; Antigens, CD ; genetics ; Antigens, Differentiation, T-Lymphocyte ; genetics ; DNA, Complementary ; Genetic Vectors ; Genotype ; Green Fluorescent Proteins ; genetics ; Lectins, C-Type ; genetics ; Mice ; Mice, Transgenic ; Plasmids ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Transfection

OBJECTIVETo clone the recombinant human islet neogenesis-associated protein (rhINGAP) gene for its secretory expression in Pichia pastoris.

METHODSINGAP gene was amplified with PCR and inserted between Xho I and EcoR I downstream sites of the alpha factor of the recombinant plasmid alpha/pUC18. The fusion gene of alpha factor and INGAP was subsequently inserted between BamH I and EcoR I sites of the plasmid pPIC9K of P. pastoris. After confirmation with restriction enzyme digestion and sequencing, the positive recombinant plasmid that integrated INGAP gene was linearized with Sal I digestion and transformed into the yeast host strain GS115 through electroporation. The yeast transformants that harbored the INGAP gene with high copies were selected with the auxotroph medium and G418, followed then by PCR verification of the positive transformants, from which the expression of recombinant human INGAP was induced with methanol as the only carbone source. The antigenic activity of the desired protein was then detected using Western blotting and enzyme-linked immunosorbent assay (ELISA).

RESULTS AND CONCLUSIONThe recombinant expression plasmid INGAP/pPIC9K was successfully constructed, and 3 positive transformants were obtained. The expressed protein showed good antigenic activity as confirmed by Western blotting and ELISA.

Antigens, Neoplasm ; genetics ; metabolism ; Biomarkers, Tumor ; genetics ; metabolism ; Blotting, Western ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Islets of Langerhans ; metabolism ; Lectins, C-Type ; genetics ; metabolism ; Pancreatitis-Associated Proteins ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; metabolism

OBJECTIVETo investigate the effect of small interfering RNA (siRNA)-mediated islet neogenesis associated protein (INGAP) gene silencing on the proliferation of islet cells.

METHODSDifferent siRNAs targeting INGAP gene were designed and transfected into INS-1 islet cells, and the expression levels of INGAP mRNA and protein following the transfection were detected using RT-PCR, flow cytometry and Western blotting. The proliferation of the transfected INS-1 cells was evaluated using MTT assay.

RESULTSCompared with those in the irrelevant siRNA, empty vector control, and un-transfected groups, the expression levels of INGAP mRNA and protein in the cells transfected with siRNA6 were reduced significantly. The cell proliferation rate significantly increased after transfection with siRNA6 (P<0.05).

CONCLUSIONsiRNA targeting INGAP can effectively down-regulate INGAP expression and inhibit the proliferation of INS-1 cells.

Animals ; Antigens, Neoplasm ; genetics ; Biomarkers, Tumor ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Insulinoma ; pathology ; Islets of Langerhans ; pathology ; Lectins, C-Type ; genetics ; Pancreatitis-Associated Proteins ; RNA Interference ; RNA, Small Interfering ; genetics ; Rats