[Comparative analysis on both high risk behaviours, infection of HIV and syphilis between married and unmarried men who have sex with men].

OBJECTIVE: To understand the distribution of marriage status among men who have sex with men (MSM) in the city of Changzhou, and to explore the impact of marriage on AIDS related high risk behaviors and HIV infection in this population. METHODS: Target sampling (snowball sampling) was adopted to carry out a cross-sectional study, and structured questionnaire-based interviews were conducted to collect information on social demography, HIV related high risk behaviors. Blood and urine samples were collected to detect HIV, syphilis, gonorrhea and Chlamydia trachomatis infections. RESULTS: Of the 655 participants, 37.4% were married. Married MSM mostly sought their sexual partners at the public bathing house (61.6%), while unmarried MSM were mainly through bars (33.6%) or internet (31.1%). The proportion of having anal sex with men during the last 6 months was lower in the married group (50.8%) than in the unmarried group (73.3%), (P < 0.001) The percentage of having sex with women in the last 6 months was significantly higher in the married group (68.9%) than that in the unmarried group (33.2%) (P < 0.001), (OR = 4.454, 95%CI: 3.168 - 6.261). The rates of condom use in the last anal sex with men in married and unmarried groups were 71.0% and 77.6%, respectively (P = 0.152). The rate of condom use in the last intercourse with women was significantly lower in the married group (44.0%) than that in the unmarried group (70.4%) (P < 0.001), (OR = 0.331, 95%CI: 0.205 - 0.535). In the sex trade, most of the married MSM would "buy" sex (66.7%), while unmarried MSM would "sell" sex (63.2%) (P < 0.05), (OR = 3.429, 95%CI: 1.255 - 9.366). The percentage of having drugs in the previous year was higher in married group (3.3%) than that in the unmarried group (0.8%) (P < 0.05). In married and unmarried groups, the infection rates of HIV, syphilis, gonorrhea and Chlamydia trachomatis appeared to be (8.6%, 8.6%), (17.1%, 12.3%), (1.6%, 2.4%), and (3.3%, 9.0%), respectively (P > 0.05). CONCLUSION: Marriage seemed to have had limited effects on reducing the high risk behaviors of MSM. Different and multiform interventions should be developed according to the different characteristics of married or unmarried MSM population.

[Construction of recombinant NF-κB reporter adenovirus and activity analysis].

OBJECTIVE: To study the feasibility of adenovirus-based nuclear factor-κB (NF-κB) reporter as a model to screen the upstream signal regulators of NF-κB. METHODS: A type 5 (E1/E3 deficient) adenovirus vector pAdxsi was used to construct the NF-κB reporter adenovirus. Multiple adherent and suspending cell lines were infected by the NF-κB reporter adenovirus, and the luciferase activity of the NF-κB reporter gene was measured. RESULTS: An NF-κB reporter adenovirus (Ad-NF-κB-luc) was successfully constructed. The virus was capable of infecting HepG2, MGC803, THP-1 and U937 cell lines and showed high activities of NF-κB-luc reporter gene when stimulated by tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS). CONCLUSION: The Ad-NF-κB-luc reporter gene transfer system can effectively infect those cells hard-transfected by conventional transfection reagents. It also produces a high activity of NF-κB-luc reporter gene with stability and reliability. Our study expands the application of NF-κB reporter gene.

In vitro biological characterization of DCUN1D5 in DNA damage response.

BACKGROUND: Novel prognostic biomarkers or therapeutic molecular targets for laryngeal squamous cell carcinoma (LSCC) are an urgent priority. We here sought to identify multiple novel LSCC-associated genes. METHODS: Using high-density microarray expression profiling, we identified multiple genes that were significantly altered between human LSCCs and paired normal tissues. Potential oncogenic functions of one such gene, DCUN1D5, were further characterized in vitro. RESULTS: Our results demonstrated that DCUN1D5 was highly expressed in LSCCs. Overexpression of DCUN1D5 in vitro resulted in 2.7-fold increased cellular migration, 67.5% increased invasive capacity, and 2.6-fold increased proliferation. Endogenous DCUN1D5 expression was decreased in a time-dependent manner after genotoxic stress, and silencing of DCUN1D5 by siRNA decreased the number of cells in the S phase by 10.2% and increased apoptosis by 11.7%. CONCLUSION: Our data suggest that DCUN1D5 in vitro might have vital roles in DNA damage response, but further studies are warranted to assess its significance in vivo.

[Study on the mechanism of C17orf62 induced cell death].

OBJECTIVE: To isolate the long coding sequence of human novel gene C17orf62 which is named as C17orf62-L by us, and analyze its effects on cell viability, subcellular localization, and expression profile in multiple cell lines. METHODS: RT-PCR (reverse transcription polymerase chain reaction ) was used to clone C17orf62-L which encoded 187 amino acids from human multi-tissue cDNA library. We used bioinformatics analysis to identify structural characteristics of C17orf62-L, and RT-PCR to detect its expression. By Laser Scanning Confocal Microscopy we identified its subcellular localization of C17orf62-L. Furthermore, flow cytometry experiment was used to validate whether overexpression of C17orf62-L could influence cell phenotypes and Western blot was used to study related mechanisms. RESULTS: C17orf62-L was cloned and constructed into the pcDNA-and pEGFP-expression plasmids. C17orf62-L had signal peptide and transmembrane domain.C17orf62-L was widely expressed in multiple cell lines and was validated partial co-localization with Golgi apparatus. Functional studies showed C17orf62-L could induce cell death accompanied with rising of cleaved PARP(poly ADP-ribose polymerase). CONCLUSION: Human C17orf62 is a novel cell death inducing gene.

Estrogen stimulates the expression of mismatch repair gene hMLH1 in colonic epithelial cells.

Estrogen is reported to have a protective effect on colon cancer; however, the underlying mechanism is unclear. Impaired mismatch repair plays an important role in colonic carcinogenesis. The purpose of this study was to investigate the association of estrogen on regulating mismatch repair expression in colonic epithelial cells. In cultured COLO205 cells, the effect of estradiol (E2) and antagonist ICI182.780 on the expression of hMLH1 and hMSH2 was studied using reverse transcription-PCR and Western blotting. The correlation between serum level E2 and the expression of hMLH1 and hMSH2 in colonic mucosal tissue of 42 healthy individuals was also examined using reverse transcription-PCR and immunohistochemical staining. E2 increased the expression of hMLH1 in COLO205 cells, which was suppressed by ICI182.780. However, the effect of E2 on hMSH2 expression was not significant in COLO205 cells. In healthy individuals, a strong positive correlation of E2 level with hMLH1 expression in normal colonic epithelial cell was observed when serum E2 level was >45 pg/mL, but no correlation was seen between E2 and hMSH2 expression. E2 affects the expression of hMLH1 but not hMSH2 in vitro, and high serum E2 level correlates with hMLH1 expression in vivo. These findings suggest that the anticolonic cancer effect of estrogen may be related to hMLH1 regulation.

[Both PIK3IP1 and its novel found splicing isoform, PIK3IP1-v1, are located on cell membrane and induce cell apoptosis].

OBJECTIVE: To find novel isoform of PIK3IP1 and analyze their effects on cell viability, subcellular localization, and expression profile in cell lines. METHODS: RT-PCR was used to clone PIK3IP1 and its novel splicing isoform PIK3IP1-v1 from multi-tissue cDNA pool. By cell-based assays, we studied how PIK3IP1 and PIK3IP1-v1 affected the activity of Renila luciferase and morphological change in the HEK 293T cells. Furthermore, flow cytometry experiment was used to validate that overexpression of both splicing isoforms could induce cell apoptosis. Bioinformatics analysis was used to identify structural characteristics of these two splicing isoforms. By fluorescence microscopy assay, we identified their subcellular localization. RT-PCR was used to detect the expression of PIK3IP1 in the cell lines. RESULTS: PIK3IP1 and its novel splicing isoform PIK3IP1-v1 were cloned and constructed into the pcDNA-and pEGFP-expression plasmids. They both had signal peptide and transmembrane domain. Nevertheless, PIK3IP1-v1 was in absence of an extracellular Kringle domain. They could inhibit the activity of Renila luciferase and induce cell apoptosis. Simultaneously, both splicing isoforms are validated with subcellular localization on cell membrane and lowly expressed in many cell lines. CONCLUSION: PIK3IP1-v1 is a novel splicing isoform of PIK3IP1. Both of them are located on cell membrane and can induce cell apoptosis.

[Molecular cloning and preliminary function study of a novel human gene AC3-33 related to suppress AP-1 activity].

Activator protein-1(AP-1) is an important transcription factor, and dysregulation of its activity has been associated with many human diseases, including various cancers. A novel human gene AC3-33 (GenBank name: c30rf33, Accession No. FLJ31139), which can suppress PMA and Ionomycin induced activation of AP-1, was identified from 650 human func-tion-known genes by using the high throughput-high content cell-based screening technology. The gene whose full cDNA length is 1391 bp containing 6 exons and 5 introns is located in the human chromosome 3q25.31. The predicted protein encoded by this gene contains 251 amino acids with a theoretical molecular weight of 29 kDa. Expression of the AC3-33 gene is widly found in adrenal glands and cervix. The amino acid sequences of AC3-33 is highly conserved, and has no homology to other known proteins. Subcellular localization studies show that the AC3-33 protein was localized in the cytoplasm. Our preliminary results showed that AC3-33 is an important novel gene related to supress AP-1 activity.

Berberine inhibits cyclin D1 expression via suppressed binding of AP-1 transcription factors to CCND1 AP-1 motif.

AIM: To verify the suppressive effect of berberine on the proliferation of the human pulmonary giant cell carcinoma cell line PG and to demonstrate the mechanisms behind the antitumoral effects of berberine. METHODS: The proliferative effects of PG cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide colorimetry. The cell cycle was examined by flow cytometry. The expression level of cyclin D1 was detected by RT-PCR. The activities of the activating protein-1 (AP-1) and NF-kappaB signaling pathways related to cyclin D1 were examined by luciferase assay. The cytoplasmic level of c-Jun was detected by Western blot analysis. An electrophoretic mobility shift assay was used to examine the binding of transcription factors to the cyclin D1 gene (CCND1) AP-1 motif. RESULTS: The results showed that the proliferation of PG cells treated with different concentrations (10, 20, and 40 microg/mL) of berberine for 24 and 48 h was suppressed significantly compared to the control group. After treatment with berberine, the proportion of PG cells at the G0/G1 phase increased, while cells at the S and G2/M phases decreased. Berberine could inhibit the expression of cyclin D1 in PG cells. Berberine inhibited the activity of the AP-1 signaling pathway, but had no significant effect on the NF-kappaB signaling pathway. Berberine suppressed the expression of c-Jun and decreased the binding of transcription factors to the CCND1 AP-1 motif. CONCLUSION: Berberine suppresses the activity of the AP-1 signaling pathway and decreases the binding of transcription factors to the CCND1 AP-1 motif. This is one of the important mechanisms behind the antitumoral effects of berberine as a regulator of cyclin D1.

Tissue engineered cartilage from hTGF beta2 transduced human adipose derived stem cells seeded in PLGA/alginate compound in vitro and in vivo.

Chondrogenic potential of human adipose derived stem cells (hASCs) makes them a possible source of seeding cells for cartilage tissue engineering. We aim to examine the chondrogenic differentiation of human transforming growth factor beta2 (hTGF beta2) transduced hASCs seeded in three-dimensional scaffold in vitro and in vivo. In this study, hASCs were isolated from human subcutaneous adipose tissue and transduced with a replication deficient adenovirus carrying hTGF beta2 (Ad5-hTGF beta2), and then the transduced cells were seeded and cultured in PLGA/alginate compounds. RT-PCR analysis revealed that Ad5-hTGF beta2 transduced hASCs produced aggrecan and collagen type II after 7-day induction in vitro and continued throughout the culture period; this was also demonstrated by the positive staining of Alcian blue and immunohistochemistry for collagen type II. For in vivo study, Ad5-hTGF beta2 transduced hASCs seeded in PLGA/alginate compounds were implanted in subcutaneous pockets of nude mice; after 12 weeks, the implants were harvested and examined by haematoxylin and eosin staining, AB-PAS staining, and immunohistochemical analysis, and the results demonstrated the formation of cartilage tissue. As a control, all these were not observed in the constructs with Ad5-EGFP transduced hASCs. In conclusion, our study demonstrates that adenovirus-mediated hTGF beta2 gene transfer is able to induce the hASCs into chondrogenic lineage both in vitro and in vivo. Ad5-hTGF beta2 transduced hASCs combined with three-dimensional PLGA/alginate compound may be a viable method in treating injuries of cartilage.

Ectopic neocartilage formation from predifferentiated human adipose derived stem cells induced by adenoviral-mediated transfer of hTGF beta2.

Chondrogenic potential of human adipose derived stem cells (hASCs) makes them a possible source of seeding cells for cartilage tissue engineering. In this study, chondrogenic differentiation of hASCs induced by transduction with replication-deficient adenovirus carrying human transforming growth factor beta2 (Ad5-hTGF beta2) was demonstrated by RT-PCR, immunohistochemistry staining, biochemical and western blot analysis. To evaluate if the in vitro differentiated hASCs could keep their chondrocytic phenotype and produce neo-cartilage in vivo, predifferentiated hASCs were seeded in different scaffolds and implanted in subcutaneous pockets on the dorsum of nude mice. After 4 and 12 weeks culture in vivo, specimens were harvested and examined by histological and immunohistochemical analysis, cartilage-like tissue formation was only found in alginate gel and PLGA/alginate compound groups, in PLGA group, fibrous tissues and angiogenesis ingrowth were observed. These findings demonstrated that adenovirus-mediated hTGF beta2 gene transfer could induce hASCs into a chondrogenic lineage in vitro, however, this predifferentiation did not guarantee ectopic cartilage formation in vivo unless appropriate three-dimensional scaffolds were used as the cell carry vehicles.

[Cloning, expression and subcellular localization of a novel human gene-RNF122].

OBJECTIVE: To clone a functionally unknown gene RNF122 and analyze its expression pattern and subcellular localization. METHODS: PCR was used to clone the novel gene-RNF122 from the mixed human tissue cDNA library. Bioinformatics analysis was used to identify structure characteristics of the gene. Northern blot was used to analyze its expression in normal tissues; RT-PCR was employed for its expression in cell lines and tumor tissues. By co-focal microscope, we identified its subcellular localization with organelle markers. RESULTS: A novel human gene-RNF122 was cloned, which was proved to have been expressed in several normal and tumor tissues and many cell lines, and localized in ER and Golgi apparatus. CONCLUSION: RNF122 is a novel gene which encodes a protein that has a classic RING domain. It is widely expressed in several tissues and cell lines. The encoded protein is localized in ER and Golgi apparatus, which indicates that it may play a role in the process of protein degradation.