LINC00675 suppresses proliferative, migration and invasion of clear cell renal cell carcinoma via the Wnt/ß-catenin pathway.

OBJECTIVE: To clarify the role of LINC00675 in affecting the progression of clear cell renal cell carcinoma (ccRCC) and its potential mechanism, thus providing effective hallmarks and therapeutic targets for the clinical treatment of ccRCC. MATERIALS AND METHODS: Differentially expressed long non-coding RNAs (lncRNAs) in renal epithelial tissues and ccRCC tissues were searched by analyzing the dataset downloaded from The Cancer Genome Atlas (TCGA) and LINC00675 was selected. LINC00675 level in ccRCC cell lines was determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Overexpression model of LINC00675 model in 786-O and 769-P cells was constructed by the transfection of pcDNA3.1(+)-LINC00675 (LV-LINC00675). Changes in proliferative, migratory, and invasive capacities of 786-O and 769-P cells overexpressing LINC00675 were assessed. At last, relative levels of ß-catenin, Vimentin, and N-cadherin in ccRCC cells overexpressing LINC00675 were detected by qRT-PCR and Western blot. RESULTS: LINC00675 was downregulated in ccRCC tissues and cell lines. Overexpression of LINC00675 attenuated proliferative, migratory, and invasive capacities of 786-O and 769-P cells. Downregulation in ß-catenin after overexpression of LINC00675, while Vimentin and N-cadherin levels did not change. CONCLUSIONS: LINC00675 is downregulated in ccRCC. Overexpression of LINC00675 attenuates ccRCC to proliferate, migrate, and invade by activating the Wnt/ß-catenin pathway.

[Association between TNFRSF11A and TNFRSF11B gene polymorphisms and the outcome of hepatitis C virus infection].

Objective: To explore the relationship between the tumor necrosis factor receptor superfamily members 11A (TNFRSF11A) and 11B (TNFRSF11B) gene polymorphisms and the outcome of hepatitis C virus (HCV) infection. Methods: In this case-control study, 749 cases of persistent HCV infection, 494 cases of spontaneous clearance and 1 486 control subjects were included from 2008 to 2016. TaqMan-MGB probe method was used to detect the genotype of TNFRSF11A rs1805034 and TNFRSF11B rs2073617. The genotypes distribution of the two single nucleotide polymorphisms (SNP) were analyzed in different populations. Results: Co-dominant model showed that individuals carrying the rs2073617 CC genotype were prone to have chronic HCV infection, compared with individuals carrying the rs2073617 TT genotype (OR=1.517, 95%CI: 1.055-2.181, P=0.024). Recessive model results showed that individuals carrying rs2073617 CC genotype were more likely to develop chronic HCV infection compared with individuals carrying rs2073617 TT or TC genotype (OR=1.435, 95%CI: 1.033-1.996, P=0.032). Additive model showed that the risk for chronic HCV infection increased with the increase of the number of rs2073617 C alleles (OR=1.204, 95%CI: 1.013-1.431, P=0.035). Conclusion: The genetic polymorphism of TNFRSF11B rs2073617 might be related with the chronicity of HCV infection.

Anti-tumor mechanism of IL-21 used alone and in combination with 5-fluorouracil in vitro on human gastric cancer cells.

To study the effect and related mechanism of IL-21 alone and in combination with 5-Fluorouracil on the proliferation and growth, transferability, and apoptosis of gastric cancer cells, we cultivated gastric cancer cell SGC-7901 and created four experimental groups with varying concentrations of IL-21 and 5-Fluorouracil: IL-21 group (IL-21 100ng/ml), semi-combination group (5-Fluorouracil 25µg/ml+IL-21 100ng/ml), 5-Fluorouracil group (5-Fluorouracil 50µg/ml), and combination group (5-Fluorouracil 50µg/ml+IL-21 100ng/ml). The MTT (3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di- phenytetrazoliumromide) assay was used to detect the inhibitory effect of each group on the proliferation and growth of gastric cancer cells. A scratch-wound assay was carried out to detect the inhibitory effect of each group on transferability. TUNEL assay was used to detect the effect of each group on the apoptosis of the gastric cancer cells, and Western blot was used to detect the expression of caspase-3, caspase-8, bcl-2, and c-myc, which are the proteins related to apoptosis, after the drug effect in each group. The results show that, compared to the 5-Fluorouracil group, the inhibitory effects after 24 h of the IL-21 group and the semi-combination group on SGC-7901 were weaker (P less than 0.001). However, if the effect lasted 48 h, then the inhibition in the semi-combination group had no significant difference compared to the 5-Fluorouracil group (P>0.05). The scarification test showed that all groups could inhibit the transferability of SGC-7901 and that the effect increased successively from the IL-21 group, the semicombination group, the 5-Fluorouracil group, to the combination group. The TUNEL assay indicated that all groups could promote the apoptosis of SGC-7901. The percentage of cell apoptosis increased, and the Western blot showed that the expression of caspase-3, caspase-8, and c-myc, respectively, in the semi-combination group, the 5-Fluorouracil group, and the combination group increased successively and that the successive increasing of c-myc was the most significant. The expression of bc1-2 tended to decrease. In conclusion, IL-21 used alone and in combination with 5-Fluorouracil are anti-tumor mechanisms in SGC-7901.

Computerized endoscopic balloon manometry to detect esophageal variceal pressure.

BACKGROUND AND STUDY AIMS: Measuring the variceal pressure is important in predicting esophageal variceal bleeding. However, current noninvasive methods of measuring variceal pressure have not gained wide popularity. We have developed a computerized endoscopic balloon manometry (CEBM) system to detect variceal pressure. The aims of the study were to test the in-vitro accuracy of CEBM and to evaluate the clinical reliability and feasibility of this method. PATIENTS AND METHODS: The CEBM system, comprising an esophageal variceal manometer and a computer, records variceal pressure and manometry images simultaneously. In the in-vitro study, variceal models were fixed inside an artificial esophagus, into which an endoscope with transparent balloon was inserted for intraluminal pressure measurement. The artificial varix was filled with water and connected to a water column to modulate the intraluminal pressure. This CEBM system was tested blindly in variceal models with different intraluminal pressures. CEBM was also used to measure variceal pressure in 23 patients with liver cirrhosis and esophageal varices, and the results were compared with the hepatic venous pressure gradient (HVPG). RESULTS: In the in-vitro study, the measured intraluminal pressure correlated significantly with the actual intraluminal pressure for different diameters (R > or = 0.993, P < 0.001). Variceal pressure measurements with CEBM were technically successful in 23 patients. Regression analysis showed a good correlation between variceal pressure measured with CEBM and the HVPG (R = 0.858, P < 0.001). CONCLUSIONS: Our preliminary results indicate that CEBM is feasible and accurate. CEBM may become a more reliable method for detecting variceal pressure.

Gene cloning, expression and vaccine testing of Schistosoma japonicum SjFABP.

A 600 bp DNA fragment was amplified by PCR from an adult Schistosoma japonicum cDNA library. Sequence analysis confirmed that this fragment contained an S. japonicum Chinese mainland strain fatty acid binding protein (Sj14FABP) gene. This gene was subsequently expressed in Escherichia coli (E. coli) and in Baculovirus/silkworm systems. The recombinant protein from E. coli was a 41 kDa GST fusion protein (rSj14/GST), which could be purified by glutathione agarose affinity chromatography, with a yield of 25 mg/L E. coli culture. The recombinant protein from the Baculovirus/silkworm system was an 18 kDa fusion protein (rSj14/His), which could be purified by Ni-NTA resin chromatography column with a yield of 3.5 mg per silkworm larva. Both rSj14/GST and rSj14/His could be recognized by S. japonicum-infected mouse sera and anti-rSj14/GST mouse sera in Western blotting. The purified recombinant protein was immunogenic in mice, rats and sheep, and 34.3%, 31.9% and 59.2% worm reductions, respectively, were obtained in vaccinated Kunming mice, Wistar rats and sheep vaccinated with Sj14/GST, compared to non-vaccinated control groups. Worm reductions of 48.8% and 49.0% were recorded in Balb/c mice immunized with Sj14/His, compared to non-vaccinated and BCG-vaccinated groups, respectively. These results indicate that rSj14FABP is a promising candidate vaccine for schistosomiasis japonica, particularly as in the rat and sheep vaccination experiments, no adjuvant was used.

Improving the diffraction quality of MTCP-1 crystals by post-crystallization soaking.

Significant improvement in the resolution and quality of the X-ray diffraction of crystals of MTCP-1 protein was observed on post-crystallization soaking. The MTCP-1 crystals grown from 1.5 M ammonium sulfate diffracted to only 3.0 A resolution with some disorder in the diffraction. After post-crystallization soaking in a solution containing 2.0 M ammonium sulfate, the disorder was eliminated and diffraction extended to better than 2.0 A resolution. Both native and selenomethionine-enriched crystals demonstrated better diffraction after soaking for several months. This simple technique may be useful to improve the diffraction quality of protein crystals generally.

Crystal structure of MTCP-1: implications for role of TCL-1 and MTCP-1 in T cell malignancies.

Two related oncogenes, TCL-1 and MTCP-1, are overexpressed in T cell prolymphocytic leukemias as a result of chromosomal rearrangements that involve the translocation of one T cell receptor gene to either chromosome 14q32 or Xq28. The crystal structure of human recombinant MTCP-1 protein has been determined at 2.0 A resolution by using multiwavelength anomalous dispersion data from selenomethionine-enriched protein and refined to an R factor of 0.21. MTCP-1 folds into a compact eight-stranded beta barrel structure with a short helix between the fourth and fifth strands. The topology is unique. The structure of TCL-1 has been predicted by molecular modeling based on 40% amino acid sequence identity with MTCP-1. The identical residues are clustered inside the barrel and on the surface at one side of the barrel. The overall structure of MTCP-1 superficially resembles the structures of proteins in the lipocalin family and calycin superfamily. These proteins have diverse functions, including transport of retinol, fatty acids, chromophores, pheromones, synthesis of prostaglandin, immune modulation, and cell regulation. However, MTCP-1 differs in the topology of the beta strands. The structural similarity suggests that MTCP-1 and TCL-1 form a unique family of beta barrel proteins that is predicted to bind small hydrophobic ligands and function in cell regulation.

Crystal structure of TNF-alpha mutant R31D with greater affinity for receptor R1 compared with R2.

Crystal structures have been determined of recombinant human tumor necrosis factor-alpha (TNF-alpha) and its R31D mutant that preferentially binds to TNF receptor R1 with more than seven times the relative affinity of binding to receptor R2. Crystals of the wild-type TNF were of space group P4(1)2(1)2 and had unit cell dimensions of a = b = 94.7 and c = 117.4 A. Refinement of the structure gave an R-factor of 22.3% at 2.5 A resolution. The crystals of TNF R31D mutant diffracted to 2.3 A resolution, and were of identical space group to the wild type with unit cell dimensions of a = b = 95.4 and c = 116.2 A, and the structure was refined to an R-factor of 21.8%. The trimer structures of the wild-type and mutant TNF were similar with a root mean square (r.m.s.) deviation of 0.56 A for Calpha atoms; however, the subunits within each trimer were more variable with an average r.m.s. deviation of 1.00 A on Calpha atoms for pairwise comparison of subunits. Model complexes of TNF with receptors R1 and R2 have been used to predict TNF-receptor interactions. Arg31 in all three subunits of wild-type TNF is predicted to form an ionic interaction with the equivalent glutamic acid in both receptors R1 and R2. Asp31 of the TNF R31D mutant is predicted to interact differently with the two receptors. The side chain of Asp31 in two subunits of the TNF mutant is predicted to form hydrogen bond interactions with Ser59 or Cys70 of R1, while it has no predicted interactions with R2. The loss of three strong ionic interactions of Arg31 and the electrostatic repulsion of Asp31 with Glu in the receptors is consistent with the reduced binding of the R31D mutant to both receptors relative to wild-type TNF. The replacement of these ionic interactions by two weaker hydrogen bond interactions between Asp31 of the R31D mutant and R1, compared with no interactions with R2, is in agreement with the observed preferential binding of the R31D mutant to R1 over R2. Analysis of the structure and function of receptor-discriminating mutants of TNF will help understand the biological role of TNF and facilitate its use as an antitumor agent.

Model complexes of tumor necrosis factor-alpha with receptors R1 and R2.

The biological activities of tumor necrosis factor-alpha (TNF-alpha) are mediated by two different receptors, TNFR1 and TNFR2. To analyze the receptor binding site(s) of TNF-alpha, molecular models have been built of the complexes of TNF-alpha with the extracellular regions of receptors R1 and R2, based on the known crystal structures of TNF-alpha and lymphotoxin bound to R1. The model structure of R2 from residues 18-160 was built by analogy to the crystal structure of R1 in complex with lymphotoxin. The amino acid sequences of R1 and R2 show 27.5% identity over this region and were aligned with five insertions and three deletions. There are 18 conserved cysteines that form disulfides. R2 has lost one pair of cysteines compared with R1, but two new cysteines were modeled as forming a new disulfide bond. Both symmetric and asymmetric trimers of TNF-alpha were used to model the complexes with TNFR1 and R2. An analysis of differences in the model complexes showed good agreement with data on the differential binding of TNF mutants to its two receptors.