[Genetic analysis of 45 patients with suspected Lynch syndrome using next-generation sequencing].

Objective: To evaluate the value of next generation sequencing (NGS) in the genetic testing of Lynch syndrome. Methods: Immunohistochemical method was used to detect the expressions of DNA mismatch repair (MMR) proteins, including MutL homolog 1 (MLH1), PMS1 homolog 2 (PMS2), MutS homolog 2 (MSH2) and MutS homolog 6 (MSH6) in colorectal cancer, gastric cancer and endometrial cancer tissues collected from Shandong Provincial Hospital between 2016 and 2018. The genomic DNA of 45 patients who were suspected with Lynch syndrome was extracted from non-cancerous tissue paraffin samples, which were postoperatively confirmed by microscope. The mutations of 12 genes including MLH1 and MSH2 were detected using NGS. The germline mutant sites and significance were analyzed by bioinformatics technology and further confirmed by using Sanger sequencing. Results: The immunohistochemical results showed that the 45 cases of suspected Lynch syndrome included 22 cases of MLH1 and PMS2 deficient expression, 16 cases of MLH2 and MSH6 deficient expression, and 7 cases of MMR proteins normal expression. The NGS result showed that 28 cases of adjacent sample from colon cancer patients included 4 cases of MLH1 pathogenic mutation, 1 case of suspected MLH1 mutation, 2 cases of MLH2 pathogenic mutation, 2 cases of suspected MLH2 mutation. No MMR gene mutation was found in adjacent samples of 6 cases of rectal cancer, 6 cases of gastric cancer and 7 cases of colorectal cancer with MMR normal expression. One case of MLH1 or MHL2 pathogenic mutation and one case of MLH1 suspected mutation was detected in adjacent samples of 5 cases of endometrial cancer. Moreover, NGS also detected many other genes mutations and unreported gene mutation sites. Pathogenic and suspected MLH1 and MSH2 mutations were verified by Sanger sequencing. Conclusions: High-throughput NGS is a quick, accurate and reliable technique to identify gene variants in suspected Lynch syndrome patients. It has a wide application prospect for gene testing of tumors associated with Lynch syndrome.

MicroRNA-129-5p inhibits invasiveness and metastasis of lung cancer cells and tumor angiogenesis via targeting VEGF.

OBJECTIVE: This study was to find out the influence of microRNA-129-5p on proliferative ability, invasiveness, and metastasis of lung tumor cells and tumor angiogenesis. Besides, the effects of microRNA-129-5p on vascular endothelial growth factor (VEGF) level and potential regulatory mechanisms were also what we were interested in. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) assay was performed to detect the microRNA-129-5p level in tumor tissues and paracancerous tissues of 50 patients with LCa, and the interaction between microRNA-129-5p expression and LCa pathological parameters was analyzed. The untreated cell group (NC) and the transfected microRNA-129-5p overexpression group (microRNA-129-5p mimics) were established, and then, the transfection efficiency of microRNA-129-5p was further verified by qRT-PCR. In H1299 and SPC-A1, cell counting kit-8 (CCK-8), Tube-formation experiments, and transwell invasion and migration tests were performed to evaluate the influence of the microRNA on the biological function of LCa cells. Finally, the potential mechanism of action of VEGF, a downstream gene of microRNA-129-5p, was explored by bioinformatics analysis and recovery experiments. RESULTS: QRT-PCR results showed that the level of microRNA-129-5p in cancer tissues of LCa patients was notably lower than that in normal tissues, and the difference was statistically significant. Compared with patients with highly expressed microRNA-129-5p, patients with low level had higher rates of lymph node or distant metastasis, and the overall survival rate was lower. Compared with NC group, cell proliferation, invasiveness and migration ability, and tumor angiogenesis capacity were strikingly decreased in microRNA-129-5p mimics group. Subsequently, VEGF expression was validated conspicuously enhanced in LCa cell line and tissue and was negatively correlated with microRNA-129-5p. In addition, the recovery experiment demonstrated that overexpression of VEGF could counteract the impact of microRNA-129-5p mimics on tumor angiogenesis and the invasive and migratory capacity of LCa cells, which then together led to the malignant progression of LCa. CONCLUSIONS: The above studies demonstrated that microRNA-129-5p was strikingly correlated with LCa lymph node or distant metastasis and poor prognosis, and it can inhibit the malignant progression of this cancer. The investigation also demonstrated that microRNA-129-5p may inhibit proliferation capacity and invasiveness of LCa cells and tumor angiogenesis via regulating VEGF.

Regulation of human hsp90alpha gene expression.

Mammalian HSP90alpha and HSP90beta are encoded by two individual genes. On the basis of the upstream sequences of the human hsp90alpha gene, GenBank accession number U25822, we have constructed CAT reporter plasmids driven by individual fragments of the hsp90alpha gene. We found that (1) the proximal heat shock element complex located at -96/-60 enhances hsp90alpha promoter expression; (2) heat shock induction depends upon the coexistence of distal heat shock element at -1031/-1022 and the proximal heat shock element complex of the hsp90alpha gene; (3) unlike hsp90beta, downstream sequences of the transcription start site inhibit hsp90alpha expression. We conclude that the regulatory mechanisms for the expression of hsp90alpha and hsp90beta genes are different.

Demonstration of shared epitopes between bacterial proteins and HLA class-I proteins using monoclonal antibodies.

Monoclonal antibodies were used to identify the presence of epitopes on bacterial proteins which are cross-reactive with HLA-B27. Anti-HLA-B27 monoclonals, B27M2 and YE-2 reacted with a major protein of approximately 35 kDa, which was found to be OMPA. On some strains a 19 KDa protein was also seen. Anti-Shigella flexneri monoclonals were also developed which reacted with a 36 kDa protein and a 19 kDa protein. The 36 kDa protein was OMPF. These antibodies also reacted with a synthetic peptide representing amino acids 63-83 of the B*2705 sequence. These results support a potential role for molecular mimicry in the spondyloarthropathies.

A monoclonal antibody that recognizes HLA-B27 in the context of peptides.

The T2 mutant cell line is unable to load peptides into the MHC class I Ags inside the cells. These "empty" MHC class I Ags are not expressed on the cell surface unless the cells are cultured at low temperatures. Expression will occur at 37 degrees C only in the presence of peptides that bind to and stabilize the class I Ags. T2 cells transfected with the B*2705 gene were tested with a panel of anti-HLA-B27 mAb. Two of the antibodies, ME1 and KS3, reacted with the "empty" HLA-B27 expressed at low culture temperatures. Three antibodies, B27.M1, B27.M2, and Ye-2, were unreactive with these "empty" HLA-B27. The cells were then incubated with a panel of HLA-B27-binding peptides. One of the antibodies, Ye-2, became reactive when the cells were incubated with a peptide derived from HIV gp120 and to a less degree with a peptide derived from histone H3.3. Mouse L cells transfected with the B*2705 and the human beta 2m genes also reacted very poorly with B27.M1, B27.M2, and Ye-2. Those two peptides were also able to induce high increase in Ye-2 reactivity. Alternately, increase in Ye-2 reactivity was also observed when the L cells were incubated with IFN-gamma or TNF-alpha. These experiments indicate that the Ye-2 anti-HLA-B27 mAb recognizes HLA-B27 in the context of certain residing peptides either added exogenously or expressed endogenously. The B27.M1 and B27.M2 antibodies might share similar characteristics.

A patient-derived cytotoxic T-lymphocyte clone and two peptide-dependent monoclonal antibodies recognize HLA-B27-peptide complexes with low stringency for peptide sequences.

HLA-B27 molecules expressed on the T2 mutant cell line do not have peptides. Such empty HLA-B27 molecules were not recognized by an HLA-B27-restricted cytotoxic T-lymphocyte (CTL) clone (auto-1) derived from synovial fluid. To test for peptide dependency of the clone, B27-T2 cells were incubated with a panel of 48 different peptides. This lack of stringency was compared with that of a peptide-dependent monoclonal antibody, B27.M2. Positive B27.M2 reactivity resulted when the B27-T2 cells were incubated with two peptides: RRKAMFEDI and RRMGPPVGHR, derived from Chlamydia HSP60 and human ribonucleoprotein, respectively. Because of the limited availability of CTL versus monoclonal antibody, the specificity of B27.M2 was studied in greater detail. The importance of the HLA-B27 heavy chain in antibody recognition of class I-peptide complexes was demonstrated by site-directed mutagenesis. The stringency of the peptide residues was tested by making analogs of each of the nine residues in RRKAMFEDI, creating a panel of 180 analogs. Although stringency was highest for the sixth position, as many as six different amino acids provided positive reactivity. These results indicate that immune recognition of HLA-B27-peptide complexes might have rather low stringency for the peptide sequences. In theory, then, pathogen-derived peptides which induce autoimmunity by generating autoreactive CTL might not share much sequence similarity with the responsible self peptides.

Construction and screening of Plasmodium falciparum cDNA library.

The HAINAN isolate of Plasmodium falciparum FCC1/HN was cultured in vitro in large quantities. The total parasite mRNA was purified and reverse transcribed into cDNA. The cDNA fragments were inserted into lambda gt11 to construct a P. falciparum FCC1/HN erythrocytic stage cDNA library. Inhibitory monoclonal antibodies (McAbs) M26-32, F6-C2, and F6-D3 were used to screen the cDNA library expressed in E. coli. A total of 27 positive clones were found to react with M26-32 alone and 34 clones with both M26-32 and F6-C2. These expressed proteins may be candidates for use in malaria vaccine.