The m6A Methyltransferase METTL3-Mediated N6-Methyladenosine Modification of DEK mRNA to Promote Gastric Cancer Cell Growth and Metastasis.

Gastric cancer (GC) is the fifth most common cancer and the third deadliest cancer in the world, and the occurrence and development of GC are influenced by epigenetics. Methyltransferase-like 3 (METTL3) is a prominent RNA n6-adenosine methyltransferase (m6A) that plays an important role in tumor growth by controlling the work of RNA. This study aimed to reveal the biological function and molecular mechanism of METTL3 in GC. The expression level of METTL3 in GC tissues and cells was detected by qPCR, Western blot and immunohistochemistry, and the expression level and prognosis of METTL3 were predicted in public databases. CCK-8, colony formation, transwell and wound healing assays were used to study the effect of METTL3 on GC cell proliferation and migration. In addition, the enrichment effect of METTL3 on DEK mRNA was detected by the RIP experiment, the m6A modification effect of METTL3 on DEK was verified by the MeRIP experiment and the mRNA half-life of DEK when METTL3 was overexpressed was detected. The dot blot assay detects m6A modification at the mRNA level. The effect of METTL3 on cell migration ability in vivo was examined by tail vein injection of luciferase-labeled cells. The experimental results showed that METTL3 was highly expressed in GC tissues and cells, and the high expression of METTL3 was associated with a poor prognosis. In addition, the m6A modification level of mRNA was higher in GC tissues and GC cell lines. Overexpression of METTL3 in MGC80-3 cells and AGS promoted cell proliferation and migration, while the knockdown of METTL3 inhibited cell proliferation and migration. The results of in vitro rescue experiments showed that the knockdown of DEK reversed the promoting effects of METTL3 on cell proliferation and migration. In vivo experiments showed that the knockdown of DEK reversed the increase in lung metastases caused by the overexpression of METTL3 in mice. Mechanistically, the results of the RIP experiment showed that METTL3 could enrich DEK mRNA, and the results of the MePIP and RNA half-life experiments indicated that METTL3 binds to the 3'UTR of DEK, participates in the m6A modification of DEK and promotes the stability of DEK mRNA. Ultimately, we concluded that METTL3 promotes GC cell proliferation and migration by stabilizing DEK mRNA expression. Therefore, METTL3 is a potential biomarker for GC prognosis and a therapeutic target.

Production of mouse monoclonal antibodies against Helicobacter pylori Lpp20 and mapping the antigenic epitope by phage display library.

Lpp20, an outer membrane protein of Helicobacter pylori (H. pylori), has been identified as an immunodominant antigen. To obtain mouse monoclonal antibodies (mAbs) against it and to map its antigenic epitope is potentially to develop a vaccine for prevention and treatment of H. pylori infection. In our study, the Lpp20 gene was obtained from H. pylori genomic DNA by PCR (GenBank accession no. DQ106902), cloned into pGEX-4T-1 vector and expressed in Escherichia coli (E. coli) as a recombinant fusion protein with glutathione-S-transferase (GST), which was purified by GST-affinity chromatography. mAbs were produced by the hybridoma technique using Lpp20-GST as the immunogen. Using mAb as the target molecule and immunoscreening phage-displayed random dodecapeptide library (Ph.D.-12), the positive phage clones were sequenced and analyzed. Phage clones were chosen to immunize mice to evaluate the potential of phagotopes as effective vaccines. One mimotope (SWPLYSDASGLG) showed a good match with the Lpp20 proteins at 114-117aa (DASG) and the serum of mice induced by the phage clone clearly recognized Lpp20 protein. Our work suggests that the antigenic epitope could be mapped through screening the phage-displayed peptide libraries with mAb and a mimotope of Lpp20 providing an alternative approach for the diagnosis and development of a vaccine for H. pylori.

[Preparation of monoclonal antibodies against multiple antigens].

OBJECTIVE: To prepare monoclonal antibodies (mAbs) against multiple antigens by single cell fusion. METHODS: BALB/c mice were immunized with the multiple antigens, namely alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), HBsAgiHBcAg and HBeAg, and hybridomas were employed using PEG as the fusing agent. The hybridoma cells were respectively screened with AFP, CEA, HBsAg, HBcAg and HBeAg by enzyme-linked immunosorbent assay and limited dilution. The mAbs were purified by protein G affinity chromatography, its subtype was identified, the affinity constants (K(a)) were determined and the specificity was analyzed by Western blotting. RESULTS: Twenty hybridoma cell lines were obtained by single cell fusion, including 5 cell lines against AFP, 6 against CEA, 3 against HBsAg, 4 against HBcAg, and 2 against HBeAg. The subtypes of some hybridoma cell lines positive for the mAbs were identified as the immunoglobulin G1 (IgG1), with K(a) ranging from 1x10(9) M(-1) to x10(11) M(-1). Western blot analysis showed that all the mAbs strongly and specifically bound to their respective antigens. CONCLUSION: The mAbs against multiple antigens have been obtained by single cell-fusion, which increases the production of mAbs and reduces the time of preparation.

Preliminary separation of the growth factors in platelet-rich plasma: effects on the proliferation of human marrow-derived mesenchymal stem cells.

BACKGROUND: Platelet-rich plasma (PRP) as a storage vehicle of growth factors has been successfully used in clinical applications, but in most cases the platelets were autologous. However, the large volume of blood withdrawn has detrimental effects on patients with anemia or poor general health. To overcome these limitations, this study was designed to separate the growth factors in homologous platelet-rich plasma. METHODS: The gel chromatography with Superdex-75 column was applied to separate PRP supernatants into 4 major fractions. Then the four fractions were vacuumed freeze-dried and re-dissolved in phosphate buffered saline. Proteins concentrations in PRP and in four fractions were detected by bicinchoninic acid protein assay; platelet derived growth factor-AB (PDGF-AB) and transforming growth factor beta1 (TGF-beta1) levels were determined by sandwich enzyme-linked immunosorbent assays. The effects of fractions on the proliferation of human marrow-derived mesenchymal stem cells (MSCs) were determined by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: PRP supernatants were separated into four major fractions by gel chromatography. The proteins recovery was 96.72%. Of the four fractions, fraction B contained the highest TGF-beta1 and PDGF-AB levels, and the highest proteins concentrations. Cell proliferation curves of MSC demonstrated that fraction B and C induced a remarkable increase of MTT values compared to the untreated culture (P < 0.05), and the effects of fraction B and C showed no significant difference compared to the PRP group (P > 0.05). Fraction A and D showed no significant difference to the negative control group (P > 0.05). CONCLUSIONS: The growth factors in PRP supernatants could be preliminarily separated into four fractions by gel chromatography, and the freeze-drying fractions retained the biological activity of growth factors. The growth factors were mostly presented in fraction B and C, and they promoted cell proliferation effectively.

Production of mouse monoclonal antibodies against Helicobacter pylori Catalase and mapping the antigenic epitope by phage display library.

The Catalase of Helicobacter pylori (H. pylori) helps bacteria to protect themselves from oxygen toxicity and damage and have been identified an immunodominant antigen. To obtain mouse monoclonal antibodies (mAbs) against Catalase and to map its antigenic epitope is potentially to develop a vaccine for prevention and treatment of H. pylori infection. In our study, MAbs were produced by the hybridoma technique using recombinant Catalase--GST as the immunogen and were immunoscreened against phage-displayed random dodecapeptide library (Ph.D.-12). After three rounds of biopanning, 34 phage clones were randomly selected and their specificity to mAb was verified by sandwich and competitive inhibition ELISA. Fifteen phage clones were sequenced and their amino acids were deduced. One mimotope (SVSLPYANLATH) showed good match with Catalase protein at 394-405aa and the serum of mice induced by the phage clone clearly recognized Catalase protein. Our work suggests that the antigenic epitope could be mapped through screening the phage-displayed peptide libraries with mAb and a mimotope of Catalase would provide an alternative approach for the development of a vaccine for H. pylori.