[Gene Polymorphisms of Patients with Lymphoma-Associated Hemophagocytic Syndrome in Longyan area, Fujian Province].

OBJECTIVE: To analyze the gene polymorphisms of patients with lymphoma-associated hemophagocytic syndrome in Longyan area, Fujian province. METHODS: A total of 125 patients with lymphoma-associated hemophagocytic syndrome in Longyan, Fujian province, admitted to Longyan First Hospital from May 2017 to November 2020 were selected. Peripheral venous blood was collected from all the patients, and the genotypes of perforin 1 (PRF1) and interleukin-10 (IL-10) gene loci were detected by PCR-fluorescence probe method, and the correlation between PRF1 and IL-10 gene polymorphisms and lymphoma-associated hemophagocytic syndrome was analyzed. RESULTS: The mutation frequencies of PRF1 gene loci rs885821 (C>T), rs885822 (C>T), rs1889490 (G>A) in patients with lymphoma-associated hemophagocytic syndrome were 10.40%, 78.8% and 64.4%, respectively. The mutation frequencies of rs1800872 (A>C), rs1800871 (C>T) and rs1800896 (G>A) of IL-10 loci were 56.0%, 45.2% and 77.6%, respectively. CONCLUSION: PRF1 and IL-10 gene loci were polymorphic in patients with lymphoma-associated hemophagocytic syndrome in Longyan area, Fujian province. Alleles C and G of PRF1 and IL-10 were risk factors, and alleles T and A were protective factors.

Metformin attenuates multiple myeloma cell proliferation and encourages apoptosis by suppressing METTL3-mediated m6A methylation of THRAP3, RBM25, and USP4.

Based on the results of epidemiological and preclinical studies, metformin can improve the prognosis of patients with malignant tumors. Studies have confirmed that metformin inhibits multiple myeloma (MM) cell proliferation and promotes apoptosis. Nevertheless, the specific mechanism remains to be elucidated. MM cells were intervened with different doses of metformin to detect cell proliferation and apoptosis. Western blotting and RT-qPCR were employed to assess the expression of METTL3, METTL14, WTAP, FTO, and ALKBH5 after metformin intervention. The microarray dataset GSE29023 was retrieved from the Gene Expression Omnibus (GEO) database and calculated using the R language (limma package) to authenticate differentially expressed genes (DEGs). The database for annotation, visualization, and integrated discovery (David) was applied for GO annotation analysis of DEGs. Subsequently, the string database and Cytoscape software were applied to construct protein-protein interaction (PPI) and DEM hub gene networks. Bioinformatics analysis and MeRIP were applied to predict and test METTL3-mediated m6A levels on mRNA of THRAP3, RBM25, and USP4 in METTL3 knocked-down cells. Then rescue experiments were performed to explore effects of METTL3 and THRAP3, RBM25, or USP4 on cell proliferation and apoptosis. The effect on MM cell xenograft tumor growth was observed by injection of metformin or/and overexpression of METTL3 in in vivo experiments. Metformin decreased cell proliferation and encouraged cell apoptosis in a dose-dependent manner. Global m6A modification was elevated in MM cells compared to normal cells, which was counteracted by metformin treatment. Furthermore, THRAP3, RBM25, and USP4 were identified as possible candidate genes for metformin treatment by GSE29023 data mining. METTL3 interference impaired m6A modification on mRNA of THRAP3, RBM25, and USP4 as well as expression levels. The mRNA stability and expression of THRAP3, RBM25, and USP4 was decreased after metformin treatment, which was reversed by METTL3 overexpression. THRAP3, RBM25 or USP4 knockdown reversed the assistance of METTL3 overexpression on the malignant behavior of MM cells. Finally, upregulation of METTL3 was shown to exert facilitative effects on xenograft tumor growth by blocking metformin injection. The present study demonstrates that metformin can repress the expression of THRAP3, RBM25, and USP4 by inhibiting METTL3-mediated m6A modification, which in turn hamper cell proliferation and promotes cell apoptosis.Abbreviations: multiple myeloma (MM), Gene Expression Omnibus (GEO), differentially expressed genes (DEGs), database for annotation, visualization and integrated discovery (David), protein-protein interaction (PPI), epithelial­mesenchymal transition (EMT), methyltransferase like 3 (METTL3), methyltransferase like 14 (METTL14), wilms tumor 1-associated protein (WTAP), methyltransferase like 16 (METTL16), acute myeloid leukemia (AML), non-small lung cancer (NSCLC), glioma stem cells (GSCs), normal bone marrow-derived plasma cells (nPCs), false discovery rate (FDR), biological process (BP), optical density (OD), horseradish peroxidase (HRP), M6A RNA immunoprecipitation assay (MeRIP).

CRISPR/Cas9 (D10A) nickase-mediated Hb CS gene editing and genetically modified fibroblast identification.

This study investigated whether CRISPR/Cas9 (D10A) nickase-mediated gene editing can correct the aberrant Hb Constant Spring mutation (Hb CS or HBA2: c.427 T > C) in fibroblasts. Vectors for repairing the α-globin-encoding gene, HBA2:c.427 T > C mutation, includingthe CRISPR/Cas9(D10A)-sg plasmid and donor with homology arms, were constructed and used to perform gene editing in patient-derived fibroblasts. We subsequently analyzed the genetic correction, the gene editing efficiency and off-target effect. Sequencing analysis and the BamHI assay showed that HB CS mutant cells were repaired with Hb CS point mutations, the editing efficiency was 4.18%~9.34% and no off-target effects were detected. The results indicate that the HB CS mutant gene is effectively repaired by the CRISPR/Cas9 (D10A)system, which may enable truly personalized therapy for precise repair of α-thalassemia.