[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).

Relationship between epigenetic changes in Wnt antagonists and acute leukemia.

The present study was designed to investigate the relationship among epigenetic changes in Wnt antagonists, histone H4K20me1 and the expression of tumor-suppressor genes in acute leukemia (AL) to better understand the pathogenesis of leukemia. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to detect messenger RNA (mRNA) expression levels of Wnt antagonists (Wnt5a, HDPR1, DKK1 and DKK3) in patients with AL and in normal controls; pyrophosphate sequencing was performed to detect the methylation status of the Wnt5a promoter; and western blotting was performed to detect the overall expression levels of Wnt5a protein and histone H4K20me1 in patients with acute myeloid leukemia (AML) and in normal controls. The relationship between Wnt5a protein expression and histone H4K20me1 was analyzed. Chromatin immunoprecipitation-qPCR (ChIP-qPCR) was performed to investigate the recruitment of H4K20me1 and SET8 to the Wnt5a promoter and coding regions. Our results demonstrated that the expression levels of Wnt antagonists were generally low in AML, but showed differential expression in acute lymphocytic leukemia (ALL). In most cases of AML, methylation of the Wnt5a promoter was observed and Wnt5a protein expression was low. In some cases of AML, the overall level of H4K20me1 protein was higher than that in normal controls. In addition, Wnt5a expression was positively correlated with H4K20me1 expression and was unrelated to the methylation status of its promoter. Moreover, H4K20me1 and SET8 were enriched in the Wnt5a promoter region and coding region. By contrast, Wnt5a expression was unrelated to H4K20me1 expression in normal controls. Moreover, we observed that the methylation of Wnt antagonists was often found in patients with AL, particularly those with AML, whereas the extent of methylation was variable in ALL patients. Wnt5a expression was positively correlated with the enrichment of H4K20me1 and SET8 at the Wnt5a promoter and coding regions. H4K20me1 increased Wnt5a expression by promoting transcription initiation and elongation.

Role of HOTAIR in the diagnosis and prognosis of acute leukemia.

HOX antisense intergenic RNA (HOTAIR), a long non-coding RNA, plays an important role in the development of many types of cancers. Its function in acute leukemia (AL), however, has not been examined. The present study investigated the role of HOTAIR and its downstream genes in AL, and determined whether it could act as a molecular marker for prediction of leukemia development and prognosis. Real-time quantitative PCR was used to examine the expression of each gene in the HOTAIR signaling pathway in AL patients. The relationship between expression of HOTAIR and downstream genes and AL prognosis was analyzed. Expression of HOTAIR in patients with acute monocytic leukemia (M5) was increased as compared to controls (P<0.05). Compared to patients with low HOTAIR expression, overall survival and event-free survival of patients with high HOTAIR expression was significantly reduced. In addition, the expression of downstream genes in the HOTAIR signaling pathway including EZH2, LSD1, DNMT3A and DNMT3B was significantly increased in AL patients, and showed a significant positive correlation with high expression of HOTAIR (P<0.05). In conclusion, HOTAIR was closely related with a poor prognosis in AL patients. It may be involved in the development of leukemia by mediating methylation of DNA and histones.

[Application of Bisulfite Sequencing PCR in Detecting the Abnormal Methylation of Suppressor Gene of Wnt Signaling Pathway in Acute Promyelocytic Leukemia].

OBJECTIVE: To detect the abnormal methylation of the CPG island in the suppressor gene promoter region of the Wnt signaling pathway in cell strain NB4 of the acute promyelocytic leukemia by using the bisulfite sequcucing PCR(BSP), to screan the hyper-methylated suppressor gene of the Wnt signaling pathway and to evaluatc the potency of BSP in the methylation study. METHODS: The strain NB4 cells of the acute promyelocytic leukemia patients were used as the object, the mononuclear cells from 20 normal persons were used as the controls. The DNA was extracted and processed by bisulfite, the target sequences were amplified with PCR, then the abnormal methylation of the suppressor genes of the Wnt signaling pathway in the NB4 cells was analyzed by BSP, and the advantage and disadvantage of BSP were evaluated by comparison with the Methylation specific PCR and Pyrosequencing. RESULTS: The methylated rate of suppressor genes of the Wnt signaling pathways in the NB4 cells detected by BSP was as follows: the gene WIF1 95.26%, the gene DKK3 86%, the gene SFRP1 81.67%, the gene SFRP2 95.71%, the gene SFRP4 85%, and the gene SFRP5 95%; while the methylations in the control group were respectively as follows: the gene WIF-1 1.5%, the gene DKK3 4.2%, the gene SFRP1 0%, the gene SFRP2 0.9%, the gene SFRP4 2.5%, and the gene SFRP5 1.75%. A more significant methylation happened in the suppressor genes promoter of the Wnt signaling pathway in the NB4 cells as compared with the control group. CONCLUSION: Many hypermethylated suppressor genes are found in the Wnt signaling pathway of the acute promyelocytic leukemia NB4 cells, which may be served as one of the early diagnosis index and therapeutic target of the acute promyelocytic leukemia.