miR-141-3p attenuates RSL3-induced ferroptosis and intestinal epithelial-mesenchymal transition via directly inhabits ZEB1 in intestinal Behçet's syndrome.

The aims of this study were to investigate whether the ferroptosis is involved in intestinal Behçet's syndrome (IBS), and to identify if miR-141-3p could attenuate RAS-selective lethal 3 (RSL3)-induced ferroptosis and intestinal epithelial to mesenchymal transition (EMT) via directly inhabits zinc fnger E-box binding homeobox 1 (ZEB1). The expressions of ferroptosis-related proteins in the intestinal tissues of patients with IBS were investigated by immunohistochemistry and quantitative real-time PCR (qRT-PCR). Malondialdehyde (MDA) contents of the intestinal tissues and cells were detected. Serum from IBS patients and RSL3 were co-cultured with intestinal epithelial cells in vitro. In order to investigate whether RSL3-induced ferroptosis can be ameliorated by miR-141-3p, the intestinal epithelial cells were firstly stimulated with RSL3 and then incubated with miR-141-3p mimics. Western blot was used to measure the expression of EMT and ferroptosis-related proteins. Expression of GPX4 (22.51% ± 2.05%, 51.75% ± 3.47%, t = - 7.77, p = 0.000) and xCT (17.49% ± 1.57%, 28.73% ± 1.75%, t = - 4.38, p = 0.003) were significantly lower in intestinal mucosal tissues of patients with IBS compared with HC group. Compared with the HC samples, the IBS specimens had significantly higher MDA (t = 4.32, p = 0.01). Moreover, the relative mRNA levels of ferritin light chain (FTL) (t = 4.07, p = 0.02) and ferritin heavy chain (FTH) (t = 8.82, p = 0.001) in the intestinal tissues were significant higher in IBS patients than in HC group. Serum from IBS patients could induce intestinal epithelial cell ferroptosis in vitro. Moreover, miR-141-3p could attenuate intestinal epithelial cell ferroptosis-induced by RSL3 and intestinal EMT via targeting ZEB1 in vitro. Ferroptosis were induced in patients with IBS. Moreover, the serum from IBS patients could induce ferroptosis in vitro. miR-141-3p could attenuate intestinal epithelial cell ferroptosis and intestinal EMT via targeting ZEB1. Therefore, miR-141-3p may open new avenues for the treatment of IBS in the future. Key Points • Ferroptosis in IBS is first reported in this study. • In this study, we explored that the serum from IBS patients could induce ferroptosis in vitro and miR-141-3p could attenuate intestinal epithelial cell ferroptosis and intestinal EMT via targeting ZEB1.

[Genotype Analysis of Pregnant Women with α- and ß- Thalassemia in Fuzhou Area of Fujian Province in China].

OBJECTIVE: To analyze the genotype of pregnant women with α- and ß- thalassemia in Fuzhou area of Fujian province in China. METHODS: Blood routine examination and hemoglobin electrophoresis were performed for pregnant women, and positive samples were examined by gap polymerase chain reaction and reverse dot blot hybridization. RESULTS: 412 cases were diagnosed as α-thalassemia (63.9%); 201 cases were diagnosed as ß-thalassemia (31.2%); 32 cases were diagnosed as α and ß-composite thalassemia. There were 12 genotypes in α-thalassemia, whose major genotypes were --SEA/αα, α3.7/αα, -α4.2/αα and αQSα/αα, with carrying rate of 64.32%, 20.14%, 7.77% and 1.94%, respectively. There were 10 genotypes in ß- thalassemia, whose major genotypes were CD41-42/N, CD17/N, IVS-II-654/N and -28/N, with carrying rate of 30.84%, 27.86%, 15.92% and 10.45%, respectively. There were 9 genotypes in α and ß-composite thalassemia, whose major genotypes were --SEA/αα composited CD41-42/N, -α3.7/αα composited CD41-42/N, --SEA/αα composited CD17/N, with carrying rate of 18.75%, 15.62%, 15.62% respectively. CONCLUSION: The major genotypes of pregnant women with α- and ß- thalassemia in Fuzhou area of Fujian province in China are --SEA/αα, α3.7/αα, CD41-42/N and CD17/N. Thalassemia screening and prenatal gene diagnosis should be strengthened in Fuzhou area of Fujian province in China.

Effect of taurine on immune function in mice with T-cell lymphoma during chemotherapy.

OBJECTIVE: To observe the effect of taurine on immune function in mice with T-cell lymphoma during chemotherapy. METHODS: A total of 40 C57BL/6 mice were selected and randomly divided into 4 groups, namely model group, chemotherapy group, taurine group and chemotherapy + taurine group, each containing 10 mice. Hypodermic injection was adopted to inoculate EL-4 cells in order to establish model of T-cell lymphoma. When the tumor achieved the size of 1 cm3, intervention treatments were given to the groups respectively. Mice in model group received 0.2 mL of normal saline which was intraperitoneally injected on Days 1, 8 and 15 with 3 weeks as a cycle; mice in chemotherapy group were administered with 80 mg/kg body weight of gemcitabine which was also intraperitoneally injected on Days 1, 8 and 15 with 3 weeks as a cycle; mice in taurine group were administered with 80 mg/kg body weight of taurine intraperitoneally injected daily for consecutive 8 d; mice in chemotherapy + taurine group were treated in the same manner as the mice in taurine group and chemotherapy group. Five mice were sacrificed at 2 and 3 weeks after intervention respectively, and the tumor tissues were collected and weighted after removal of auxiliary tissue, then the tumor inhibition rate was calculated. The thymus and spleen of mice sacrificed at 3 weeks after intervention were collected and weighted, and thymus and spleen indexes were calculated. Enzyme linked immunosorbent assay was used to detect the serum levels of IL-4, IL-10, IL-12 and IFN-γ in mice of each group. RESULTS: The tumor weights in chemotherapy group, taurine group and chemotherapy + taurine group after 2 and 3 weeks of treatment were significantly lower than that in model group (P < 0.05); the tumor weight in chemotherapy + taurine group after 2 and 3 weeks of treatment was significantly lower than that in chemotherapy group (P < 0.05); the tumor inhibition rate in chemotherapy + taurine group was significantly higher than that in chemotherapy group and taurine group (P < 0.05); the thymus and spleen indexes in taurine group and chemotherapy + taurine group were significantly higher than those in chemotherapy group and model group (P < 0.05); the thymus and spleen indexes in chemotherapy group were significantly lower than those in model group (P < 0.05); after 3 weeks of treatment, the serum levels of IL-4, IL-12 and IFN-γ in chemotherapy group, taurine group and chemotherapy + taurine group were significantly lower than those in model group (P < 0.05); the IL-4 level in taurine group and chemotherapy + taurine group was significantly lower than that in chemotherapy group (P < 0.05); the serum level of IL-10 in chemotherapy group and chemotherapy + taurine group was significantly higher than that in model group and taurine group (P < 0.05); the serum level of IFN-γ in taurine group and chemotherapy + taurine group was significantly lower than that in model group and chemotherapy group (P < 0.05); after treatment of 3 weeks, the serum levels of IL-4 and IL-10 in chemotherapy group, taurine group and chemotherapy + taurine group were significantly lower than those in model group (P < 0.05), and IL-12 level was significantly higher than that in model group (P < 0.05); the level of IFN-γ in taurine group and chemotherapy + taurine group was significantly higher than that in model group (P < 0.05), while the level of IFN-γ in chemotherapy group was significantly lower than that in the other 3 groups (P < 0.05). CONCLUSIONS: Taurine can effectively enhance the immune function of mice with T-cell lymphoma during chemotherapy, reduce the toxicity of chemotherapy.

[Expression and Prognostic Significance of H3K27 Trimethylation Protein in DLBCL].

OBJECTIVE: To investigate the expression and prognostic effect of H3K27 trimethylation protein (H3K27me3) in diffuse large B-cell lymphoma(DLBCL). METHODS: A total of 102 DLBCL patients from Fujian Provincial Cancer Hospital were enrolled in this study. No therapy had been given before specimen collection. Tissue microarray(TMA) technique and immunohistochemistry(IHC) method were used for H3K27me3 immunostaining. Clinicopathologic and suvival data were carefully collected. The association between tested markers, clinicopathologic characteristics and prognosis were evaluated. Survival rates were analyzed by the Kaplan-Meier method, and prognostic factor were analyzed by the Cox proportional hazards model, the relation of different expression levels with clinical feature and prognosis of patients was compared. RESULTS: The quality of TMA was perfect and meet the standard of analysis. Among all DLBCL patients, 59.8% were characterized with high expression of H3K27me3, correlated with age, ECOG≥2, extranodular disease number≥2, elevation of LDH, medium-high risk IPI. Patients with high H3K27me3 expression manifested that the complete remission rate(CR) and overall remission rate (OR) were lower than those of patients with low expression, i.e., 20% vs 57.5% and 41.8% vs 90%, respectively (P<0.001). In addition, patients with high H3K27me3 expression showed shorter median survival time, i.e., 21.5 mon (P<0.0001). Multivariate analysis indicated that H3K27me3 was an independent risk factor for DLBCL patients (P=0.007). CONCLUSION: TMA technique is valid for the construction of DLBCL tissue chips. Patients with high expression of H3K27me3 indicates little response to treatment, worse outcome and shorter overall survival. The detection of H3K27me3 expression possesses a certain clinical value for prediction of DLBCL outcome.

[Over-expression of DLL4/Notch1 ligand inhibits proliferation of K562 cells].

This study was aimed to explore the effect of DLL4/Notch1 ligand on cell growth in leukemia cell line K562 and its relevant mechanism. The pBudCE4.1-DLL4 plasmid was transfected into K562 cells by lipofectamine 2 000, RT-PCR and Western blot were applied to monitor the mRNA and the protein expression of exogenous DLL4 gene, as well as the expression of Notch1-ICD and target gene Hes1. Expression levels of Rb, YY1 and C-MYC protein in K562 cells were also detected by Western blot. Cell counting Kit-8 was used to detect the proliferation of K562 cells, and flow cytometry with Annexin V staining was used to detect the cell apoptosis. The results showed that the mRNA and protein expression levels of DLL4, Notch1-ICD and Hes1 in cells of experimental group were significantly higher than those of control groups (P < 0.05), indicating the successful activation of the Notch1 signaling pathway. The protein expression levels of Rb, YY1 and C-MYC in cells of experimental group significantly increased when compared with that of control group cells (P < 0.05). After transfection, the proliferation of K562 cells was obviously inhibited, and apoptosis rate in DLL4-transfected cells was significantly enhanced. DLL4 transfection significantly increased the number of cells in G1 phase and decreased that in S phase. It is concluded that the over-expression of DLL4 ligand gene in K562 cells results in successful activation of the Notch1 signaling pathway, increases expression of Rb, YY1 and C-MYC genes, which induces apoptosis and reduces proliferation.

[Effects of DLL4 gene on YY1 and c-Myc protein expression and cell proliferation in leukemia cell line K562].

This study was aimed to explore the effects of Notch ligand DLL4 on the protein expression of the transcription factor YY1 and proto-oncogene c-Myc, as well as K562 cell proliferation. The experiment was divided into 3 groups: normal control, negative control (pBudCE4.1-transfected) and experimental (pBudCE4.1-DLL4-transfected) groups. At 48 hours after transfection, the expression level of DLL4, YY1 and c-Myc proteins in K562 cells of each group were detected by Western blot and indirect immunocytochemical method; the CCK-8 method was used to detect proliferation of K562 cells; at 48 hours after transfection, cell cycle distribution and apoptosis of K562 cells were detected by flow cytometry. The results showed that the protein expression of DLL4, YY1 and c-Myc in K562 cells of every group were found. The protein expression levels of DLL4, YY1 and c-Myc in the experimental group cells were significantly higher than that in control groups (p < 0.05). The cell number in G(0)/G(1) phase increased in the experimental group and was higher than that in the control groups (p < 0.001), and the number of apoptotic cells were also increased (p < 0.001). It is concluded that DLL4 gene was successfully transfected into K562 cells, which increased the protein expression levels of transcription factor YY1 and proto-oncogene c-Myc, leading to the cell proliferation slower in experiment group, inducing the cell cycle arrested in G(0)/G(1) phase and increasing apoptosis.

[Arresting effect of p16 and dll4 transfection on cell cycle of K562 cells].

This study was purposed to investigate the expression and role of eukaryotic expression vector containing p16, dll4 genes in leukemia K562 cells. A vector pBudCE4.1-16-dll4 containing wild type p16cDNA and dll4cDNA was designed and constructed, then this vector was transfected into leukemia K562 cells by using lipofectamine 2000. The expression of p16 and dll4 genes was detected by Western blot, the cell growth curve and cell cycle were determined by CCK-8 kit and flow cytometry respectively. The results showed that the recombinant plasmid pBudCE4.1-16-dll4 was constructed and transfected into K562 cells in vitro successfully. The expression of exogenous P16 and Dll4 proteins could be detected in K562 cells. After transfection for 48 hours, the K562 cells were arrested in G(1) phase, the cell count increased in G(0)/G(1) phase and reduced in S phase, the cell proliferation decreased as compared with control. It is concluded that the p16 and dll4 genes can simultaneously express in K562 cells transfected with recombinant plasmid pBudCE4.1-16-dll4 in vitro which results in G(0)/G(1) arrest and reduces cell proliferation.

[Inhibitory effect of p16, p53 transfection on leukemic cell lines K562 and HL-60].

This study was purposed to construct a vector containing human suppressor gene p53 and p16, and to investigate their expression and effect on K562 and HL-60 cells. pBudCE4.1-53-16 is a vector designed for simultaneous expression of human suppressor gene p53 and p16 in mammalian cell line. After transfection into K562 cells with lipofectamine(TM) 2000, the expression of p53 and p16 genes was detected by Western blot and immunocytochemical method. The growth curve, apoptosis, cell cycle were assayed by CCK-8 and flow cytometry. The results showed that the recombinant plasmid pBudCE4.1-53-16 was constructed successfully and were verified by PCR and restriction analysis. The expression of P53 and P16 protein could be detected after transfection into leukemia cells (K562 and HL-60) for 48 hours. As compared with control group, the cell proliferation in experimental group was inhibited, the cells were arrested in G0 phase and apoptotic cells increased (p<0.001). It is concluded that the recombinant plasmid pBudCE4.1-53-16 has been established. p16 and p53 in the recombinant plasmid pBudCE4.1-53-16 synchronously express in leukemic cells after transfection in vitro for 2 days and results in reduced proliferation, G0 arrest and apoptosis increase.

Co-transfection of p16(INK4a) and p53 genes into the K562 cell line inhibits cell proliferation.

BACKGROUND AND OBJECTIVES: The tumor suppressor genes p53 and p16(INK4a), both of which act in tumor surveillance, are homozygously deleted in the human leukemia cell line K562. This study was performed to assess whether co-transfection of the p16(INK4a) and p53 genes could inhibit K562 cell proliferation. DESIGN AND METHODS: p16(INK4a) and p53 genes were co-transfected into K562 cells with liposome, and the expression of the transfected genes was detected by Western-immunoblotting and immunocytochemistry. The effect of the p16(INK4a) and p53 transfected cell culture was quantified by trypan blue staining, and the number of recovered viable cells was assessed every day after transfection. Cells were analyzed for expression of annexin V in order to detect apoptosis. Differentiation of transfected K562 cells was measured by the benzidine oxidation test, and the cell cycle was analyzed by flow cytometry. RESULTS: After co-transfection, there were 23% and 28% p53 and p16(INK4a) positive cells respectively. Co-transfection with p16(INK4a) and p53 genes significantly inhibited cell proliferation when compared to transfection with either p16(INK4a) or p53 gene. The percentage of cells expressing the apoptosis-related cell surface antigen annexin V was significantly higher in p53 and p16(INK4a) transfected cells than in p53 or p16(INK4a) transfected cells (6.24+/-0.37% vs 4.88+/- 0.17%, p<0.05 and vs 2.78+/-0.26%, p<0.05, respectively). p16(INK4a) and p53 co-transfection significantly increased the number of cells in G1 phase and decreased that in S phase. INTERPRETATION AND CONCLUSIONS: Expression of wild-type p16(INK4a) and p53 genes in K562 cells results in reduced proliferation and apoptosis. Introduction of exogenous p16(INK4a) and p53 genes into K562 cells might contribute to the clinical treatment of leukemia.

[Inhibition of K562 cell proliferation by wild type p16 and p53 genes co-transfection].

The tumor suppressor gene p53 and p16, both of which play an important role in inhibition of tumorigenesis, are homozygously deleted in human myeloid leukemia cell line K562. To explore the inhibition of K562 cell proliferation by wild type p16 and p53 genes, both p16 and p53 genes were co-transfected into K562 cells mediated by liposome. The expression of the two genes was measured by immunocytochemical method, the cell cycle was analysed by flow cytometry, and the number of recovered viable cells was assessed after transfection. After co-transfection, the p53 and p16 positive cells were 23% and 28%, respectively. The results showed that co-transfection of p16 and p53 genes significantly inhibits cell proliferation comparing with transfection either by p16 gene or by p53 gene (P < 0.05). Expression of p16 and p53 proteins increased the cell number in G(1) phase but decreased the cell number in S phase. It is concluded that co-transfection of p16 and p53 genes has a stronger growth-inhibitory effect on K562 cell growth than that of transfection only by p16 gene or by p53 gene, may be a pathway for gene therapy in leukemia.