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Objective@#To construct the mmu-miR-155 eukaryotic overexpression vector pmR-155 and to investigate its effect on HBV replication and expression of PTEN in vivo.@*Methods@#The mmu-mir-146a precursor gene fragment pre-mmu-mir-146a was amplified by PCR, then connected to the pmR-mCherry plasmid vector after double enzyme digestion, the accuracy of recombinant vector was verified by colony PCR、double enzyme digestion and sequencing; then the recombinant vector was transfected HBV transgene mice(Experimental Group)with hydrodynamics-based injection via vena caudalis, and pmR-mCherry plasmid、PBS were respectively transfected into the mice as Empty plasmid Group、Blank Group. The concentration of IFN-γ in the serum was detected by ELISA. The expression of SOCS1、PTEN mRNA in the liver was detected by qPCR at 30d post-transfectioned. The Western blot was performed to detect the changes in SOCS1、PTEN、HBX in the liver tissue at 30 d post-transfectioned. The results were analyzed with Student’s t-test, or one-way analysis of variance and the least significant difference test.@*Results@#the colony PCR、double enzyme digestion and sequencing verified that the gene was inserted into the pmR-mCherry vector. Compared with Blank Group, the expression of miR-155 in the Experimental Group was significantly increased(t = 8.90, P < 0.01); the concentration of IFN-γ in the Experimental Group was significantly increased(F = 26.58, P < 0.01); the mRNA(FSOCS1 mRNA = 19.72, P < 0.01; FPTEN mRNA = 7.38, P < 0.05) and protein(FSOCS1 = 50.30, P < 0.01; FPTEN = 129.00, P < 0.01) expression of COCS1、PTEN was significantly decreased in the Experimental group and the protein of HBX was also significantly(FHBX = 77.97, P < 0.01).@*Conclusion@#The pmR-155 eukaryotic overexpression vector is successfully constructed, this recombinant vector can express miR-155 stably; miR-155 can down-regulate cocs1、PTEN gene expression and up-regulate the expression of IFN-γ, it can inhibit the replication of HBV and a potential targets to treating hepatocellular carcinoma.
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Objective To construct the has-miR 146a eukaryotic overexpression vector pmR 146a and to explore its effect on the expression of c-Myc gene in HepG2.2.15 cells.Methods The has-miR-146a precursor gene fragment pre-has-miR-146a was amplified by PCR,then connected to the pmR-mCherry plasmid vector after double enzyme digestion,the accuracy of recombinant vector was verified by colony PCR,double enzyme digestion and sequencing;then the recombinant vector was transfected into HepG2.2.15 cells as the experimental group,meanwhile the empty vector group (transfecting pmR-mCherry empty plasmid group) and blank group(transfecting reagent lip2000+PBS),then the fluorescent protein expression amount was observed under the fluorescence microscopy at 24,48 h;the expression of has miR-146a was evaluated by qPCR;at 24,48 h after transfection,the expression levels of c-Myc gene mRNA were detected by qPCR,and the c-Myc protein expression level after 48 h was detected by Western blot.Results The colony PCR,double enzyme digestion and sequencing verified that the pre-has-miR-146a gene fragment was inserted into the pmR-mCherry vector;at 24,48 h after transfection in the experimental group and empty vector group,intracellular strong fluorescence was seen by fluorescent microscope,the transfection efficiency was at 50％-60％ contrasting without fluorescence;the has-miR-146a expression level in the experimental group was significantly higher than that in the empty vector group and blank group (P＜0.01);the c-Myc mRNA expression at 24,48 h after tranfection was significantly lower than that in the empty vector group and blank group (P＜0.05);the protein expression amount at 48 h after transfection was lower than that in the empty vector group and blank group (P＜0.01).Conclusion The pmR-146a eukaryotic overexpression vector is successfully constructed,this recombinant vector can express miR-146a stably;miR-146a can down-regulate c-Myc cancer gene expression,which can serve as one of potential targets for treating hepatocellular carcinoma.
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Objective To construct the miRNA‐21 eukaryotic overexpression vector pmR‐21 and to explore its regulation effect on the expression of c‐myc gene in HepG2 .2 .15 cells .Methods The miRNA‐21 precursor gene fragment pre‐miRNA‐21 was amplified by PCR ,then connected to the pmR‐mCherry plasmid vector after double enzyme digestion ,the accuracy of the recombi‐nant vector was verified by double enzyme digestion and sequencing ;then the recombinant vector was transfected into HepG2 .2 .15 cells ,the fluorescent protein expression was observed under the fluorescence microscopy at 24 h and the transfection efficiency was detected by flow cytometry ;the expression of miRNA‐21 was evaluated by real‐time quantitative PCR;at 72 h after transfection ,the expression levels of c‐myc gene were detected by RT‐PCR and Western blot ;CCK‐8 was used to detect the cell proliferation in each group .Results The double enzyme digestion and Western blot verified that the target gene fragment was inserted into the pmR‐mCherry vector;at 24 h after transfection ,intracellular strong fluorescence was seen ,the transfection efficiency was higher than 50% ;miRNA‐21 expression level of the pmR‐21 recombinant vector group was significantly increased;c‐myc gene expression was increased in the pmR‐21 recombinant vector group at 72 h after transfection ,the cell proliferation in the pmR‐21 recombinant group was faster than that in the control group(P<0 .05) .Conclusion The pmR‐21 eukaryotic overexpression vector is successfully con‐structed ,this recombinant vector can express miRNA‐21 stably ;miRNA‐21 can up‐regulate c‐myc gene expression ,c‐myc gene is one of miR‐21′s targets for playing a cancer‐promoting action .
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Objective To investigate the effects of overexpression of heat shock protein 22(HSP22) in hematopoietic malignant tumor cell lines.Methods A lentiviral system was used to mediate transduction of HSP22 complementary DNA-containing expression vector or empty vector into K562 and Namalwa cells.The transduction effeciency was tested by fluorescence microscope scan and flow cytometry.Semi-quantitative RT-PCR and Western blot were used to identify the expression levels of HSP22 mRNA and protein.Growth curve analysis,cell cycle analysis,colony-forming assay,tumor growth in nude mice and apoptosis analysis were used to evaluate the role of HSP22 in K562 and Namalwa cells.Results Lentivector expression systemmediated delivery of HSP22 into K562 and Namalwa cells can inhibit colony forming of K562 and Namalwa cells,the average numbers of colonies per well were 108,72,125 and 80 for K562-V,K562-H,Namalwa-V and Namalwa-H respectively (P =0.000 16 and 0.000 37 for K562 and Namalwa respectively).HSP22 transduction can also inhibit proliferation of Namalwa cells in vitro (P =0.015,0.042 and 0.048 for day 5,6 and 7 respectively) and K562 cells in vivo (P =0.022 for day 21).No significant difference in cell cycle and apoptosis was found in K562 and Namalwa cells compared with controls (all P ＞ 0.05).Conclusion Overexpression of HSP22 could inhibit the growth of hematopoietic malignant tumor cell lines K562 and Namalwa.
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Objective To investigate the expression of FBXW7 during the development of Notch1induced murine leukemia.Methods Notch1 over-expressing murine model of T-cell acute lymphoblastic leukemia was used to study the expression of FBXW7 by real-time PCR methods.Bone marrow mononuclear cells (BMNC) were isolated on different days after transplantation and CD45.2+ GFP+ leukemia cells were sorted by flow cytometry at late stage.The expression changes of FBXW7 were tested by real-time PCR.Results The mouse bone marrow cells both from leukemia and control groups expressed FBXW7.Different expression patterns of FBXW7 were observed during the development of leukemia. The expression of FBXW7 was gradually increased in control group, whereas the expression level of FBXW7 in leukemia group was decreased steadily and reached one-sixth of that in control group on 12th day.Furthermore,lower expression level of FBXW7 was observed in CD45+.2 GFP+ leukemia cells.Conclusion Decreased expression of FBXW7 is observed in Notch1-induced mouse leukemia model,suggesting that the abnormal ubiquitin degradation pathway mediated by FBXW7 might contribute to the leukemogenesis in Notch1-induced murine leukemia model.
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Objective To explore the mechanism of the cell-cycle arrest induced by human melanoma differentiation associated gene-7 (mda-7/IL-24) in chronic myelocytic leukemia cell line K562. Methods Microarray analysis was performed to determine the genes that were differentially regulated by mda-7/IL-24 in K562 cells, and was validated by realtime PCR. The phosphorylated pRb were detected by Western blotting analysis. Results A microarray analysis showed that G_0/G_1 phase-associated genes p21~(WAF-1) and BCCIP were up-egulated, while cdk6 and Smurf2 were down-regulated. The directional change in the expression of the four genes was successfully validated with real-time quantitative RT-PCR. pRb Set~(795) phosphorylation was observed with no modification of the pRb protein level. Conclusion These results suggest that IL-24/mda-7 may inhibit K562 proliferation and induce G_0/G_1 cell cycle angst by up-regulating p21~(WAF-1) and BCCIP, down-regulating cdk6 and Smurf2.
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Objective To investigate the anti-inflammatory/immune modulatory effects of high-expressing membrane bound M-CSF-hematopoietic malignant cells on macrophages. Methods After coculturing RAW264.7 and murine macrophage cell line with Namalwa-M, a cell line stably expressing mM-CSF, and companing with Nainalwa-V, a cell line stably transfected with the empty vector as the control, flow cytometry was used to detect the expression of the marker of alternatively activated macrophages, CD206, and intracellular expression of IL-10, IL-12, IL-6 and TNF-α to study the immunophenotype of RAW264.7; phagocytic assays to investigate their functional activity in vitro. Results RAW264.7 cocultured with Namalwa-M consistently showed high-level expression of CD206, which indicated activities of these macrophage cells were increased. Furthermore, these RAW264.7 expressing high levels of IL-10, TNF-a and low levels of IL-12, IL-6, as determined by intracellular staining were suggested that the phagocytic activity was increased. Functionally, RAW264.7 cocultured with Namalwa-M showed a higher level of phagocytic activity. Conclusion Macrophage generated in vitro after cocultured with mM-CSF-expressing hematopoietic malignant cell line could be transformed into abnormal macrophage with an immunophenotype defined as IL-10-high, IL-12-low, IL-6-low and TNF-α-high.
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Objective To investigate the antitumor activity of IL-24 delE5 in human leukemia cell line K562. Methods The expression of mda-7/IL-24 and its splice variant induced by TPA in leukemic cell lines, U937 and HL-60, was evaluated. The effects of IL-24 delE5 in K562 on cell proliferation, colony-forming ability, cell cycle, apoptosis, and tumor growth in vivo by using MTr assay, colony forming assay, flow cytometry, Annexin-V/PI and tumor xenograft models in nude mice were assessed. Meantime, the effects of IL-24 delE-5 and mda-7/IL-2A were compared. Results The expression of IL-24 dciE5 was detected in differentiated U937 and HL-60 cells. Transfection with IL-24 delE5 significantly reduced tumor cell viability, inhibited colony formation. Comparing with the control, G_0/G_1 stage add from (24.46±3.99) % to (42.69±3.04) %, caused cell cycle arrest in G_0/G_1 stage and significantly inhibited the growth of K562 transplantation tumor. No significant differences in the aforementioned antileukemia characteristics between IL-24 delE5 and mda-7/IL-24 was found. Conclusion Similar with mda-7/IL-24, IL-24 delE5 can efficiently inhibit the proliferation of K562 in vitro and in vivo, probably through induction of G_0/G_1 cell cycle arrest.
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<p><b>OBJECTIVE</b>To investigate the levels of IL-18 in the bone marrow of both normal subjects and patients with hematological diseases and to determine the possible significance of IL-18 in pathogenesis of some hematological malignancies.</p><p><b>METHODS</b>The IL-18 mRNA levels in the bone marrow of 140 patients with hematological diseases and 15 normal donors were determined by the semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Immunohistochemical method was used to detect IL-18 protein in 12 patients with acute myeloid leukemia (AML). The possible regulation of IL-18 for proliferation of some leukemia cells was investigated using antisense techniques.</p><p><b>RESULTS</b>IL-18 mRNA levels were obviously higher in the patients with leukemia or other malignant hematological diseases (OMHD) than in normal donors. However, no significant difference was found in the level of transcription between patients with iron deficiency anemia (IDA) and normal controls. Immunohistochemical method confirmed the presence of IL-18 protein in 10 out of 12 AML cases with positive transcription. By 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, IL-18 antisense oligodeoxynucleotides (ASON) clearly inhibited the growth of J6-1 and HL-60 cells (42% and 12% inhibited, respectively) in a dose-dependent manner.</p><p><b>CONCLUSIONS</b>IL-18 was detected at elevated levels in the bone marrow of patients with some hematological malignancies, and might be involved in the proliferation of certain leukemic cells in vivo through an autocrine mechanism.</p>