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ABSTRACT: This study was aimed to construct miRNA sponge targeting miR-20a and to establish a stable cell line Jurkat-S, paving the way for further research on function of miR-20a and application of RNAi in gene therapy. One pair of two-repeated oligonucleotide sequences containing bulged sites that are mispaired opposite miR-20a positions 9-12 was designed and synthesized with enzyme cutting sites. The annealed oligonucleotide fragments were subcloned into pCDNA3.0-L expressing vector. After double-enzyme cutting, the vector was ligated to the annealed oligonucleotide fragments again. Enzyme cutting and luciferase activity assay were performed for identification after four repeats. Then the ligated fragment was subcloned to lentivirus expressing vector. Virus particles were collected after the control or sponge vectors were co-transfected with the psPAX2 packaging plasmid and the envelope plasmid pMD2.G into HEK-293T cells using Lipofectamine2000. The Jurkat cells were transdused with recombinant lentivirus-transdusing units plus 6 µg/ml of Polybrene. Real-time PCR and Western blot were used to detect the mRNA and protein expression of P21 and E2F1 after lentivirus transdusion respectively. As a result, luciferase activity assay demonstrated that the sponge targeting miR-20a was constructed successfully and the virus was packaged in 293T. The titer of virus was 5×10(7) TU/ml. Stable transfected Jurkat-S cell line was established. As was expected, the mRNA and protein level of P21 and E2F1 was upregulated significantly in Jurkat-S cells. It is concluded that the miR-20a sponge is constructed successfully, and Jurkat-S stable cell line is established, in which the expression of miR-20a is inhibited stably.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 10/2012; 20(5):1056-62.
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ABSTRACT: This study was aimed to establish a high-throughput luciferase reporter system, through which to screen and identify miRNAs directly targeting p21, and to explore the biological function and significance of these miRNAs. Molecular cloning technique was used to construct and identify two lentivirus-expressing vectors-pWPXL-Luc and pWPXL-Luc-P21-3'UTR, virus particles were collected after the pWPXL-Luc or pWPXL-Luc-P21-3'UTR vectors were co-transfected with the psPAX2 packaging plasmid and the envelope plasmid pDM2G into HEK-293T cells. Furthermore, two stable cell lines expressing luciferase singly or co-expressed luciferase and P21-3'UTR were established by transducing HEK-293 cells with recombinant lentivirus; the former was used as control in the following experiments. Finally luciferase activity of the latter stable cells was measured by using the luciferase reporter assay system. The results showed that high-titre recombinant lentivirus was produced and two stable cell lines were constructed, also to some certain, the luciferase activity was in direct proportion to the number of cells. In conclusion, the high-throughput luciferase reporter system is established successfully; using this system, the 28 miRNA that directly target P21 Cip1/Waf1 are screened experimentally.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 01/2012; 20(1):159-63.
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ABSTRACT: This study was aimed to explore the synergistic effect of 2-methoxyestradiol (2-ME2) and bortezomib (Bor) on the proliferative inhibition and apoptosis of U266 cell line and its possible mechanism. The cells were treated with 2-ME2, Bor alone and 2-ME2 combined with Bor, respectively. The cell viability and proliferative curve were detected by CCK8, the cell apoptosis was detected by caspase 3/7 activity test, cell cycle status was analyzed by flow cytometry, and real-time PCR was used to detect the mRNA expression of P21, BAX and BCL-2. The results showed that compared with cells treated with 2-ME2 or Bor alone, the proliferative potential of cells in combination group was significantly inhibited (p < 0.05), and apoptosis rate markedly increased (p < 0.05), cell cycle was arrested at G(1)-S phase, the mRNA expressive level of P21 and BAX increased, while the expression of BCL-2 decreased. It is concluded that 2-ME2 combined with Bor synergistically inhibits cell proliferation and induces apoptosis in U266 cell line. The possible mechanism may be associated with its effect of up-regulating P21 and BAX expressions.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 12/2011; 19(6):1424-8.
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ABSTRACT: MicroRNAs (miRNAs) are small non-coding RNA molecules that are often located in genomic breakpoint regions and can act as oncogenes or tumor suppressor genes in human cancer. Our previous study showed that microRNA-423 (miR-423), which localized to the frequently amplified region of chromosome 17q11, was upregulated in hepatocellular carcinoma (HCC). However, the potential functions and exact mechanistic roles of miR-423 in hepatic carcinogenesis remain unknown. Here, we demonstrated that miR-423 significantly promotes cell growth and cell cycle progression at the G(1)/S transition in HCC cells. In particular, we found that miR-423-3p contributes to these effects, whereas miR-423-5p does not. Further studies revealed that p21Cip1/Waf1 is a downstream target of miR-423 in HCC cells, as miR-423 bound directly to its 3' untranslated region and reduced both the messenger RNA and protein levels of p21Cip1/Waf1. Moreover, enforced expression of p21Cip1/Waf1 abrogated miR-423-induced effects on HCC cell proliferation and cell cycle progression. These findings indicate that miR-423 exerts growth-promoting effects in hepatic carcinogenesis through the suppression of tumor suppressor p21Cip1/Waf1 expression. The results of this study define miR-423 as a new oncogenic miRNA in HCC.
Carcinogenesis 09/2011; 32(11):1641-7. · 5.70 Impact Factor
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ABSTRACT: microRNA (miRNA) are small non-coding RNA molecules of 19 - 25 nucleotides in a variety of eukaryotic systems, that control gene expression at the post-transcriptional level by degrading or translational repressing target messenger RNA (mRNA). Many studies have addressed the role of miRNA in normal hematopoiesis, giving an interpretative key to the aberrant expression observed in human hematological diseases. Here, the advances of main studies on the role of miRNA in normal hematopoiesis, and identify the association of miRNA with the development, progression of myeloproliferative diseases, including miRNA and lymphopoiesis, miRNA and erythropoiesis, miRNA and megakaryopoiesis, miRNA and myelopoiesis, miRNA and myeloproliferative neoplasm with positive BCR-ABL-chronic myeloid leukemia, miRNA and myeloproliferative neoplasm with negative PCR-ABL (PV.IME, ET), and so on are reviewed.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 07/2011; 19(4):1071-4.
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ABSTRACT: This study was aimed to quantitatively detect the expression level of lysophosphatide acid acyltransferase β (lpaat β) mRNA in leukemia patients so as to provide theoretical basis for the target therapy of lpaat β in leukemia. Real-time fluorescently quantitative PCR was used to detect the relative expression level of lpaat β mRNA to analyze its expression change in various type of leukemia. The results showed that the lpaat β mRNA expression level in acute leukemia (AL) patients was significantly higher than that in normal controls (p < 0.05); lpaat β mRNA expression level in acute myeloid leukemia (AML) patients was significantly higher than that in normal controls (p < 0.05) and was positively correlated with white blood cell count (≥ 20.0 × 10(9)/L) (p < 0.05) and CD34 expression level of leukemia, but was not related with extramedullary infiltration. Except for acute promyelocytic leukemia (APL), the lpaat β mRNA expression level was negatively correlated with chemotherapy sensitivity in chronic myeloid leukemia (AML) patients. lpaat β mRNA expression level in chronic myeloid leukemia (CML) patients was significantly higher than that in normal controls (p < 0.05). lpaat β mRNA expression level in acute lymphoid leukemia (ALL) patients was not higher than that in normal controls (p > 0.05). It is concluded that the lpaat β gene overexpression exists in both AML and CML patients. lpaat β produced by AML cells probably plays an important role in abnormal proliferation and drug-resistance of AML cells.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 11/2010; 18(6):1422-6.
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ABSTRACT: MicroRNAs (miRNAs) are small endogenous, non-coding RNAs that specifically bind to the 3' untranslated region (3'UTR) of target genes in animals. However, some recent studies have demonstrated that miRNAs also target the coding regions of mammalian genes. Here, we show that miRNA-181a downregulates the expression of a large number of zinc finger genes (ZNFs). Bioinformatics analysis revealed that these ZNFs contain many miR-181a seed-matched sites within their coding sequences (CDS). In particular, miR-181a 8-mer-matched sequences were mostly localized to the regions coding for the ZNF C2H2 domain. A series of reporter assays confirmed that miR-181a inhibits the expression of ZNFs by directly targeting their CDS. These inhibitory effects might be due to the multiple target sites located within the ZNF genes. In conclusion, our findings indicate that some miRNA species may regulate gene family by targeting their coding regions, thus providing an important and novel perspective for decoding the complex mechanism of miRNA/mRNA interplay.
Nucleic Acids Research 11/2010; 38(20):7211-8. · 8.03 Impact Factor
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ABSTRACT: This study was aimed to quantitatively detect the expression levels of gfi-1 gene in leukemia patients, and to investigate the effect of gfi-1 gene silenced by short hairpin RNA (shRNA) on proliferation of leukemia cell line K562. Quantitative real-time PCR and Western blot were used to detect the mRNA and protein expression levels of GFI-1 in newly diagnosed patients with leukemia. One pair of oligonucleotide sequences targeted at human gfi-1 mRNA were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pLVTHM vector. Virus particles were collected when the control and shRNA vectors had been co-transfected with the psPAX2 packaging plasmid and the envelope plasmid pMD2 G into HEK-293T cells using Lipofectamine 2000. The K562 cells were transfused with 1 x 10⁶ recombinant lentivirus-transfusing units plus 6 microg/ml of polybrene. Rea-time PCR and Western blot were used to detect the expressions of gfi-1 and bax mRNA after lentivirus transfusion. CCK-8 assays was used to evaluate the proliferation potential of cells. The results showed that the gfi-1 expression level in all leukemia patients was significantly higher than that in normal group (p < 0.05); the gfi-1 mRNA expression in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL) patients was significantly higher than that in normal group (p < 0.05); but the difference of gfi-1 mRNA expression between AL and CML or ALL and AML was not significant. Notably, the gfi-1 mRNA expression level had a positive correlation with high white blood cell count of > 20.0 x 10⁹/L (p < 0.05). As was expected, the mRNA and protein level of gfi-1 was reduced significantly in K562 cells after lentivirus transfusion, whereas the mRNA and protein level of bax was upregulated. And CCK-8 assay showed that gfi-1 gene silencing can inhibit K562 proliferation. It is concluded that gfi-1 expression is upregulated in leukemia patients and may contribute to leukemogenesis. The gfi-1 specific shRNA mediated by lentivirus can effectively down-regulate the expression of gfi-1 and inhibit the proliferation of K562 cells, which lay a basis for further research on gene therapy in leukemia.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 08/2010; 18(4):849-54.
