Detection of Exosomal PML-RARA Fusion Gene Expression Level by Droplet Digital PCR / 中国实验血液学杂志

OBJECTIVE@#To establish a method for detecting the exosomal PML-RARA fusion gene expression by droplet digital PCR (ddPCR).@*METHODS@#By using Taqman probe-based ddPCR technique, the method that able to detect both long and short isoforms of PML-RARA fusion gene transcripts was established. RNA from PML-RARA negative cell line HL-60 as negative control was used to set the limit of blank (LOB), while the RNA from PML-RARA positive cell line NB4 and the recombinant plasmid pSG5-PML-RARA(S) were used to set the limit of detection (LOD) for long and short PML-RARA transcripts, respectively. Furtherly, the expression of exosomal PML-RARA fusion gene in NB4 cell culture supernatant and serum of patients with acute promyelocytic leukemia (APL) was analyzed by ddPCR technique.@*RESULTS@#The LOB of ddPCR assay for long and short PML-RARA transcripts were 0.0725 and 0.083 copies per microliter of PCR reaction system, respectively, while the LOD of long and short PML-RARA transcripts were 0.19 and 0.21 copies per microliter of PCR reaction system, respectively. In addition, the expression of exosomal PML-RARA fusion gene derived from both NB4 cell culture supernatant and serum of APL patients was successfully detected.@*CONCLUSION@#A ddPCR-based technique for detecting fusion gene transcripts has been established, which can be used to analyze absolute quantification in the minimal quantity of PML-RARA transcripts derived from exosomes. It suggests the possibility of this technique to non-invasively and dynamicly monitore the exosomal PML-RARA transcripts from APL patients' serum.

Experimental Study on Apoptosis of Kasumi-1 Cells Induced by Sertraline and Its Molecular Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the biological effects of sertraline, one of psychotropic drugs, on actue myeloid leukemia cell line Kasumi-1.</p><p><b>METHODS</b>Cells were treated by different concentrations of sertraline for different times. The effects of sertraline were evaluated by cell growth, cell morphology, cell cycle distribution and markers of cell apoptosis, respectively. Western blot was used to detect the expression change of related proteins.</p><p><b>RESULTS</b>Sertraline could inhibit cell proliferation and induce apoptosis. After treatment with 15 µmol/L and 20 µmol/L sertraline for 24 h, the inhibitory rate of Kasumi-1 cell proliferation was (19.00 ± 7.37)% and (47.90 ± 11.19)%, respectively. Meanwhile, compared with the control group, the percentage of Annexin V positive cells in Kasumi-1 cells treated with sertraline for 24 h raised obviously from (9.71 ± 2.12)% to (20.54 ± 2.52)% and (45.37 ± 7.88)% (P < 0.01), respectively. The cells were arrested in G0/G1 and G2/M phase. In addition, it was found that sertraline could also down-regulate the level of translationally controlled tumor protein (TCTP) in Kasumi-1 cells.</p><p><b>CONCLUSION</b>Sertraline can significantly induce the apoptosis of Kasumi-1 cells, that probably is associated with the down-regulation of TCTP protein expression.</p>

Role of the interferon-stimulated response elements I/II in expression regulation of the retinoic acid induced gene G / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the regulatory role of interferon-stimulated response elements (ISREs) located on the retinoic acid-induced gene G (RIG-G) promoter in RIG-G expression.</p><p><b>METHODS</b>By using point mutation technique, the authors constructed the wide type and site mutant reporter gene plasmids according to the ISRE sequence on RIG-G promoter, and detected the functional activities by luciferase reporter assay.</p><p><b>RESULTS</b>Mutation in ISRE II alone had no obvious effect on the expression of the reporter gene, whereas mutation in ISRE I dramatically inhibited the transactivity of RIG-G promoter. Mutation in both ISRE I and ISRE II resulted in complete loss of its response to the transcription factors for the reporter gene.</p><p><b>CONCLUSION</b>Both ISRE I and ISRE II on the RIG-G promoter are the binding sites for the complex of transcription factors. They are required for RIG-G expression, and ISRE I has a preferential role over ISRE II.</p>

Regulation mechanism for rig-g gene expression induced by all-trans retinoic acid / 中国实验血液学杂志

To investigate the molecular mechanisms of all-trans retinoic acid (ATRA)-induced rig-g gene expression and to better understand the signal transduction of ATRA during acute promyelocytic leukemia (APL) cell differentiation, the luciferase reporter assay, co-immunoprecipitation and chromatin immunoprecipitation were used to clarify the basic transcriptional factors, which directly initiated the expression of rig-g gene. The results showed that the expression of STAT2, IRF-9 and IRF-1 could be upregulated by ATRA with different kinetics in NB4 cells. IRF-9 was able to interact with STAT2 to form a complex, which could bind the rig-g gene promoter and trigger the rig-g expression. IRF-1 alone could also activate the reporter gene containing rig-g gene promoter, but C/EBPalpha could strongly inhibit this transcription activity of IRF-1. It is concluded that during ATRA-induced APL cell differentiation, IRF-1 is first upregulated by ATRA, and then IRF-1 increases the protein levels of IRF-9 and STAT2 with the downregulation of C/EBPalpha. The complex of IRF-9 and STAT2 is the primary transcriptional factor for rig-g gene induction. This study will be helpful for better understanding the signal transduction networks of ATRA during the course of APL cell differentiation.

A novel molecular mechanism of interferon alpha-regulated expression of retinoic acid-induced gene G / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanisms by which IFN-alpha regulated retinoic acid-induced gene G (RIG-G) expression.</p><p><b>METHODS</b>The expression of STAT1, p-STAT1 and RIG-G in IFN-alpha-treated NB4 cells was detected by Western blot. The roles of STAT1, STAT2 and IRF-9 in IFN-alpha-induced RIG-G expression were analyzed in STAT1-null U3A cells by cell transfection, reporter gene assay, co-immunoprecipitation and chromatin immunoprecipitaion.</p><p><b>RESULTS</b>In U3A cells, only when STAT2 and IRF-9 were co-transfected, the luciferase activities of RIG-G promoter-containing reporter gene could be highly increased about 8-fold compared with that in the control group. Moreover, in the absence of IFN-alpha, similar effects were observed in either IRF-9 co-transfected with wild type or mutant form of STAT2, whereas IFN-alpha could increase the transactivation activity of wild type STAT2 and IRF-9 by 6-fold compared with that without IFN-alpha, but had no effect on mutant STAT2. In addition, STAT2 could interact with IRF-9 and bind to the RIG-G promoter.</p><p><b>CONCLUSION</b>STAT2 may interact with IRF-9 in a STAT1-independent manner. The complex STAT2/IRF-9 is the key factor mediating the expression of RIG-G gene regulated by IFN-alpha. This is a novel signal transduction cascade for IFN which is different from the classical JAK-STAT pathway.</p>

Effects of PML-RARalpha on cAMP-induced AML cell differentiation / 中国实验血液学杂志

To explore the molecular mechanisms of acute promyelocytic leukemia cell differentiation induced by cAMP combined with low-dose As2O3, the PR9 cell line, which was stably transfected by PML-RARa fusion gene, was used as in vitro model. The effects of PML-RARa on cAMP-induced AML cell differentiation were evaluated according to cell growth, cell morphology, cell surface antigen as well as luciferase reporter gene assay, in the cells before and after the treatment with cAMP and/or As2O3. The results showed that cAMP alone could slightly increase the expression of CD11b in the PR9 cells expressing the PML-RARa fusion protein, but could not induce these cells to differentiate. The cells presented the terminal differentiation morphology and significantly increased CD11b expression only under the treatment of cAMP combined with As2O3. In addition, PML-RARa had strong inhibitory activity on the transcription of the reporter gene containing cAMP response elements. In conclusions, the PML-RARa fusion protein could dramatically block the signaling pathway of cAMP during the AML cell differentiation.

In vitro study of the effects of CDA-II combined with cAMP on apoptosis induction in retinoic acid resistant acute promyelocytic leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of CDA-II alone or combined with cAMP on the retinoic acid (RA)-resistant acute promyelocytic leukemia (APL) cells.</p><p><b>METHODS</b>The RA-resistant cell line NB4-R2 was used as an in vitro model and treated with CDA-II alone or in combination with cAMP. Cell apoptosis was assessed by morphology observation, distribution of cellular DNA contents and sub-G1 cell population. The level of Bcl-2 was detected by flow cytometry, DNA "ladder" was detected by agarose-electrophoresis.</p><p><b>RESULTS</b>CDA-II could induce NB4-R2 cell apoptosis through decreasing the level of cellular anti-apoptotic protein Bcl-2. cAMP could significantly enhance the role of CDA-II. Bcl-2 positive cell rates decreased to (15.1 +/- 4.8)% and (7.3 +/- 2.9)% in NB4-R2 cells treated with 1 mg/ml CDA-II plus 100 micromol/L cAMP for 48 h and 72 h, respectively. While 100 micromol/L of cAMP could decrease Bcl-2 positive NB4-R2 cells from (92.0 +/- 0.6)% to (75.3 +/- 2.0)%.</p><p><b>CONCLUSIONS</b>CDA-II combined with cAMP could exert potent apoptotic effect on RA-resistant APL cells.</p>

Utilization of the stable ectopic expression cell line as a model for the investigation of RIG-G gene / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the biological function of RIG-G gene by establishing a cell line stably expressing RIG-G protein.</p><p><b>METHODS</b>Ectopic RIG-G gene was transfected into U937 cells by using Tet-off expression system. Changes before and after RIG-G expression were detected for cell growth, cell morphology, cell surface antigen and cell cycle regulating proteins.</p><p><b>RESULTS</b>RIG-G protein arrested the cells at G0/G1 phase and inhibited cell growth by increasing the cell cycle inhibitors P21 and P27. As compared to that in control group, the proportion of cells at G0/G1 phase in RIG-G-expressing cell group increased from (43.9 +/- 5.6)% to (63.9 +/- 2.3)% (P < 0.01). The rate of growth inhibition was (68.7 +/- 0.2)%. In addition, an increase in CD11C expression [(61.3 +/- 1.1)% vs. (18.0 +/- 0.4)% (P < 0.01)] and in cells with morphologic features of partial differentiation (smaller cell size, reduced nucleus-cytoplasm ratio, notched nucleus and coarse chromatin) was also observed in RIG-G-expressing cells.</p><p><b>CONCLUSIONS</b>RIG-G has potential abilities to inhibit cell proliferation and promote cell differentiation.</p>

Study on mechanisms of the expression regulation of interferon-induced gene RIG-G / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanisms of the expression regulation of retinoic acidinduced gene G (RIG-G) by interferon alpha (IFNalpha).</p><p><b>METHODS</b>RIG-G promoter region was analyzed by bioinformatics. The functional activities of RIG-G promoter with or without IFNalpha were detected by luciferase reporter assay and electrophoretic mobility shift assay (EMSA).</p><p><b>RESULTS</b>RIG-G promoter region contained two well-conserved IFN-stimulated response elements (ISREs). Both ISRE I and ISRE II showed their effective binding abilities with signal transducer and activator of transcription 1 (STAT1). In HT1080 cells, in contrast with the empty plasmid pXP2, pXP2-A reporter construct containing intact ISRE I and ISRE II showed a significant higher baseline expression (1741.2 +/- 517.5) which could be further enhanced up to three-four folds by IFNalpha (5338.7 +/- 1226.9, P < 0.05). However, the luciferase activity of pXP2-A as well as its IFNalpha inducibility could be abrogated in STAT1-deficient U3A cells (from 1741.2 +/- 517.5 to 406.1 +/- 103.2, P < 0.05), indicating that the STAT1 protein was a prerequisite for the activities of ISRE I and ISRE II.</p><p><b>CONCLUSION</b>ISREs present in RIG-G promoter region are molecular basis of IFNalpha induced RIG-G expression. RIG-G is a target gene directly regulated by STAT1 protein and should play a key role in IFNalpha signaling pathways.</p>