OBJECTIVETo screen the protein interacting with retinoic acid receptor variant protein (RAR alpha-V) via the yeast two-hybrid technique (YTHT) and to find out the targets protein and study its biological function.

METHODSThe bait vector of pGBKT7-RAR alpha-V was constructed for screening proteins interacting with RAR alpha-V in K562 cell cDNA expression library via YTHT. The protein-protein interaction was confirmed with re-transformation in yeast and GST pull-down in vitro.

RESULTSThe bait vector was successfully constructed without toxicity, leakage and self-activation. Sixteen proteins were screened by YTHT and eight positive clones were identified by re-transformation in yeast. The interaction between RAR alpha-V and JTV-1 was confirmed by GST pull-down in vitro.

CONCLUSIONSThere are some kinds of proteins interacting with RAR alpha-V in cell. The biological dysfunction caused by certain protein-protein interaction may be involved in the pathogenesis of leukemia.

Gene Library ; Humans ; K562 Cells ; Protein Interaction Mapping ; Receptors, Retinoic Acid ; metabolism ; Retinoic Acid Receptor alpha ; Two-Hybrid System Techniques

Humans ; Leukemia, Promyelocytic, Acute ; Oncogene Proteins, Fusion ; Retinoic Acid Receptor alpha ; STAT3 Transcription Factor ; Tretinoin

Humans ; Leukemia, Promyelocytic, Acute ; Oncogene Proteins, Fusion ; Retinoic Acid Receptor alpha ; Translocation, Genetic ; Tretinoin

Humans ; Leukemia, Promyelocytic, Acute ; genetics ; Oncogene Proteins, Fusion ; genetics ; Receptors, Retinoic Acid ; genetics ; Retinoic Acid Receptor alpha ; STAT5 Transcription Factor ; genetics

OBJECTIVETo explore the effects of TBLR1-RARα on the differentiation induction of leukemia cell line K562 cells into erythroid lineage and to investigate its related mechanisms.

METHODSTet-Off inducible system was used to construct the conditional expression vector of TBLR1-RARα fusion gene by cloning the TBLR1-RARα fragment into lentivirus vector pLVX-Tight-Puro, the expression of TBLR1-RARα fusion gene was induced by doxycycline (Dox). Then, K562 cells were transfected with lentivirus pLVX-Tight-Puro-TBLR1-RARα-flag, and the expression of fusion proteins was verified by Western blot. After treatment of K562 with all-trans retinoid acid (ATRA), real time RT-PCR was performed to test the expression of erythroid differentiation-related CD71 and α, ε, γ-globins gene. Flow cytometry was used also to analyze the expression of erythroid differentiation markers CD71 and CD235a. Benzidine staining was used to detect the production of hemoglobin in K562 cells.

RESULTSqRT-RCR showed that ATRA could increase the expression level of CD71 and α, ε, γ-globin genes when TBLR1-RARα was expressed. After treatment of ATRA, the proportion of CD71(+) cells detected by the flow cytometry also increased. Benzidine staining showed that ATRA could induce hemoglobin production in K562 cells with TBLR1-RARα fusion gene expression.

CONCLUSIONThe expression of TBLR1-RARα fusion gene contribute to ATRA-inducing differentiation of K562 cells into erythroid lineage.

Cell Differentiation ; Erythrocytes ; Hemoglobins ; Humans ; K562 Cells ; Nuclear Proteins ; Receptors, Cytoplasmic and Nuclear ; Receptors, Retinoic Acid ; Repressor Proteins ; Retinoic Acid Receptor alpha ; gamma-Globins

OBJECTIVETo investigate the effect of all-trans retinoid acid (ATRA) and granulocyte colony-stimulating factor (G-CSF) on the growth, apoptosis, differentiation and expression of RARα2 of myeloma cells.

METHODSMyeloma cell lines OPM2 (RARα2 positive) and U266 (RARα2 negative) were treated with ATRA in the presence or absence of G-CSF. The cells were divided into 6 groups: control groups, G-CSF groups (treated with 1000 U/ml and 2000 U/ml), ATRA groups (treated with 1.0 μmol/L ATRA) and combined groups (treated with 1000 U/mL or 2000 U/mL G-CSF plus 1.0 μmol/L ATRA). The cell viability, growth and apoptosis were examined by MTT method, inverted microscopy and Annexin-V/PI staining, respectively; RARα2 expression was detected by reverse transcription PCR; morphology change was evaluated by Wright-Giemsa staining; CD49e expression were analyzed by flow cytometry.

RESULTSThe proliferation of OPM2 cells was inhibited by ATRA treatment (P<0.05) . The growth inhibition rates in combined groups were higher than corresponding single ATRA groups (P<0.05). However, the above effects in U266 cells were not significant (P >0.05). The OPM2 cell stained by Wright-Giemsa in ATRA groups showed that the cell nucleus became smaller, chromatin condensed, number of nucleolus reduced, the volume of cytoplasm increased and the cytoplasm became dark blue. Expression rates of CD49e were low in both U266 and OPM2 cells. Expression of RAR&alpha;2 in OPM2 cells of combination groups were higher than those of control group and corresponding single groups (P<0.05); and there was no significant difference between control group and G-CSF groups (P>0.05). Expression of RAR&alpha;2 in U266 cells of control group and G-CSF groups was not detected; and ATRA groups and combination groups had weak expression.

CONCLUSIONATRA can induce proliferation inhibition in RAR&alpha;2-expressing myeloma cells, and it may also play a certain role in promoting differentiation of RAR&alpha;2 positive myeloma cells.

Cell Differentiation ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Humans ; Multiple Myeloma ; metabolism ; pathology ; Receptors, Retinoic Acid ; metabolism ; Retinoic Acid Receptor alpha ; Tretinoin ; pharmacology

OBJECTIVETo investigate the effect of retinoic acid on the differentiation and maturation of B cells from lymph node of children, and relational changes of the expression levels of retinoic acid receptor genes.

METHODSTwenty-four patients with digestive tract malformation underwent surgical operation in the surgical ward of our hospital. They were divided into 3 groups according to age: < 1 yr, 1 - 3 yr, -5 yr, 8 cases in each age group. The lymph nodes in the margin of excised tissues were obtained. The cells separated from lymph nodes were cultured in vitro. The cells were divided into 5 groups: Retinoic acid (RA), RA plus Ro41-5253, RA receptor antagonist (RA+Ro), lipopolysaccharide (LPS), lipopolysaccharide plus RA (LPS+RA) and control, and were subjected to the corresponding treatments. After 24 h and 48 h of cell culture, the surface markers on the B cells were detected by flow cytometer to observe the maturation and activation of B cells. The expression levels of retinoic acid receptor genes were quantitatively analyzed by RT- fluorescent quantitative PCR.

RESULTSAfter culture, in < 1 yr group, the number of the mature B cells (IgM(+)IgD(+)CD25(-)) in the RA group was significantly higher than that in the control (24 h: 23% +/- 5% vs. 17% +/- 3%; 48 h: 28% +/- 6% vs. 22% +/- 4%) (P < 0.05), and that in the RA + RO group was not significantly different from that in the control (24 h: 16% +/- 4%; 48 h: 20% +/- 9%) (P > 0.05). The number of activated B cells (IgM(+)CD25(+)) in the LPS + RA group was obviously higher than that in the LPS group (24 h: 82% +/- 10% vs. 76% +/- 8%; 48 h: 83% +/- 8% vs. 78% +/- 10%)(P < 0.05). The level of RARalpha gene expression of B cells (lg copies/50 ng RNA) in the RA group was significantly higher than that in the control (24 h: 7.03 +/- 1.36 vs. 5.79 +/- 2.05; 48 h: 7.91 +/- 1.60 vs. 6.21 +/- 1.88) (P < 0.05), and that in the LPS + RA group was significantly higher than that in the LPS group (24 h: 7.29 +/- 1.53 vs. 5.98 +/- 1.48; 48 h: 7.83 +/- 1.66 vs. 5.79 +/- 2.36)(P < 0.05). In 1 - 3 yr group the changes of maturation and activation of B cells in the lymph nodes were the same as the < 1 yr group. In -5 yr group, such changes were not significant.

CONCLUSIONRA can promote the development of B cell from lymph node in vitro culture in retinoic acid receptor genes may take part in the ontogenesis of B cell, and play a key role in the regulation of retinoic acid.

B-Lymphocytes ; drug effects ; physiology ; Cells, Cultured ; Child, Preschool ; Female ; Humans ; Infant ; Lymph Nodes ; drug effects ; Lymphocyte Activation ; drug effects ; Male ; Receptors, Retinoic Acid ; genetics ; Retinoic Acid Receptor alpha ; Tretinoin ; pharmacology

This study was aimed to investigate the mechanism of all-trans retinoic acid (ATRA) up-regulating apelin expression in vascular smooth muscle cells (VSMCs). The effect of ATRA on apelin expression in the VSMCs was investigated by RT-PCR, real-time PCR and Western blot analysis. To further define whether retinoic acid receptor &alpha; (RAR&alpha;) mediated the induction of apelin by ATRA, endogenous RAR&alpha; was down regulated by transfection of siRNA against RAR&alpha; (si-RAR&alpha;) or RAR&alpha; was over-expressed by infection of the adenovirus vector pAd-GFP-RAR&alpha; in the VSMCs. The results showed that ATRA significantly induced apelin expression in a time- and dose-dependent manner in the VSMCs. Although RAR&alpha; expression was increased in a time-dependent manner, the expressions of RARβ and RARγ were little changed by the ATRA treatment. When VSMCs were treated with a RAR&alpha; antagonist Ro 41-5253 prior to the addition of ATRA, or si-RAR&alpha; was used to down regulate endogenous RAR&alpha; expression, the blockade of RAR&alpha; signaling partially reduced the response of apelin to ATRA. Moreover, RAR&alpha; over-expression, induced by infection of pAd-GFP-RAR&alpha;, further increased the induction of apelin by ATRA. In conclusion, ATRA may up-regulate apelin expression in VSMCs, and the mechanism may be RAR&alpha; dependent.
Benzoates ; Chromans ; Gene Expression Regulation ; Intercellular Signaling Peptides and Proteins ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptors, Retinoic Acid ; metabolism ; Retinoic Acid Receptor alpha ; Signal Transduction ; Transfection ; Tretinoin ; metabolism ; Up-Regulation

OBJECTIVES: To study the mechanism of retinoic acid receptor α (RARα) signal change to regulate neurexin 1 (NRXN1) in the visual cortex and participate in the autistic-like behavior in rats with vitamin A deficiency (VAD). METHODS: The models of vitamin A normal (VAN) and VAD pregnant rats were established, and some VAD maternal and offspring rats were given vitamin A supplement (VAS) in the early postnatal period. Behavioral tests were performed on 20 offspring rats in each group at the age of 6 weeks. The three-chamber test and the open-field test were used to observe social behavior and repetitive stereotyped behavior. High-performance liquid chromatography was used to measure the serum level of retinol in the offspring rats in each group. Electrophysiological experiments were used to measure the long-term potentiation (LTP) level of the visual cortex in the offspring rats. Quantitative real-time PCR and Western blot were used to measure the expression levels of RARα, NRXN1, and N-methyl-D-aspartate receptor 1 (NMDAR1). Chromatin co-immunoprecipitation was used to measure the enrichment of RARα transcription factor in the promoter region of the NRXN1 gene. RESULTS: The offspring rats in the VAD group had autistic-like behaviors such as impaired social interactions and repetitive stereotypical behaviors, and VAS started immediately after birth improved most of the behavioral deficits in offspring rats. The offspring rats in the VAD group had a significantly lower serum level of retinol than those in the VAN and VAS groups (P<0.05). Compared with the offspring rats in the VAN and VAS groups, the offspring rats in the VAD group had significant reductions in the mRNA and protein expression levels of NMDAR1, RARα, and NRXN1 and the LTP level of the visual cortex (P<0.05). The offspring rats in the VAD group had a significant reduction in the enrichment of RARα transcription factor in the promoter region of the NRXN1 gene in the visual cortex compared with those in the VAN and VAS groups (P<0.05). CONCLUSIONS RARα affects the synaptic plasticity of the visual cortex in VAD rats by regulating NRXN1, thereby participating in the formation of autistic-like behaviors in VAD rats.
Animals ; Autistic Disorder ; Female ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate ; Retinoic Acid Receptor alpha ; Visual Cortex ; Vitamin A ; Vitamin A Deficiency

Objective: To investigate the genetic screening methods for cryptic acute promyelocytic leukemia (APL) to further explore its clinical prognosis. Methods: From June 2016 to November 2018, we collected 373 newly diagnosed APL cases. The patients were retrospected by the results of PML-RARα detections both by RT-PCR and i-FISH, those who harbored positive PML-RARα detection by RT-PCR and negative by i-FISH were chosen. Metaphase FISH and Sanger sequencing were further performed to verify these results. Results: A total of 7 cryptic APL cases were discovered. These cases had tiny fragment of RARα inserted into PML in chromosome 15, formed ins (15;17) . The 7 cryptic APL cases had no PML-RARα gene subtype specificity, involving 5 cases in L subtype, 1 case in S subtype and 1 case in V subtype respectively. After the treatment of retinoic acid and arsenic or anthracyclines, 6 cases achieved complete remission, 1 case died of intracranial hemorrhage on the 6th day of therapy. Conclusion: The size and covering position of PML-RARα probe should be taken into account when PML-RARα was performed by FISH on APL patients. Furthermore, combination with Metaphase FISH could improve the recognition of cryptic APL. There were no differences between the cryptic and common APL patients in terms of clinical features and treatment choices. Cryptic APL patients also had a good response to the therapy of retinoic acid and arsenic or anthracyclines.
Chromosomes, Human, Pair 15 ; Chromosomes, Human, Pair 17 ; Cytogenetics ; Humans ; In Situ Hybridization, Fluorescence ; Leukemia, Promyelocytic, Acute/genetics* ; Oncogene Proteins, Fusion ; Retinoic Acid Receptor alpha ; Tretinoin