Effect of Apollon siRNA combined with tetramethylpyrazine on proliferation and apoptosis of leukemia K562 cells / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the effect of small interfering RNA (siRNA) silencing Apollon gene combined with tetramethylpyrazine (TMP) on the proliferation and apoptosis of human chronic myeloid leukemia cell line K562.</p><p><b>METHODS</b>K562 cells were divided into blank control, negative control, and RNA interference (RNAi) group. For the RNAi group, the pGPHI-GFP-Neo-Apollon eukaryotic expression vector based on the best Apollon siRNA fragments screened out in previous experiments was constructed; the blank control group received no treatment, and the negative control group was transfected with negative plasmid vector. The mRNA and protein expression of Apollon was measured by RT-PCR and cell immunofluorescence, respectively. Additionally, TMP (320 μg/mL) was applied to set TMP, TMP+negative control, and TMP+RNAi groups. The cell viability and apoptosis rate were determined by MTT assay and flow cytometry, respectively.</p><p><b>RESULTS</b>The constructed vector was stably expressed in K562 cells. The RNAi group had significantly lower mRNA and protein expression of Apollon than the blank control group and negative control (P<0.05). The RNAi group had significantly increased proliferation inhibition rate and apoptosis rate, as compared with the blank contorl group (P<0.05). The TMP+RNAi group had significantly increased proliferation inhibition rate and apoptosis rate, as compared with the RNAi, and TMP groups (P<0.05).</p><p><b>CONCLUSIONS</b>Apollon siRNA can significantly inhibit the proliferation and promote the apoptosis of K562 cells, and the addition of TMP can further increase the proliferation inhibition rate and apoptosis rate, suggesting that siRNA technology combined with drugs has a significant potential value in the treatment of leukemia.</p>

Expression of homeobox gene HOXA9 in childhood acute leukemia, and its clinical significance / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the expression of homeobox gene HOXA9 in the bone marrow mononuclear cells of children with acute leukemia (AL) and its clinical significance.</p><p><b>METHODS</b>Forty-six children with AL were divided into acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) groups. Fifteen children with idiopathic thrombocytopenic purpura were selected as a control group. The mRNA expression of HOXA9 was measured by reverse transcription polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>HOXA9 expression was detected in 63% of the 52 bone marrow samples from 46 AL children. The positive HOXA9 expression rate in the AML group was significantly higher than in the ALL and control groups (86% vs 35% and 13%; P<0.05). The mRNA expression of HOXA9 in the AML group was significantly higher than in the ALL and control groups (P<0.05). Among the children with AML, those with M5 AML had the highest HOXA9 mRNA level, followed by children with M4 AML and children with M1 and/or M2 AML, but HOXA9 expression was not detected in children with M3 AML. The high-risk subgroup of AML children had relatively high levels of HOXA9 expression. In the children with AML, the initial treatment subgroup had significantly higher positive HOXA9 expression rate and HOXA9 mRNA levels than in the remission subgroup and control group (P<0.05), but there were no significant differences between the latter two groups (P>0.05). The non-remission subgroup had significantly higher HOXA9 expression than the remission subgroup and control group (P<0.05).</p><p><b>CONCLUSIONS</b>High expression of HOXA9 is associated with the occurrence of AL, and its expression level is significantly higher in children with AML than in those with ALL. There is a positive correlation between the expression level of HOXA9 and the risk of childhood leukemia, and high expression of HOXA9 suggests poor prognosis. Therefore, HOXA9 can be used as one of the indices in the diagnosis, treatment and prognosis prediction of childhood AL.</p>

Effect of astragalus injection on U937 leukemia cells proliferation and apoptosis and relevant molecular mechanisms / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the effect of astragalus injection on U937 leukemia cells proliferation and apoptosis and relevant molecular mechanisms.</p><p><b>METHODS</b>Leukemia cell line U937 cells were treated with different concentrations of astragalus (62.5, 125, 250, 500, 1 000 μg/mL). The U937 cells without astragalus treatment were used as the control group. The ability of cell proliferation was measured by MTT method. Flow cytometry was used to explore cell apoptosis. The cell morphology changes were observed under a fluorescent microscope by dyeing Hoechst33258. mRNA expression of c-myc and p27 in U937 cells which was exposed in 1 000 μg/mL astragalus after 0, 12, 24 and 48 hours was detected by reverse transcription polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>Various concentrations of astragalus injection inhibited U937 cell proliferation effectively compared with the control group (P<0.05). They also induced U937 cells apoptosis and the apoptosis rate reached to (63 ± 4)% in the 1 000 μg/mL astragalus treatment group. mRNA expression level of c-myc was gradually declined and p27 mRNA expression was gradually increased with astragalus treatment time (P<0.01).</p><p><b>CONCLUSIONS</b>Astragalus injection may inhibit proliferation and induce apoptosis of leukemia cell line U937 in vitro. This contributes to down-regulation of c-myc expression and up-regulation of p27 expression.</p>

Effects of Apollon antisense oligonucleotide on proliferation and apoptosis of K562 cells / 中华实用儿科临床杂志

Objective To investigate the effects of Apollon antisense oligonucleotide (ASODN) on proliferation,apoptosis and drug resistance of human leukemia(K562) cells.Methods Specific phosphorothioate ASODN and missense oligonucleotide (MSODN) of Apollon mRNA were synthesized and transfected into K562 cells following cationic liposome.The proliferation inhibition of K562 cells was assessed by MTT.The apoptosis rate was detected by Annexin V-FITC.The sensitivity of K562 cells to etoposide and vincristine was detected by MTT.Results Apollon antisense oligonucleotide inhibition of K562 cells with the concentration and time increased.ASODN at a final concentration of 600 nmol/L could significantly inhibit the K562 cell proliferation.The apoptosis rate was apparently increased (P ＜ 0.01).Conclusions Apollon ASODN may decrease Apollon gene expression,suppress K562 cells proliferation effectively,and induce significant apoptosis of K562 cells.Apollon ASODN is able to reverse the drug resistance via inhibition of Apollon expression and inducement of apoptosis.

Effect of HOXA10 gene silenced by shRNA on proliferation and apoptosis of U937cell line / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the effects of lentivirus-mediated RNA interference targeting HOXA10 gene on the proliferation, apoptosis and morphology of leukemic cell line U937.</p><p><b>METHODS</b>Four different shRNA plasmids were designed and built to interfere with HOXA10 gene. The four interference plasmids were transfected into 293T cells with the HOXA10 over expression plasmid and then the RNAi efficiency of the four interference plasmids was determined by Western blot. The best one was chosen to transfect 293T cells with lentiviral helping plasmids to produce packaged lentivirus (lenti-shHOXA10). U937 cells were divided into interference group (lenti-shHOXA10), negative control group and untreated group. After infection with the packaged lentivirus, infection efficiency of lentivirus for U937 was detected by flow cytometry, and the expression of HOXA10 gene mRNA and protein was detected by real-time PCR and Western blot. Cell survival was determined by MTT assay. Apoptosis rate was detected by flow cytometry.</p><p><b>RESULTS</b>Lentiviral-shRNA vector of HOXA10 gene was successfully constructed. Compared with the negative control and untreated groups, mRNA level of HOXA10 decreased by (92.3±1.3)%, protein levels decreased by 91.1%, and the inhibition rate of U937 cells [(43.9±0.7)%] increased in the interference group (P<0.05). Wright's staining showed that the ratio of karyon to cytoplasm was reduced and mitotic phase was rare in the interference group. Apoptosis rate in the interference group [(27.1±1.4)%] was significantly higher than in the negative [(19.4±1.9)%] and untreated groups [(5.5±1.3)%] (P<0.05).</p><p><b>CONCLUSIONS</b>Lentivirus mediated RNAi can reduce the expression level of HOXA10, effectively inhibit proliferation and promote apoptosis of U937 cells. HOXA10 gene is expected to become a new target for the treatment of leukemia at gene level.</p>

Effects of bone marrow stromal cells and VLA-4 antibody on apoptosis of childhood leukemia cells / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the protective effect of bone marrow stromal cells (BMSCs) upon childhood leukemia cells and the influence of VLA-4 antibody in vitro on leukemia cell apoptosis.</p><p><b>METHODS</b>BMSCs from children with acute leukemia-were isolated by human lymphocyte separation medium. BMSCs (adherent) and leukemia cells (suspended) were cultured in vitro. This study included four groups: leukemia cells alone (control), leukemia cells+BMSCs, leukemia cells+BMSCs supernatant and leukemia cells+BMSCs+VLA-4 antibody. The apoptosis rate of leukemia cells in the four groups was determined by Annexin Ⅴ-FITC double-labeled flow cytometry. The expression of survivin and bcl-2 genes in leukemia cells was ascertained by RT-PCR.</p><p><b>RESULTS</b>The apoptosis rate of leukemia cells in the leukemia cells+BMSCs and the leukemia cells+BMSCs supernatant groups was lower than that in the control group (P<0.05). Compared with the leukemia cells+BMSCs and the leukemia cells+BMSCs supernatant groups, the apoptosis rate of leukemia cells in the VLA-4 antibody group increased significantly (P<0.05). In the VLA-4 antibody group, the apoptosis rate of leukemia cells increased with prolonged culture time. There were significant differences in the apoptosis rate between 12 hrs and 24 hrs after VLA-4 antibody treatment (P<0.01). The expression of survivin and bcl-2 genes in leukemia cells from the VLA-4 antibody groups was reduced compared with that from the leukemia cells+BMSCs and the leukemia cells+BMSCs supernatant groups (P<0.05).</p><p><b>CONCLUSIONS</b>BMSCs play protective roles on leukemia cells. VLA-4 antibody can block the adhesion between BMSCs and leukemia cells and promote leukemia cell apoptosis.</p>