| BackgroundAcute leukemia is a kind of tumor diseases that are seriously harmful to the health of children, which is divided into acute lymphocytic leukemia (ALL) and acute myelogenous leukemia (AML) according to the proliferation of different types of leukemia cells. About 25-30% of acute leukemia in children are AML. Although the therapy strategies haved greatly improved in the past decades, but its chemotherapy effect, remission rates and 5-year disease-free survival rate are still poor, except the acute promyelocytic leukemia. So, the developing new therapeutic method to improve the prognosis of AML becomes a urgent problem for pediatric homological doctors.Nuclear factor of kappa B (NF-κB) is a kind of transcription factor found recent years which is closely related with cell proliferation and apoptosis. NF-κB is composed by the Rel protein family submits in the form of homologous or heterologous dimmer which exists in cytoplasm. Among them, p50/p65 is one kind of NF-κB which is found earliest, distributes widest and plays important roles. It can induce transcription by specifically combine with diverse promoters'or enhancers' sequence and work in many pathophysiological processes such as immune response, inflammatory reaction, the proliferation and apoptosis of cells and so on. Recent years, the inappropriate activation of NF-κB is closely related with the occurrence, proliferation,migrationn,and drug resistence in hematologic systemic tumor, and how to regulate and control the NF-κB is becoming a hotspot in anti-tumor therapy.Recent years, the newly RNA interference (RNAi) technology provides a new means to research the function of NF-κB. RNA interference, as one means of gene therapy, is a process that the mRNA degrades specifically and the gene which the mRNA codes cannot express, becomes silence through transfecting the complementary double-strand RNA with the endogenous mRNA into the cell artificially. The RNAi technology owns the character of high efficiency, specificity, economical and so on.Acute monocytic leukemia is one type of AML in children whose complete remission rate is low and the prognosis is bad. It mainly manifests malignant proliferation and blocked apoptosis of abnomal original and naive mononuclear cells. In the preliminary experiment of our research group, we found that NF-κB is activated continually in THP-1 cell lines. That gives us an idea that we can use RNAi technology to inhibit the expression of NF-κB to induce the THP-1 cell lines apoptosis, and it may become a new gene target for the therapeutic method to treat AML in vitro experiment.ObjectiveUse the specific NF-κB p65 siRNA to interference the THP-1 cell lines, and observe the effect and possible mechanism of cell proliferation and apoptosis on THP-1 cell lines.MethodsThe THP-1 cell was cultured in vitro and divided the cell when they are in exponential growth phase into three groups①blank group, dealing with PBS solution;②negative control group, dealing with control siRNA;③experimental group, dealing with specific NF-κB p65 siRNA. The expression level of p65, IκB and CyclinD1 mRNA were determined by reverse transcription polymerase chain reaction (RT-PCR), and the expression level of p65, IκB and CyclinDl were detected by western blot. The apoptosis of the THP-1 cell lines was detected by Annexin V/PI and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL). The proliferation of the THP-1 cell was obtained by cell count kit-8.Results1 Control siRNA to monitor the efficiency in THP-1 cell lines:A fluorescein-labeled control siRNA was used to monitor the efficiency in THP-1 cell, demonstrating approximately 60% transfection efficiency after transfection at 24h.2 The expression level of NF-κB p65 mRNA and protein decreased in THP-1 cell after p65 siRNA transfection:the result of the RT-PCR indicated that NF-κB p65 mRNA expression of the experimental group decreased and the decrease was highest at 48 hours after interference, the inhibition ratio is (75.52±4.02)%. The expression level of the blank group and negative group had not visible change. The result of the Western Blot indicated that after interference 48 hours, the NF-κB p65 protein expression in blank group, negative group and experimental group are2.41±0.11 2.33±0.09,0.82±0.10 respectively. In the experimental group, the expression level of p65 protein decreased most obvious, and the inhibition ratio is (74.68±5.27)%. Both the results manifest that NF-κB p65 siRNA can inhibit the expression of p65 mRNA and protein, and the most inhibitory ratio reaches peak at 48 hours.3 The expression level of CyclinD1 mRNA and protein decreased in THP-1 cell after p65 siRNA transfection:after interference 48 hours, the expression level of CyclinDl mRNA and protein in experimental group decreased obviously, thus in blank and negative group didn't change distinctly. We can get a conclusion that the expression level of CyclinD1 mRNA and protein decreased simultaneously following that the change of p65 mRNA and protein after transfection with NF-κB p65 siRNA.4 The expression level of IκBαmRNA and protein increased in THP-1 cell after p65 siRNA transfection:after interference 48 hours, the expression level of IκBαmRNA in experimental group decreased obviously, and the expressional relative content is (1.25±0.05), thus in blank and negative group didn't change distinctly, the IκBαprotein expression in blank°group, negative group and experimental group are0.76±0.06,0.81±0.02,1.14±0.06 respectively. From the results we find that the expression level of IκBαmRNA and protein increased after transfection with NF-κB p65 siRNA.5 The cell apoptosis rate raised in THP-1 cell after p65 siRNA transfection:the apoptosis of THP-1 cell lines at 48 hours in the experiment group increased, the apoptosis ratio is (8.49±0.14)%, thus in the blank group and negative group didn't change any more(P<0.05), by the method of flow cytometry. Also in the outcome of TUNEL displayed that the apoptosis of THP-1 cell lines in the experiment group increased after the NF-κB p65 siRNA inference, the apoptosis ratio is (16.47±1.75)%, yet the apoptosis in blank group (4.30±1.47)% and the negative group (3.69±0.86)% did not change markedly at 48 hours (P<0.05).6 The growth of THP-1 cell become slower and the proliferation was inhibited after p65 siRNA interference:in the result of cell count kit-8 used to detect the vitality of THP-1 cell indicated that in the 2nd day, in the experiment group the proliferation became slower than that in the blank group and negative group, lacking the character of exponential growth, thus this situation did not happen in the blank group and negative group. After the 2nd day, the cell proliferated as normal, showing up the character of exponential growth. This result shows that NF-κB p65 siRNA can inhibit the proliferation of THP-1 cell lines.Conclusion:The specific NF-κB p65 siRNA can inhibit the expression of NF-κB p65 effectively, and inhibit the proliferation and induce the apoptosis of THP-1 cell lines; NF-κB p65 may become a potential target in gene therapy for acute monocytic leukemia in children. |
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