ABSTRACT
OBJECTIVE@#To explore the effects and molecular mechanism of circ-SFMBT2 on the proliferation, migration and invasion of acute myeloid leukemia (AML) cells.@*METHODS@#Bone marrow samples from 35 pediatric AML patients and 35 healthy controls in Henan Provincial Children's Hospital from April 2015 to April 2017 and human bone marrow stromal cell lines (HS-5) and AML cell lines (HL-60, THP-1, U-937 and Kasumi-1) were collected. The expressions of circ-SFMBT2, miR-491-5p and homeobox A9 (HOXA9) in bone marrow samples and cells were detected by RT-qPCR and Western blot. The Pearson method was used to analyze the correlation of circ-SFMBT2, miR-491-5p and HOXA9 mRNA expression levels in bone marrow samples of AML patients. HL-60 cells were cultured in vitro and divided into 5 groups: Control, si-NC, si-circ-SFMBT2, si-circ-SFMBT2+anti-NC and si-circ-SFMBT2+anti-miR-491-5p, HL-60 cells were transfected with si-NC, si-circ-SFMBT2, anti-NC, and miR-491-5p inhibitor with Lipofectamine™ 3000. RT-qPCR and Western blot were performed to detect the expression levels of circ-SFMBT2, miR-491-5p and HOXA9 in cells of each group. The proliferation activity of HL-60 cells in each group was detected by CCK-8 assay at 24, 48 and 72 h after transfection, respectively. The apoptosis rate was detected by flow cytometry. The migration and invasion abilities of cells were detected by Transwell assay. The regulatory roles of circ-SFMBT2, miR-491-5p and HOXA9 in AML cells were verified by dual-luciferase reporter gene assay, RNA pull-down and RNA-binding protein immunoprecipitation (RIP) experiments.@*RESULTS@#The expression levels of circ-SFMBT2 and HOXA9 mRNA were increased in bone marrow samples and cell lines (HL-60, THP-1, U-937 and Kasumi-1) of children with AML (P <0.001), while the expression level of miR-491-5p was significantly decreased (P <0.001). Pearson correlation analysis showed that the expression levels of circ-SFMBT2 and miR-491-5p in bone marrow samples of AML children were negatively correlated (r =-0.905), miR-491-5p was also negatively correlated with HOXA9 mRNA (r =-0.930), while the expression levels of HOXA9 mRNA and circ-SFMBT2 was positively correlated (r =0.911). The overall survival rate of AML children with high expression of circ-SFMBT2 was significantly decreased than those with low expression of circ-SFMBT2 (P <0.05). Silencing of circ-SFMBT2 could greatly up-regulate the expression of miR-491-5p, decrease the expression of HOXA9, inhibit the proliferation, migration and invasion of AML cells, and promote cell apoptosis (P <0.05). Down-regulation of miR-491-5p expression greatly attenuated the inhibitory effects of circ-SFMBT2 silencing on cell proliferation, migration and invasion (P <0.05). Dual-luciferase reporter gene assay, RNA pull-down and RIP experiments confirmed that circ-SFMBT2 could target miR-491-5p and negatively regulate the expression of miR-491-5p in AML, and HOXA9 was the target of miR-491-5p.@*CONCLUSION@#Silencing of circ-SFMBT2 may inhibit the proliferation, migration and invasion of AML cells by regulating the miR-491-5p/HOXA9 axis.
Subject(s)
Child , Humans , Cell Line, Tumor , Cell Proliferation , Genes, Homeobox , HL-60 Cells , Leukemia, Myeloid, Acute , Luciferases , MicroRNAs , Repressor Proteins , RNA, Messenger , RNA, Circular/geneticsABSTRACT
Objective To investigate the effects of small interference RNA (siRNA) targeting HOXA9 on the proliferation and apoptosis of human acute monocytic leukemia U937 cell line.Methods Effective and specific siRNA oligo targeting HOXA9 was designed and compounded.It was transfected transiently into U937 cells by cationic liposome.The cells was divided into three groups:experimental group(siRNA targeting HOXA9 was transfected by liposome),negative control group (negative siRNA was transfected by liposome) and cell control group (add equal cells and medium).The expression of HOXA9 mRNA and protein were detected by reverse transcription PCR and Western blot.The cell proliferation was assessed by MTT.The apoptosis of each group were measured by Annexin V-FITC.Results Aftcr transfected by siRNA targeting HOXA9,the relative mRNA expression levels of HOXA9 in the experimental group,negative control group and cell control group were (22.980±0.548) ％,(82.371±1.517) ％ and (84.637±2.252) ％,respectively (P ＜ 0.05),and the relative protein expression levels were (50.377±2.773).％,(105.500±3.900) ％ and (111.392±3.905) ％,respectively (P ＜ 0.05).The inhibitory rates of cell proliferation and the apoptosis rates of the experimental group were significantly increased.The inhibitory rates of cell proliferation of 24 h,48 h and 72 h were (41.909±4.333) ％,(54.470±3.756) ％ and (65.835±1.024) ％,respectively,and the apoatosis rate was (26.800±2.081) ％.Compared with 2 controls,the experimental group differences had statistically significance (P ＜ 0.05).Conclusion siRNA targeting HOXA9 can effectively silence HOXA9 gene expression in U937 cell,suppress cell proliferation and induce cell apoptosis obviously,which providing experimental basis for clinical lenkemia therapy by targeting HOXA9 gene.
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AIM: To observe whether transfection of mammalian expression vector pEGFP containing the gene of B-cell specific moloney leukemia virus insertion site 1(BMI-1) could express in human cervix cancer cell line HeLa, and to detect its effect on HOX family expression and cell cycle.METHODS: pEGFP-BMI-1 was transfected into HeLa cells with Lipofectamine 2000. The expression of pEGFP-BMI-1 was determined by EGFP fluorescence and Western blotting. SYBR green I real-time RT-PCR was used to quantitate mRNA expression of P16~(INK4a), hTERT, HOXA9, HOXB4 and HOXC13. FACS analysis was used to detect the change of cell cycle.RESULTS: In HeLa cells transfected with pEGFP-BMI-1, the results of real-time RT-PCR showed that the mRNA expressions of P16~(INK4a), HOXA9 and HOXC13 were reduced to 9.2%, 10.9% and 69.7%, respectively, as compared to control HeLa cells (P<0.01). However, hTERT and HOXB4 mRNA expressions did not change significantly (P>0.05). FACS analysis showed a decrease from 65.68 % to 50.53% in G_1 population and a significant increase from 27.17% to 39.59 % in S population after transfection (P<0.01).CONCLUSION: BMI-1 over-expression in HeLa cells down-regulates mRNA expressions of P16~(INK4a), HOXA9 and HOXC13, decreases G_1 population and increases S population. Therefore, BMI-1 may be involved in carcinogenesis and cancer development.