lncRNA C9ORF139在调节急性髓系白血病细胞增殖、侵袭、迁移和凋亡中的作用及机制

Role of lncRNA C9ORF139 in regulating the proliferation,invasion,migration and apoptosis of acute myeloid leukemia cells and its mechanism

目的探讨长链非编码RNA(lncRNA)C9ORF139对急性髓系白血病(AML)细胞增殖、侵袭、迁移、凋亡的影响及可能机制。方法回顾性收集2017年11月至2021年8月常州市第二人民医院血液内科30例初发AML患者和30名健康对照者骨髓标本,采用实时荧光定量聚合酶链反应(qRT-PCR)检测各标本C9ORF139转录水平相对表达量。合成针对C9ORF139基因靶点的小干扰RNA(siRNA)序列,转染至人急性早幼粒细胞白血病HL-60细胞、人单核细胞白血病THP-1细胞,为C9ORF139敲减组,以转染阴性对照序列的siRNA的细胞为对照组;采用qRT-PCR检测各组细胞C9ORF139转录水平相对表达量,计算干扰效率。CCK8法检测各组细胞增殖能力(以吸光度值表示细胞增殖能力,吸光度值越高,细胞增殖能力越强),Transwell实验检测细胞的迁移、侵袭能力,流式细胞术检测细胞凋亡情况。依据miRanda数据库预测miRNA-24-3p(miR-24-3p)与C9ORF139和TAOK1有结合位点,双荧光素酶报告基因实验检测miR-24-3p分别与C9ORF139、TAOK1的靶向关系。结果qRT-PCR检测示,初发AML患者骨髓标本中C9ORF139的相对表达量高于健康对照组,差异有统计学意义(P<0.001)。C9ORF139敲减组HL-60、THP-1细胞C9ORF139相对表达量均低于相对应的对照组细胞,差异均有统计学意义(均P<0.01)。C9ORF139敲减组HL-60细胞和THP-1细胞增殖能力(培养72 h吸光度值:1.04±0.13比1.78±0.23,1.05±0.04比1.79±0.13)、侵袭能力(培养48 h细胞侵袭率:0.06±0.02比1.00±0.02,0.22±0.09比1.01±0.01)、迁移能力(培养48 h细胞迁移率:0.37±0.07比1.01±0.01,0.21±0.03比1.00±0.01)均低于对应的对照组,细胞凋亡率[(6.35±0.26)%比(0.58±0.55)%,(18.75±1.83%)比(2.07±0.19)%]均高于对应的对照组,差异均有统计学意义(均P<0.01)。双荧光素酶报告基因实验显示,在THP-1、HL-60细胞中,转染miR-24-3p+C9ORF139-野生型(WT)的细胞相对荧光素酶活性均低于转染miR-24-3p-阴性对照(NC)+C9ORF139-WT的细胞,差异均有统计学意义(均P<0.001),而转染miR-24-3p+C9ORF139-突变型(MUT)的细胞与转染miR-24-3p-NC+C9ORF139-MUT的细胞相对荧光素酶活性差异均无统计学意义(均P>0.05);转染miR-24-3p+TAOK13'UTR-WT的细胞荧光素酶活性均低于miR-24-3p-NC+TAOK13'UTR-WT细胞,差异均有统计学意义(均P<0.001),而转染miR-24-3p+TAOK13'UTR-MUT的细胞与转染miR-24-3p-NC+TAOK13'UTR-MUT的细胞相对荧光素酶活性差异均无统计学意义(均P>0.05)。结论lncRNA C9ORF139可能通过与miR-24-3p相互作用来调控TAOK1基因表达,进而促进AML细胞增殖、侵袭和迁移,抑制AML细胞凋亡,可为AML治疗新靶点提供参考。

Objective To explore the effects of long non-coding RNA(lncRNA)C9ORF139 on the proliferation,invasion,migration and apoptosis of acute myeloid leukemia(AML)cells and the possible mechanisms.Methods The bone marrow specimens from 30 patients with primary AML and 30 healthy controls in Changzhou No.2 People's Hospital from November 2017 to August 2021 were retrospectively collected,and the transcript level relative expression of C9ORF139 in each specimen was detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).Small interfering RNA(siRNA)sequences targeting C9ORF139 gene were synthesized,and transfected to human acute promyelocytic leukemia HL-60 cells and human monocytic leukemia THP-1 cells as C9ORF139 knockdown group,and cells transfected with negative control sequence siRNA were used as control group.The transcript level relative expression of C9ORF139 in each group was detected by qRT-PCR,and the interference efficiency was calculated.CCK8 method was used to detect the proliferation ability of cells in each group(the absorbance value indicated the proliferation ability of cells,the higher the absorbance value,the stronger the proliferation ability of cells);Transwell assay was used to detect the migration and invasion abilities of cells,and cell apoptosis was detected by flow cytometry.Based on the miRanda database,miRNA-24-3p(miR-24-3p)was predicted to have binding sites with C90RF139 and TAOK1,the dual-luciferase reporter gene assay was used to analyze the targeting relationship of miR-24-3p with C9ORF139 and TAOK1.Results qRT-PCR showed that the relative expression of C9ORF139 in bone marrow specimens of patients with primary AML was higher than that of healthy controls,and the difference was statistically significant(P<0.001).The expressions of C9ORF139 in HL-60 and THP-1 cells of the C9ORF139 knockdown group were lower than those in the corresponding control cells,and the differences were statistically significant(both P<0.01).The proliferation ability(absorbance value of cells cultured for 72 h:1.04±0.13 vs.1.78±0.23,1.05±0.04 vs.1.79±0.13),invasion ability(invasion rate of cells cultured for 48 h:0.06±0.02 vs.1.00±0.02,0.22±0.09 vs.1.01±0.01)and migration ability(migration rate of cells cultured for 48 h:0.37±0.07 vs.1.01±0.01,0.21±0.03 vs.1.00±0.01)in HL-60 and THP-1 cells of the C9ORF139 knockdown group were lower and the apoptosis rate[(6.35±0.26)%vs.(0.58±0.55)%,(18.75±1.83)%vs.(2.07±0.19)%]were higher than the corresponding control group,and the differences were all statistically significant(all P<0.01).Dual-luciferase reporter gene assay showed that in THP-1 and HL-60 cells,the luciferase activities of cells transfected with miR-24-3p+C9ORF139-wild type(WT)were all lower than those of cells transfected with miR-24-3p-negative control(NC)+C9ORF139-WT,and the differences were statistically significant(both P<0.001),while those of cells transfected with miR-24-3p+C9ORF139-mutant(MUT)were not statistically different from the relative luciferase activities of cells transfected with miR-24-3p-NC+C9ORF139-MUT(both P>0.05).The relative luciferase activities of cells transfected with miR-24-3p+TAOK13'UTR-WT were lower than those of cells transfected with miR-24-3p-NC+TAOK13'UTR-WT,and the differences were statistically significant(both P<0.001),while the differences in relative luciferase activities between cells transfected with miR-24-3p+TAOK13'UTR-MUT and cells transfected with miR-24-3P-NC+TAOK13'UTR-MUT were not statistically significant(both P>0.05).Conclusions lncRNA C9ORF139 may regulate TAOK1 gene expression by interacting with miR-24-3p and promote the cell proliferation,invasion and migration,and inhibits apoptosis of AML cells,which provides a reference for the development of new targets for the treatment of AML.