lncRNA RBM5-AS1在急性髓系白血病中的表达及其临床意义

doi:10.3969/j.issn.1673-8640.2023.01.008

摘要/Abstract

摘要：

目的探讨长链非编码RNA结合基序蛋白5-反义链1（lncRNA RBM5-AS1）在急性髓系白血病（AML）中的表达及其临床意义。方法选取2017年1月—2019年1月南阳市中心医院AML患者72例（AML组），其中经化疗得到完全缓解（CR）43例（AML-CR组）；另选取缺铁性贫血患者45例（贫血组）。采用实时荧光定量聚合酶链反应（RT-qPCR）检测各组骨髓lncRNA RBM5-AS1水平；采用受试者工作特征（ROC）曲线分析lncRNA RBM5-AS1对AML的诊断价值；采用Kaplan-Meier曲线分析lncRNA RBM5-AS1水平与AML患者预后的关系；采用Cox回归分析评估AML患者预后的影响因素。体外培养人AML细胞系HL-60，并将其随机分为对照组、sh-NC组、sh-lncRNA RBM5-AS1组，采用RT-qPCR检测各组HL-60细胞中lncRNA RBM5-AS1水平；采用CCK-8和Transwell试验检测各组HL-60细胞的增殖、侵袭、迁移情况；采用免疫印迹法检测Wnt/β-catenin通路相关蛋白（Wnt5a、β-catenin、c-Myc、Cyclin D1）表达。结果 AML组lncRNA RBM5-AS1表达高于AML-CR组和贫血组（P<0.05），AML-CR组与贫血组lncRAN RBM5-AS1表达差异无统计学意义（P>0.05）。ROC曲线分析结果显示，lncRNA RBM5-AS1诊断AML的曲线下面积为0.853，敏感性为87.50%，特异性为84.40%。根据AML患者lncRNA RBM5-AS1水平的平均值分为高表达组（37例）和低表达组（35例），2个组法美英合作组（FAB）分型、骨髓原始细胞比例、白细胞计数差异均有统计学意义（P<0.05）。lncRNA RBM5-AS1高表达组3年累计生存率显著低于低表达组（P<0.05）。多因素分析结果显示，FAB分型M4和M5型、lncRNA RBM5-AS高表达是影响AML患者预后的危险因素（P<0.05）。干扰lncRNA RBM5-AS1表达后，HL-60细胞的增殖、侵袭、迁移能力和Wnt5a、β-catenin、c-Myc、Cyclin D1蛋白表达均被抑制（P<0.05）。结论 AML患者骨髓中lncRNA RBM5-AS1水平呈高表达，可能通过调控Wnt/β-catenin通路影响细胞的增殖、迁移和侵袭，在AML诊断和预后评估中有一定的作用。

关键词: 长链非编码RNA结合基序蛋白5-反义链1, 急性髓系白血病, 预后

Abstract:

Objective To investigate the expression and clinical significance of long non-coding RNA binding motif protein 5-antisense strand 1（lncRNA RBM5-AS1） in acute myeloid leukemia（AML）. Methods Totally，72 AML patients diagnosed and treated in Nanyang Central Hospital from January 2017 to January 2019 were enrolled as AML group，43 AML patients with complete remission（CR） after chemotherapy were regarded as AML-CR group，and 45 patients with iron deficiency anemia were enrolled as anemia group. The level of lncRNA RBM5-AS1 in bone marrow of patients in each group was determined by real-time quantitative polymerase chain reaction（RT-qPCR）. The diagnostic value of lncRNA RBM5-AS1 in AML was analyzed by receiver operating characteristic（ROC） curve. The relationship between the level of lncRNA RBM5-AS1 and the prognosis of AML patients was analyzed by Kaplan-Meier curve. The influencing factors of the prognosis of AML patients were analyzed by Cox regression analysis. Human AML cell line HL-60 was cultured in vitro and randomly classified into control group，sh-NC group and sh-lncRNA RBM5-AS1 group. The level of lncRNA RBM5-AS1 in HL-60 cells was determined by RT-qPCR. CCK-8 and Transwell assay were used to determine the proliferation，invasion and migration of HL-60 cells. The expressions of Wnt/β-catenin pathway related proteins（Wnt5a，β-catenin，c-Myc and Cyclin D1） were determined by western blot. Results The expression level of lncRNA RBM5-AS1 in AML group was higher than those in AML-CR group and anemia group（P<0.05），while there was no statistical significance for lncRNA RBM5-AS1 between AML-CR group and anemia group（P>0.05）. The results of ROC curve analysis showed that the area under curve of lncRNA RBM5-AS1 in the diagnosis of AML was 0.853，with a sensitivity of 87.50% and a specificity of 84.40%. According to the average level of lncRNA RBM5-AS1，AML patients were classified into high expression group（37 cases） and low expression group（35 cases）. There was statistical significance in French-American-British （FAB）type，the proportion of bone marrow blasts and white blood cell counts between the 2 groups（P<0.05）. The 3-year cumulative survival rate of patients in high expression group of lncRNA RBM5-AS1 was lower than that in low expression group（P<0.05）. Multivariate analysis showed that FAB M4-M5 classification and high expression of lncRNA RBM5-AS1 were risk factors affecting the prognosis of AML patients（P<0.05）. Interference with lncRNA RBM5-AS1 expression inhibited the proliferation，invasion and migration，the protein expressions of Wnt5a，β-catenin，c-Myc，Cyclin D1 of HL-60 cells（P<0.05）. Conclusions The level of lncRNA RBM5-AS1 is highly expressed in the bone marrow fluid of AML patients，which may affect cell proliferation，migration and invasion by regulating Wnt/β-catenin pathway，and thus plays a role in the diagnosis and prognostic evaluation of AML.

Key words: Long non-coding RNA binding motif protein 5-antisense strand 1, Acute myeloid leukemia, Prognosis

中图分类号:

R446.7

王瑞娟, 李超, 段丽娟, 尚淼, 杨如玉. lncRNA RBM5-AS1在急性髓系白血病中的表达及其临床意义[J]. 检验医学, 2023, 38(1): 39-45.

WANG Ruijuan, LI Chao, DUAN Lijuan, SHANG Miao, YANG Ruyu. Expression and clinical significance of lncRNA RBM5-AS1 in acute myeloid leukemia[J]. Laboratory Medicine, 2023, 38(1): 39-45.

图/表 10

参考文献 23

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基因名称	正向引物（5'~3'）	反向引物（5'~3'）
lncRNA RBM5-AS1	TGGGAATGGGGAAGAGAAC	TCTGTTCCTGGGTCTGTGTGC
GAPDH	AACGGATTTGGTCGTATTG	GGAAGATGGTGATGGGATT

基因名称	正向引物（5'~3'）	反向引物（5'~3'）
lncRNA RBM5-AS1	TGGGAATGGGGAAGAGAAC	TCTGTTCCTGGGTCTGTGTGC
GAPDH	AACGGATTTGGTCGTATTG	GGAAGATGGTGATGGGATT

组别	例数	lncRNA RBM5-AS1
AML组	72	1.91±0.34^*#
AML-CR组	43	1.13±0.21
贫血组	45	1.04±0.18
F值		185.890
P值		0.000

组别	例数	lncRNA RBM5-AS1
AML组	72	1.91±0.34^*#
AML-CR组	43	1.13±0.21
贫血组	45	1.04±0.18
F值		185.890
P值		0.000

组别	例数	性别		年龄		NCCN分级		FAB分型
组别	例数	男/例	女/例	<45岁/例	≥45岁/例	低、中危/例	高危/例	M1+M2+M3/例	M4+M5/例
高表达组	37	22	15	14	23	14	23	16	21
低表达组	35	19	16	19	16	20	15	27	8
χ²值		0.196		1.96		2.69		8.593
P值		0.658		0.162		0.101		0.003
组别	CR			骨髓原始细胞比例		白细胞计数/（×10⁹/L）	红细胞计数/（×10¹²/L）	血小板计数/（×10⁹/L）	血红蛋白/（g/L）
组别	是/例	否/例		<50%/例	≥50%/例	白细胞计数/（×10⁹/L）	红细胞计数/（×10¹²/L）	血小板计数/（×10⁹/L）	血红蛋白/（g/L）
高表达组	13	24		20	17	16.42±2.38	2.49±0.27	28.35±4.53	43.82±5.69
低表达组	17	18		8	27	10.17±1.84	2.59±0.33	30.51±5.12	46.11±6.43
χ²值	1.336			7.366		12.416	1.411	1.898	1.602
P值	0.248			0.007		0	0.163	0.062	0.114