miR-181a-5p和BACH2表达对白血病CCRF-CEM细胞凋亡和侵袭的影响

doi:10.13481/j.1671-587X.20230403

摘要/Abstract

摘要：

目的探讨微小RNA-181a-5p （miR-181a-5p）靶向BTB与CNC同源基因2（BACH2）对急性淋巴细胞白血病（ALL）细胞凋亡和侵袭的调控作用，并阐明其作用机制。方法体外构建BACH2过表达重组质粒（overExpBACH2）、miR-181a-5p inhibitor和inhibitor-NC，转染人ALL T淋巴细胞CCRF-CEM，实时荧光定量PCR（RT-qPCR）法和免疫荧光染色检测转染效果。将CCRF-CEM细胞分为对照组、inhibitor-NC组、miR-181a-5p inhibitor组、BACH2过表达（overExpBACH2）组、miR-181a-5p inhibitor+空载体（EV）组和miR-181a-5p inhibitor+ overExpBACH2组。CCK-8法检测各组细胞增殖活性，流式细胞术检测G₂期细胞百分率和细胞凋亡率。Western blotting法检测各组细胞中细胞周期蛋白D3（CyclinD3）、B细胞淋巴瘤2（Bcl-2）、Bcl-2相关X蛋白（Bax）和含半胱氨酸的天冬氨酸蛋白水解酶3（Caspase-3）蛋白表达水平，Transwell小室实验检测各组细胞中侵袭细胞数，RT-qPCR法检测各组细胞中基质金属蛋白酶9（MMP-9）、基质金属蛋白酶14（MMP-14）和BACH2 mRNA及miR-181a-5p表达水平。结果 miR-181a-5p inhibitor和overExpBACH2质粒均成功转染CCRF-CEM细胞。与对照组比较，miR-181a-5p inhibitor组、overExpBACH2组、miR-181a-5p inhibitor+EV组和miR-181a-5p inhibitor+overExpBACH2组细胞增殖活性明显降低（P<0.05），G₂期细胞百分率和细胞凋亡率明显升高（P<0.05），细胞中CyclinD3蛋白表达水平明显降低（P<0.05），Caspase-3蛋白表达水平和Bax/Bcl-2比值明显升高（P<0.05），侵袭细胞数明显减少（P<0.05），细胞中MMP-9和MMP-14 mRNA及miR-181a-5p表达水平明显降低（P<0.05），BACH2 mRNA表达水平明显升高（P<0.05）。与miR-181a-5p inhibitor组和overExpBACH2组比较，miR-181a-5p inhibitor+overExpBACH2组细胞中上述指标变化趋势更明显。结论 miR-181a-5p可靶向BACH2进而调控ALL细胞侵袭和凋亡，为临床靶向治疗ALL提供了新的靶点。

关键词: 急性淋巴细胞白血病, 微小RNA-181a-5p, BTB与CNC同源基因2, 细胞凋亡, 细胞侵袭

Abstract:

Objective To discuss the regulatory effect of microRNA-181a-5p（miR-181a-5p） targeting BTB and CNC homology 2 （BACH2） on the apoptosis and invasion of the acute lymphoblastic leukemia （ALL） cells，and to clarify its mechanism. Methods The BACH2 over-expression recombinant plasmid（overExpBACH2）， miR-181a-5p inhibitor，and inhibitor-NC were constructed in vitro，and the transfection effect after transfected with human ALL T lymphocytes CCRF-CEM was detected by real-time fluorescence quantitative PCR （RT-qPCR） and immunofluorescence staining. The CCRF-CEM cells were divided into control group， inhibitor-NC group， miR-181a-5p inhibitor group， BACH2 over-expression（overExpBACH2） group， miR-181a-5p inhibitor+empty vector（EV） group， and miR-181a-5p inhibitor+ overExpBACH2 group. The proliferation activities of cells in various groups were detected by CCK-8 assay； the percentages of cells at G₂ phase and apoptotic rates of cells in various groups were detected by flow cytometry； the expression levels of CyclinD3， B-cell lymphoma-2（Bcl-2），Bcl-2-associated X protein（Bax）， and cysteinyl aspartate specific proteinase-3（Caspase-3） proteins in cells in various groups were detected by Western blotting method； the numbers of invasion cells in various groups were detected by Transwell chamber test； the expression levels of matrix metalloproteinase-9（MMP-9），matrix metalloproteinase 14（MMP-14）， BACH2 mRNA， and miR-181a-5p in cells in various groups were detectd by RT-qPCR method. Results Both miR-181a-5p inhibitor and overExpBACH2 vector were successfully transfected into the CCRF-CEM cells. Compared with control group，the proliferation activities of the cells in overExpBACH2 group and miR-181a-5p inhibitor+EV group were decreased （P<0.05）， and the percentages of the cells at G₂ phase and the apoptotic rates were increased （P<0.05）； the expression levels of CyclinD3 protein in the cells were decreased （P<0.05）， the expression levels of Caspase-3 protein and the ratios of Bax/Bcl-2 were increased （P<0.05）， the numbers of invasion cells were decreased （P<0.05）， the expression levels of MMP-9， MMP-14 mRNA，and miR-181a-5p were decreased （P<0.05），and the BACH2 mRNA expression levels were increased （P<0.05）. Compared with miR-181a-5p inhibitor group and overExpBACH2 group， the change trends of the above indexes in miR-181a-5p inhibitor+overExpBACH2 group were more obvious. Conclusion miR-181a-5p can target BACH2 and regulate the apoptosis and invasion of the ALL cells， which provides the new ideas for the clinical target therapy of ALL.

Key words: Acute lymphoblastic leukemia, MicroRNA-181a-5p, BTB and CNC homology 2, Apoptosis, Cell invasion

中图分类号:

R733.7

王旭颖,荆明箴,余谨,付荣,杨茹. miR-181a-5p和BACH2表达对白血病CCRF-CEM细胞凋亡和侵袭的影响[J]. 吉林大学学报(医学版), 2023, 49(4): 840-849.

Xuying WANG,Mingzhen JING,Jin YU,Rong FU,Ru YANG. Effects of miR-181a-5p and BACH2 expressions on apoptosis and invasion of leukemic CCRF-CEM cells[J]. Journal of Jilin University(Medicine Edition), 2023, 49(4): 840-849.

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Primer	Sequence (5'－3')
MMP-9	F：GTCCTCGCCCTGAACCTG
MMP-9	R：GGCACAGTAGTGGCCGTAGA
MMP-14	F：ATCATTGAGGTGGACGAGG
MMP-14	R：GGGAACGCTGGCAGTAG
BACH2	F：AGCAAGAACCGCATCGC
BACH2	R：TGGGAAAGGCAGGAGAAG
GAPDH	F：GGGAAACTGTGGCGTGAT
GAPDH	R：GAGTGGGTGTCGCTGTTGA
miR-181a-5p	F：GGAACATTCAACGCTGTCG
miR-181a-5p	R：CTGGTGTCGTGGAGTCGG
U6	F：CTCGCTTCGGCAGCACA
U6	R：AACGCTTCACGAATTTGCGT

Group	Proliferation activity
Group	(t/h) 24	48	72
Control	0.79±0.02	0.95±0.01	1.05±0.01
Inhibitor-NC	0.79±0.02	0.97±0.01	1.05±0.01
MiR-181a-5p inhibitor	0.69±0.01	0.76±0.02^*	0.79±0.14^*
OverExpBACH2	0.62±0.01	0.70±0.01^*	0.74±0.03^*
MiR-181a-5p inhibitor+EV	0.69±0.00	0.77±0.02^*	0.81±0.04^*
MiR-181a-5p inhibitor+overExpBACH2	0.57±0.01	0.64±0.01^*△#	0.65±0.09^*△#

Group	Percentage of cells at G₂ phase	Apoptotic rate
Control	10.72±0.26	6.58±0.52
Inhibitor-NC	10.42±0.26	7.14±0.36
MiR-181a-5p inhibitor	16.76±0.47^*	24.93±0.61^*
OverExpBACH2	19.11±0.38^*	28.35±1.69^*
MiR-181a-5p inhibitor+EV	16.45±0.24^*	24.39±0.67^*
MiR-181a-5p inhibitor+overExpBACH2	24.16±0.27^*△#	38.56±0.68^*△#

Group	MMP-9 mRNA	MMP-14 mRNA	BACH2 mRNA	miR-181a-5p
Control	1.00±0.02	1.00±0.02	1.00±0.05	1.00±0.01
Inhibitor-NC	1.07±0.10	1.03±0.10	0.90±0.10	0.98±0.03
MiR-181a-5p inhibitor	0.45±0.06^*	0.50±0.06^*	1.82±0.15^*	0.52±0.02^*
OverExpBACH2	0.28±0.02^*	0.26±0.02^*	10.13±0.32^*	0.29±0.02^*
MiR-181a-5p inhibitor+EV	0.43±0.02^*	0.41±0.02^*	2.01±0.27^*	0.49±0.06^*
MiR-181a-5p inhibitor+overExpBACH2	0.08±0.01^*△#	0.10±0.01^*△#	17.03±1.36^*△#	0.13±0.01^*△#

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