微管蛋白2A在肝细胞癌中的表达及其在缺氧条件下对肝癌细胞恶性生物学行为的影响

doi:10.3969/j.issn.1006-5725.2025.20.005

摘要/Abstract

摘要：

目的分析微管蛋白2A（TUBB2A）在肝细胞癌中的表达及预后价值，检测TUBB2A在缺氧微环境中对肝癌细胞恶性生物学行为的影响。方法利用癌症基因组图谱（TCGA）数据库和定量PCR分析TUBB2A在肝细胞癌组织和癌旁组织中的表达及其与肝细胞癌预后的关系。在常氧和缺氧条件下处理肝癌细胞HepG2和Hep3B，采用定量PCR和Western blot检测TUBB2A的mRNA和蛋白表达水平。通过慢病毒感染技术构建稳定过表达和敲除TUBB2A的肝癌细胞株HepG2和Hep3B，在缺氧条件利用CCK-8、集落形成实验、Transwell迁移与侵袭实验检测TUBB2A的表达变化对肝癌细胞增殖、迁移与侵袭的影响。Western blot检测TUBB2A对肝癌细胞中糖酵解关键蛋白，葡萄糖转运蛋白1（GLUT1）、M2型丙酮酸激酶（PKM2）和乳酸脱氢酶A（LDHA）表达的影响。结果 TCGA数据库分析显示，TUBB2A在肝癌中的表达显著高于癌旁组织（P < 0.01），TUBB2A高表达的肝细胞癌患者的总生存率更低（P < 0.01）。TUBB2A在30例临床肝细胞癌组织标本中的mRNA表达水平高于相应癌旁组织（P < 0.05），同时TUBB2A的表达与肝癌患者的临床分期（Ⅰ期和Ⅳ期）和甲胎蛋白（AFP）水平呈正相关（P均< 0.05）。在缺氧诱导的肝癌细胞HepG2和Hep3B中，TUBB2A mRNA和蛋白表达水平均显著上升（P < 0.05，P < 0.01）。缺氧条件下，在肝癌细胞HepG2和Hep3B中过表达TUBB2A，能够促进肝癌细胞的增殖、迁移与侵袭，同时上调糖酵解关键蛋白GLUT1、PKM2和LDHA的表达；而敲除TUBB2A后，两种肝癌细胞的增殖、迁移与侵袭能力均受到抑制，GLUT1、PKM2和LDHA的表达也显著降低（P < 0.05，P < 0.01）。结论 TUBB2A在肝细胞癌中高表达，与患者预后不良呈正相关。TUBB2A促进肝癌细胞增殖、迁移、侵袭与糖酵解的作用可能与缺氧微环境相关，其可能成为肝细胞癌预后与治疗的潜在靶点。

关键词: TUBB2A, 肝细胞癌, 缺氧, 增殖与转移, 有氧糖酵解

Abstract:

Objective This study aimed to investigate the expression and prognostic significance of Tubulin Beta 2A Class IIa （TUBB2A） in hepatocellular carcinoma （HCC） and to explore its roles in modulating malignant behavior of HCC cells under hypoxia. Methods The expression of TUBB2A in HCC tissues and its correlation with patient prognosis were analyzed using data from The Cancer Genome Atlas Program （TCGA） and validated by Real-time quantitative PCR （qPCR）. HepG2 and Hep3B cells were cultured under both normoxia and hypoxia， and followed by assessment of mRNA and protein levels of TUBB2A by qPCR and Western blot. Stable TUBB2A overexpressing and knockout cell models （HepG2 and Hep3B） were established using lentiviral infection technology. The biological function of TUBB2A in HCC cell proliferation， mobility， and invasion of HCC cells under hypoxia were assessed by CCK-8 assay， colony formation assay， and Transwell migration and invasion assays. Western blot was performed to examine the impact of TUBB2A on the expression of key glycolytic proteins， including glucose transporter 1 （GLUT1）， pyruvate kinase M2 （PKM2）， and lactate dehydrogenase A （LDHA） in HCC cells under hypoxia. Results TCGA analysis revealed that TUBB2A expression is highly upregulated in HCC tissues compared to that in adjacent tissues and TUBB2A expression is associated with poorer overall survival （P < 0.01）. The mRNA expression of TUBB2A in HCC tissues from 30 HCC patients was higher than that in corresponding adjacent tissues， and TUBB2A expression is positively associated with the Ⅰ and Ⅳ clinic stage and AFP level of HCC patients （P < 0.05）. Hypoxia significantly increased TUBB2A mRNA and protein expression in HepG2 and Hep3B cells. Functional studies demonstrated that TUBB2A overexpression under hypoxia enhanced HCC cells proliferation， mobility and invasion， as well as increased the expression of GLUT1、PKM2 and LDHA （P < 0.05 or P < 0.01）. On the contrary， TUBB2A knockout under hypoxia showed opposite effects， suppressed these malignant phenotypes and downregulated glycolytic markers （P < 0.05 or P < 0.01）. Conclusions TUBB2A is significantly overexpressed in HCC and it is closely associated with poor prognosis. Our findings demonstrated that TUBB2A promotes proliferation， migration， invasion and glycolysis in HCC cells under hypoxia， suggesting its potential as a prognostic biomarker and therapeutic target of HCC.

Key words: TUBB2A, hepatocellular carcinoma, hypoxia, proliferation and metastasis, glycolysis

中图分类号:

R735.7

杨伟,梁念孩,夏会东,摆茹. 微管蛋白2A在肝细胞癌中的表达及其在缺氧条件下对肝癌细胞恶性生物学行为的影响[J]. 实用医学杂志, 2025, 41(20): 3175-3184.

Wei YANG,Xiahai LIANG,Huidong XIA,Ru. BAI. The expression of TUBB2A in HCC and its effect on malignant biological behaviors of HCC cells under hypoxia[J]. The Journal of Practical Medicine, 2025, 41(20): 3175-3184.

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临床特征	例数	TUBB2A表达		P值
临床特征	例数	低	高	P值
年龄				0.313
≥ 60岁	13	4	9
≤ 60岁	17	9	8
性别				0.502
男	23	11	12
女	7	4	3
临床分期				0.025
Ⅰ期	25	4	21
Ⅳ期	5	1	4
HBV感染				0.158
是	24	12	12
否	6	2	4
AFP				0.012
> 400 ng/mL	10	2	8
< 400 ng/mL	20	5	15
肝硬化				1.359
是	19	9	10
否	11	7	4

细胞系	TUBB2A mRNA	TUBB2A 蛋白
HepG2常氧	1.17 ± 0.21	1.01 ± 0.00
HepG2缺氧	5.94 ± 0.45^**	1.97 ± 0.31^*
t值	4.42	1.54
P值	< 0.01	< 0.05
Hep3B常氧	0.90 ± 0.15	0.99 ± 0.00
Hep3B缺氧	4.05 ± 0.36^**	2.06 ± 0.31^**
t值	3.17	1.70
P值	< 0.01	< 0.01

组别	HepG2	Hep3B
Vector	1.17 ± 0.21	1.01 ± 0.00
TUBB2A-OE	5.94 ± 0.45^**	1.97 ± 0.31^**
t值	-28.77	-11.93
P值	< 0.01	< 0.01
LacZ	0.96 ± 0.15	0.92 ± 0.04
TUBB2A-KO1	0.06 ± 0.03^***	0.03 ± 0.02^***
t值	10.54	33.32
P值	< 0.001	< 0.001
TUBB2A-KO2	0.59 ± 0.08^*	0.48 ± 0.04^***
t值	3.90	13.09
P值	< 0.05	< 0.001
TUBB2A-KO3	0.52 ± 0.04^*	0.31 ± 0.14^***
t值	5.00	7.26
P值	< 0.05	< 0.001

组别	HepG2细胞活力		Hep3B细胞活力		集落形成数量
组别	24 h	48 h	24 h	48 h	HepG2	Hep3B
Vector	0.72 ± 0.01	0.86 ± 0.02	0.56 ± 0.01	0.70 ± 0.02	35.00 ± 8.89	30.67 ± 4.16
TUBB2A-OE	0.81 ± 0.03^**	0.91 ± 0.01^*	0.63 ± 0.04^*	0.73 ± 0.03^*	74.67 ± 7.02^**	76.00 ± 4.00^**
t值	-3.72	-3.15	-2.89	-5.00	-6.12	-14.221
P值	< 0.01	< 0.05	< 0.05	< 0.05	< 0.01	< 0.01
LacZ	0.83 ± 0.02	1.13 ± 0.03	0.58 ± 0.02	0.77 ± 0.01	79.10 ± 4.45	64.90 ± 1.61
TUBB2A-KO1	0.76 ± 0.02^*	0.97 ± 0.02^**	0.53 ± 0.02^*	0.72 ± 0.01^*	68.08 ± 1.31^*	61.05 ± 2.08^*
t值	3.39	4.21	3.05	2.98	2.15	1.12
P值	< 0.05	< 0.01	< 0.01	< 0.05	< 0.05	< 0.05

组别	迁移细胞数		侵袭细胞数
组别	HepG2	Hep3B	HepG2	Hep3B
Vector	117.33 ± 15.01	20.33 ± 4.73	202.33 ± 34.85	56.33 ± 16.80
LV-TUBB2A	366.00 ± 28.69^**	38.67 ± 5.03^**	382.33 ± 16.50^**	119.67 ± 17.95^**
t值	-12.89	-5.67	-7.23	-4.56
P值	< 0.01	< 0.01	< 0.01	< 0.01
LacZ	872.67 ± 25.66	526.00 ± 33.51	32.00 ± 3.61	45.00 ± 4.36
TUBB2A-KO1	655.00 ± 9.85^**	322.00 ± 41.07^**	20.67 ± 2.52^*	26.67 ± 4.73^*
t值	4.67	5.02	3.41	3.89
P值	< 0.01	< 0.01	< 0.05	< 0.05