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

Abstract

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

CLC Number:

R735.7

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.

Figures/Tables 13

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References 27

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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

The expression of TUBB2A in HCC and its effect on malignant biological behaviors of HCC cells under hypoxia

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组别	相对表达量
组别	GLUT1	PKM2	LDHA
Vector	0.58 ± 0.01	0.76 ± 0.01	0.46 ± 0.01
TUBB2A-OE	0.82 ± 0.01^*	0.86 ± 0.00^*	0.90 ± 0.02^**
t值	-18.95	-4.48	-23.07
P值	< 0.05	< 0.05	< 0.01
LacZ	0.94 ± 0.01	1.21 ± 0.02	0.90 ± 0.01
TUBB2A-KO1	0.61 ± 0.01^***	0.96 ± 0.02^**	0.58 ± 0.02^***
t值	70.20	14.59	43.42
P值	< 0.001	< 0.01	< 0.001

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