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

YANG Wei; LIANG Xiahai; XIA Huidong; BAI Ru

doi:10.3969/j.issn.1006-5725.2025.20.005

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.