Shi Juan-Juan, Wang Yi-Kai, Wang Mu-Qi, Deng Jiang, Gao Ning, Li Mei, Li Ya-Ping, Zhang Xin, Jia Xiao-Li, Liu Xiong-Tao, Dang Shuang-Suo, Wang Wen-Jun
Department of Infectious Diseases, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, Shaanxi Province, China.
Department of Operating Room, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, Shaanxi Province, China.
World J Gastrointest Oncol. 2024 Feb 15;16(2):398-413. doi: 10.4251/wjgo.v16.i2.398.
Prohibitin 1 (PHB1) has been identified as an antiproliferative protein that is highly conserved and ubiquitously expressed, and it participates in a variety of essential cellular functions, including apoptosis, cell cycle regulation, proliferation, and survival. Emerging evidence indicates that PHB1 may play an important role in the progression of hepatocellular carcinoma (HCC). However, the role of PHB1 in HCC is controversial.
To investigate the effects of PHB1 on the proliferation and apoptosis of human HCC cells and the relevant mechanisms .
HCC patients and healthy individuals were enrolled in this study according to the inclusion and exclusion criteria; then, PHB1 levels in the sera and liver tissues of these participates were determined using ELISA, RT-PCR, and immunohistochemistry. Human HepG2 and SMMC-7721 cells were transfected with the pEGFP-PHB1 plasmid and PHB1-specific shRNA (shRNA-PHB1) for 24-72 h. Cell proliferation was analysed with an MTT assay. Cell cycle progression and apoptosis were analysed using flow cytometry (FACS). The mRNA and protein expression levels of the cell cycle-related molecules p21, Cyclin A2, Cyclin E1, and CDK2 and the cell apoptosis-related molecules cytochrome C (Cyt C), p53, Bcl-2, Bax, caspase 3, and caspase 9 were measured by real-time PCR and Western blot, respectively.
Decreased levels of PHB1 were found in the sera and liver tissues of HCC patients compared to those of healthy individuals, and decreased PHB1 was positively correlated with low differentiation, TNM stage III-IV, and alpha-fetoprotein ≥ 400 μg/L. Overexpression of PHB1 significantly inhibited human HCC cell proliferation in a time-dependent manner. FACS revealed that the overexpression of PHB1 arrested HCC cells in the G0/G1 phase of the cell cycle and induced apoptosis. The proportion of cells in the G0/G1 phase was significantly increased and the proportion of cells in the S phase was decreased in HepG2 cells that were transfected with pEGFP-PHB1 compared with untreated control and empty vector-transfected cells. The percentage of apoptotic HepG2 cells that were transfected with pEGFP-PHB1 was 15.41% ± 1.06%, which was significantly greater than that of apoptotic control cells (3.65% ± 0.85%, < 0.01) and empty vector-transfected cells (4.21% ± 0.52%, < 0.01). Similar results were obtained with SMMC-7721 cells. Furthermore, the mRNA and protein expression levels of p53, p21, Bax, caspase 3, and caspase 9 were increased while the mRNA and protein expression levels of Cyclin A2, Cyclin E1, CDK2, and Bcl-2 were decreased when PHB1 was overexpressed in human HCC cells. However, when PHB1 was upregulated in human HCC cells, Cyt C expression levels were increased in the cytosol and decreased in the mitochondria, which indicated that Cyt C had been released into the cytosol. Conversely, these effects were reversed when PHB1 was knocked down.
PHB1 inhibits human HCC cell viability by arresting the cell cycle and inducing cell apoptosis activation of the p53-mediated mitochondrial pathway.
禁止素1（PHB1）已被鉴定为一种抗增殖蛋白，高度保守且广泛表达，它参与多种重要的细胞功能，包括细胞凋亡、细胞周期调控、增殖和存活。新出现的证据表明，PHB1可能在肝细胞癌（HCC）进展中起重要作用。然而，PHB1在HCC中的作用存在争议。
研究PHB1对人肝癌细胞增殖和凋亡的影响及其相关机制。
根据纳入和排除标准招募HCC患者和健康个体；然后，使用酶联免疫吸附测定（ELISA）、逆转录-聚合酶链反应（RT-PCR）和免疫组织化学法测定这些参与者血清和肝组织中的PHB1水平。将人HepG2和SMMC-7721细胞用pEGFP-PHB1质粒和PHB1特异性短发夹RNA（shRNA-PHB1）转染24 - 72小时。用MTT法分析细胞增殖。使用流式细胞术（FACS）分析细胞周期进程和凋亡。分别通过实时PCR和蛋白质免疫印迹法测量细胞周期相关分子p21、细胞周期蛋白A2（Cyclin A2）、细胞周期蛋白E1（Cyclin E1）和细胞周期蛋白依赖性激酶2（CDK2）以及细胞凋亡相关分子细胞色素C（Cyt C）、p53、Bcl-2、Bax、半胱天冬酶3（caspase 3）和半胱天冬酶9的mRNA和蛋白质表达水平。
与健康个体相比，HCC患者血清和肝组织中PHB1水平降低，且PHB1降低与低分化、TNM III - IV期和甲胎蛋白≥400μg/L呈正相关。PHB1的过表达以时间依赖性方式显著抑制人肝癌细胞增殖。FACS显示，PHB1的过表达使HCC细胞停滞在细胞周期的G0/G1期并诱导凋亡。与未处理的对照细胞和空载体转染细胞相比，用pEGFP-PHB1转染的HepG2细胞中G0/G1期细胞比例显著增加，S期细胞比例降低。用pEGFP-PHB1转染的凋亡HepG2细胞百分比为15.41%±1.06%，显著高于凋亡对照细胞（3.65%±0.85%，P<0.01）和空载体转染细胞（4.21%±0.52%，P<0.01）。SMMC-7721细胞也获得了类似结果。此外，当PHB1在人肝癌细胞中过表达时，p53、p21、Bax、caspase 3和caspase 9的mRNA和蛋白质表达水平升高，而Cyclin A2、Cyclin E1、CDK2和Bcl-2的mRNA和蛋白质表达水平降低。然而，当PHB1在人肝癌细胞中上调时，Cyt C在细胞质中的表达水平升高，而在线粒体中的表达水平降低，这表明Cyt C已释放到细胞质中。相反，当PHB1被敲低时，这些作用被逆转。
PHB1通过使细胞周期停滞并诱导细胞凋亡，激活p53介导的线粒体途径来抑制人肝癌细胞活力。
