Department of Biochemistry and Molecular Biology, Guangxi Medical University, 22 Shuangyong Road, Nanning 530021, the Guangxi Zhuang Autonomous Region, China.
Guangxi Key Laboratory of Biological Targeting Diagnosis and Therapy Research, Guangxi Medical University, 22 Shuangyong Road, Nanning 530021, the Guangxi Zhuang Autonomous Region, China.
Int J Med Sci. 2018 Jan 1;15(1):69-76. doi: 10.7150/ijms.21113. eCollection 2018.
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with a high rate of mortality. Our previous study shows the expression of calponin 2 (CNN2) is up-regulated in hepatocellular carcinoma tissues, especially in metastatic ones. To better understand the role of CNN2 in HCC, RNA interference (RNAi) was used to explore its role in tumor growth and metastasis. Lentivirus-mediated CNN2-shRNA was transfected into SK-hep-1 cells, and the efficacy of CNN2 expression, cell migration, invasion, proliferation and cell cycles were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB), Transwell assay, methyl thiazol tetrazolium assay and flow cytometry, respectively. SK-hep-1 cells transfected with Lentivirus-CNN2 shRNA were xenografted in Balb/C nude mice to explore the effect of CNN2-shRNA in tumor growth. Xenograft tumor tissues were examined for their histopathology, cell apoptosis, the expression of total protein and their corresponding phosphorylated protein of MEK1/2, ERK1/2, AKT, by hematoxylin and eosin stain (H & E staining), TUNEL assay, immunohistochemical technique, respectively. Our research shows it is evident that CNN2 shRNA can effectively down-regulate the expressions of CNN2 mRNA and protein, inhibit cell proliferations, arrest cell cycles at the S phase and reduce cell migration and invasion. SK-hep-1 cells with CNN2 down-regulation have markedly attenuated tumor growth in nude mice. Xenograft tumor tissues have displayed typical tumor characteristics and no apoptosis is detected in shRNA group or in control group. No metastatic tumor was found in any group of nude mice. With CNN2 protein down-regulation, the protein of pMEK1/2 and pERK1/2 are effectively down-regulated, except pAKT, AKT, MEK1/2 and ERK1/2. CNN2 plays an important role in tumor growth and metastasis, possibly through MEK1/2-ERK1/2 signaling pathway. Our study illustrate that CNN2 might be a potential target in HCC molecular target therapy.
肝细胞癌（HCC）是最常见的恶性肿瘤之一，死亡率很高。我们之前的研究表明，钙调节蛋白 2（CNN2）在肝癌组织中表达上调，尤其是在转移性肝癌组织中。为了更好地了解 CNN2 在 HCC 中的作用，我们使用 RNA 干扰（RNAi）技术来探索其在肿瘤生长和转移中的作用。慢病毒介导的 CNN2-shRNA 转染 SK-hep-1 细胞，通过实时定量聚合酶链反应（qRT-PCR）、Western blot（WB）、Transwell 实验、噻唑蓝比色法（MTT 法）和流式细胞术分别评估 CNN2 表达、细胞迁移、侵袭、增殖和细胞周期的变化。将转染慢病毒-CNN2 shRNA 的 SK-hep-1 细胞移植到 Balb/C 裸鼠中，探讨 CNN2-shRNA 对肿瘤生长的影响。对移植瘤组织进行苏木精-伊红（H & E）染色、TUNEL 检测、免疫组化技术，分别检测组织病理学变化、细胞凋亡、MEK1/2、ERK1/2、AKT 总蛋白及其相应磷酸化蛋白的表达。我们的研究表明，CNN2 shRNA 可有效下调 CNN2 mRNA 和蛋白的表达，抑制细胞增殖，将细胞周期阻滞在 S 期，并减少细胞迁移和侵袭。下调 CNN2 表达的 SK-hep-1 细胞在裸鼠体内显著抑制肿瘤生长。移植瘤组织显示出典型的肿瘤特征，shRNA 组和对照组均未检测到细胞凋亡。裸鼠各组均未发现转移性肿瘤。CNN2 蛋白下调后，pMEK1/2 和 pERK1/2 蛋白表达也明显下调，而 pAKT、AKT、MEK1/2 和 ERK1/2 蛋白表达则无明显变化。CNN2 在肿瘤生长和转移中发挥重要作用，可能通过 MEK1/2-ERK1/2 信号通路。我们的研究表明，CNN2 可能是 HCC 分子靶向治疗的潜在靶点。
