Monzavi Nastaran, Zargar Seyed Jalal, Gheibi Nematollah, Azad Mahdi, Rahmani Babak
Department of Cell & Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran, Iran.
Cellular & Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran.
Iran J Basic Med Sci. 2019 Oct;22(10):1166-1171. doi: 10.22038/ijbms.2019.36612.8764.
Hepatocellular carcinoma (HCC) is one of the leading fatal neoplasms and the most common primary liver malignancy worldwide. Peptide hormone ANGPTL8 (betatrophin) may act as an important regulator in HCC development through the Wnt/β-catenin pathway. We aimed to evaluate the effects of recombinant ANGPTL8 on Wnt/β-catenin signaling in human liver carcinoma cells (HepG2) and their viability.
The expression of ANGPTL8 was conducted in the pET-21b-E. coli Bl21 (DE3) system and the produced peptide was purified. HepG2 cells were treated with different concentrations of ANGPTL8 (25, 50, 100, 150, 200, and 250 ng/ml) for 24, 48, and 72 hr. MTT assay was performed to detect the viability of treated cells, and apoptotic induction by ANGPTL8 was measured by flow cytometry assay. Finally, using qRT-PCR the mRNA levels of Wnt signaling modulators WIF-1 and β-catenin were evaluated in treated cells.
MTT assay showed that ANGPTL8 inhibits proliferation of HepG2 cells moderately in a time-independent manner. The highest concentration of the ANGPTL8, 250 ng/ml, reduced cell proliferation after 24, 48, and 72 hr similarly about 30%. In the same concentration of ANGPTL8, after 24 hr of treatment flow cytometry assay revealed a mild increase in early and late apoptosis up to 7.7 and 14.3%, respectively. The qRT-PCR showed that in a concentration-dependent and time-independent fashion, the expression of WIF-1 and β-catenin genes respectively increased and decreased significantly (<0.05).
Our findings suggest that ANGPTL8 may act as a moderate suppressor against HCC cell proliferation possibly via affecting Wnt signaling modulators.
肝细胞癌（HCC）是全球主要的致命性肿瘤之一，也是最常见的原发性肝脏恶性肿瘤。肽激素血管生成素样蛋白8（ANGPTL8，即betatrophin）可能通过Wnt/β-连环蛋白信号通路在肝癌发生发展中发挥重要调节作用。我们旨在评估重组ANGPTL8对人肝癌细胞（HepG2）中Wnt/β-连环蛋白信号传导及其活力的影响。
在pET-21b-大肠杆菌BL21（DE3）系统中表达ANGPTL8，并对产生的肽进行纯化。用不同浓度（25、50、100、150、200和250 ng/ml）的ANGPTL8处理HepG2细胞24、48和72小时。进行MTT试验以检测处理后细胞的活力，通过流式细胞术检测ANGPTL8诱导的凋亡情况。最后，使用qRT-PCR评估处理后细胞中Wnt信号调节因子WIF-1和β-连环蛋白的mRNA水平。
MTT试验表明，ANGPTL8以时间非依赖性方式适度抑制HepG2细胞的增殖。ANGPTL8的最高浓度250 ng/ml在24、48和72小时后同样使细胞增殖降低约30%。在相同浓度的ANGPTL8处理24小时后，流式细胞术检测显示早期和晚期凋亡分别轻度增加至7.7%和14.3%。qRT-PCR表明，WIF-1和β-连环蛋白基因的表达分别以浓度依赖性和时间非依赖性方式显著增加和降低（<0.05）。
我们的研究结果表明，ANGPTL8可能通过影响Wnt信号调节因子，作为肝癌细胞增殖的中度抑制剂。
