Zhao Ge, Wang Ting, Huang Qi-Ke, Pu Meng, Sun Wei, Zhang Zhuo-Chao, Ling Rui, Tao Kai-Shan
Ge Zhao, Qi-Ke Huang, Meng Pu, Wei Sun, Zhuo-Chao Zhang, Kai-Shan Tao, Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.
World J Gastroenterol. 2016 Jun 21;22(23):5364-73. doi: 10.3748/wjg.v22.i23.5364.
To investigate whether Tg737 is regulated by microRNA-548a-5p (miR-548a-5p), and correlates with hepatocellular carcinoma (HCC) cell proliferation and apoptosis.
Assays of loss of function of Tg737 were performed by the colony formation assay, CCK assay and cell cycle assay in HCC cell lines. The interaction between miR-548a-5p and its downstream target, Tg737, was evaluated by a dual-luciferase reporter assay and quantitative real-time polymerase chain reaction. Tg737 was then up-regulated in HCC cells to evaluate its effect on miR-548a-5p regulation. HepG2 cells stably overexpressing miR-548a-5p or miR-control were also subcutaneously inoculated into nude mice to evaluate the effect of miR-548a-5p up-regulation on in vivo tumor growth. As the final step, the effect of miR-548a-5p on the apoptosis induced by cisplatin was evaluated by flow cytometry.
Down-regulation of Tg737, which is a target gene of miR-548a-5p, accelerated HCC cell proliferation, and miR-548a-5p promoted HCC cell proliferation in vitro and in vivo. Like the down-regulation of Tg737, overexpression of miR-548a-5p in HCC cell lines promoted cell proliferation, increased colony forming ability and hampered cell apoptosis. In addition, miR-548a-5p overexpression increased HCC cell growth in vivo. MiR-548a-5p down-regulated Tg737 expression through direct contact with its 3' untranslated region (UTR), and miR-548a-5p expression was negatively correlated with Tg737 levels in HCC specimens. Restoring Tg737 (without the 3'UTR) significantly hampered miR-548a-5p induced cell proliferation, and rescued the miR-548a-5p induced cell proliferation inhibition and apoptosis induced by cisplatin.
MiR-548a-5p negatively regulates the tumor inhibitor gene Tg737 and promotes tumorigenesis in vitro and in vivo, indicating its potential as a novel therapeutic target for HCC.
研究Tg737是否受微小RNA-548a-5p（miR-548a-5p）调控，以及与肝细胞癌（HCC）细胞增殖和凋亡的相关性。
通过集落形成试验、CCK试验和细胞周期试验，在肝癌细胞系中进行Tg737功能缺失分析。采用双荧光素酶报告基因试验和定量实时聚合酶链反应，评估miR-548a-5p与其下游靶标Tg737之间的相互作用。然后在肝癌细胞中上调Tg737，以评估其对miR-548a-5p调控的影响。将稳定过表达miR-548a-5p或miR对照的HepG2细胞皮下接种到裸鼠体内，评估上调miR-548a-5p对体内肿瘤生长的影响。最后，通过流式细胞术评估miR-548a-5p对顺铂诱导的凋亡的影响。
作为miR-548a-5p靶基因的Tg737下调，加速了肝癌细胞增殖，miR-548a-5p在体外和体内均促进了肝癌细胞增殖。与Tg737下调类似，在肝癌细胞系中过表达miR-548a-5p可促进细胞增殖、增加集落形成能力并阻碍细胞凋亡。此外，miR-548a-5p过表达增加了体内肝癌细胞生长。miR-548a-5p通过直接与其3'非翻译区（UTR）接触下调Tg737表达，且miR-548a-5p表达与肝癌标本中Tg737水平呈负相关。恢复Tg737（无3'UTR）显著阻碍miR-548a-5p诱导的细胞增殖，并挽救了miR-548a-5p诱导的细胞增殖抑制和顺铂诱导的凋亡。
miR-548a-5p负向调控肿瘤抑制基因Tg737，并在体外和体内促进肿瘤发生，表明其作为肝癌新型治疗靶点的潜力。
