Department of Internal Medicine of Chinese Medicine, Henan University of Chinese Medicine , Zhengzhou, Henan, China.
The First Affiliated Hospital of Henan University of Chinese Medicine , Zhengzhou, Henan, China.
Bioengineered. 2021 Dec;12(1):578-588. doi: 10.1080/21655979.2021.1882133.
Reportedly, long non-coding RNAs (lncRNAs) are implicated in hepatocellular carcinoma (HCC) progression, yet little is known concerning the biological functions of TTN antisense RNA 1 (TTN-AS1) in HCC. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) was performed for detecting TTN-AS1, SPOCK1 mRNA, and miR-139-5p expressions in HCC cells and tissues. After TTN-AS1 was overexpressed or knocked down in HCC cells, CCK-8 and 5-Ethynyl-2'-deoxyuridine (EdU) assays were carried out for examining cell multiplication. Transwell assays were conducted for evaluating HCC cell migration and invasion. Dual-luciferase reporter assay was employed for verifying the binding relationships between miR-139-5p and TTN-AS1, and between SPOCK1 3'UTR and miR-139-5p. Western blot was employed to measure SPOCK1, E-cadherin, N-cadherin, and Vimentin protein expressions. We demonstrated that, TTN-AS1 and SPOCK1 expression levels were remarkably enhanced in HCC cells and tissues, whereas miR-139-5p expression was observably reduced. Functional experiments suggested that TTN-AS1 knockdown markedly repressed HCC cell multiplication, migration, epithelial-mesenchymal transition (EMT), and invasion. In addition, TTN-AS1 interacted with miR-139-5p and decreased its expression. Moreover, SPOCK1 was a miR-139-5p target, and miR-139-5p inhibitors were able to reverse TTN-AS1 knockdown-induced inhibitory effect on SPOCK1 expression. SPOCK1 overexpression plasmid could counteract TTN-AS1 knockdown-induced inhibiting impact on HCC cell multiplication, migration, invasion, and EMT. In conclusion, TTN-AS1 expression level is remarkably enhanced in HCC, and TTN-AS1 can promote the multiplication, migration, invasion, and EMT of HCC cells via regulating miR-139-5p/SPOCK1 axis.
据报道，长链非编码 RNA（lncRNA）参与了肝细胞癌（HCC）的进展，但对于 TTN 反义 RNA 1（TTN-AS1）在 HCC 中的生物学功能知之甚少。在本研究中，通过定量实时聚合酶链反应（qRT-PCR）检测 HCC 细胞和组织中 TTN-AS1、SPOCK1 mRNA 和 miR-139-5p 的表达。在 HCC 细胞中转染 TTN-AS1 过表达或敲低后，通过 CCK-8 和 5-乙炔基-2'-脱氧尿苷（EdU）实验检测细胞增殖。通过 Transwell 实验评估 HCC 细胞迁移和侵袭。双荧光素酶报告实验用于验证 miR-139-5p 与 TTN-AS1 之间以及 SPOCK1 3'UTR 与 miR-139-5p 之间的结合关系。Western blot 用于测量 SPOCK1、E-钙黏蛋白、N-钙黏蛋白和波形蛋白的蛋白表达。结果表明，TTN-AS1 和 SPOCK1 的表达水平在 HCC 细胞和组织中显著升高，而 miR-139-5p 的表达水平显著降低。功能实验表明，TTN-AS1 敲低显著抑制 HCC 细胞增殖、迁移、上皮间质转化（EMT）和侵袭。此外，TTN-AS1 与 miR-139-5p 相互作用并降低其表达。此外，SPOCK1 是 miR-139-5p 的靶基因，miR-139-5p 抑制剂能够逆转 TTN-AS1 敲低诱导的 SPOCK1 表达抑制作用。SPOCK1 过表达质粒可以抵消 TTN-AS1 敲低诱导的对 HCC 细胞增殖、迁移、侵袭和 EMT 的抑制作用。总之，TTN-AS1 在 HCC 中表达水平显著升高，TTN-AS1 可以通过调节 miR-139-5p/SPOCK1 轴促进 HCC 细胞的增殖、迁移、侵袭和 EMT。
