Tangshan Maternal and Child Health Care Hospital, North China University of Science and Technology, Tangshan, 063000, China.
School of Basic Medical Sciences, Peking University Health Science Center, Beijing, 100191, China.
Sci Rep. 2023 May 9;13(1):7500. doi: 10.1038/s41598-023-33976-5.
Hepatocellular carcinoma (HCC) is a symptomatic disease involed multi-stage program. Here, we elucidated the molecular mechanism of LncTUG1 in the regulation of HCC evolvement. And that may in all likelyhood supply a innovative latent target for HCC's diagnoses and prognosis. LncRNA TUG1, miR-144-3p, RRAGD and mTOR signaling pathway were screened as target genes in the database, and their expression levels at the cytological level were verified utilized qRT-PCR, Western Blot and immunohistochemistry. Then, we adopted CCK-8, Transwell and flow cytometry assays to estimate cell proliferation, invasion and apoptosis. By use of luciferase reporter assay, the relationships of LncRNA TUG1, miR-144-3p and RRAGD was confirmed. In addition, the LncRNA TUG1-miR-144-3p-RRAGD-mTOR signaling pathway in HCC cells was verified adopted rescue experiment and confirmed by xenotransplantation animal experiment. LncTUG1 in HCC tissues from three databases were identified and further verified through qRT-PCR in HCC cells (Huh7, Hep3B). Knockdown the LncTUG1 could increase apoptosis and inhibite invasion and proliferation in HCC cells. Using inhibitors and activators of the mTOR/S6K pathway, LncTUG1 was confirmed to regulate HCC progression by the mTOR/S6K pathway. Luciferase reporter assay demonstrated that TUG1 negatively regulates miR-144-3p. Furthermore, miR-144-3p negativly regulates RRAGD by way of interacting with the 3'UTR of the RRAGD mRNA in HCC utilized luciferase reporter assay. In vivo, we also discovered that neoplasm weight and tumor volume reduced significantly in subcutaneous xenograft nude mouse models derived from sh-LncTUG1-expressing Huh7 cells. And the expressions of p-mTOR, p-S6K and RRAGD were decreased obviously while the miR144-3p increased in subcutaneous xenograft nude mouse models. In a word, the research suggests that LncTUG1 targets miR-144-3p while miR-144-3p binds to RRAGD mRNA, which induces mTOR/S6K pathway activation and promotes the progression of HCC.
肝细胞癌（HCC）是一种涉及多阶段程序的症状性疾病。在这里，我们阐明了长链非编码 RNA TUG1 在 HCC 演变调节中的分子机制。这很可能为 HCC 的诊断和预后提供了一个创新的潜在靶点。在数据库中筛选 LncRNA TUG1、miR-144-3p、RRAGD 和 mTOR 信号通路作为靶基因，并利用 qRT-PCR、Western Blot 和免疫组织化学验证其在细胞学水平的表达水平。然后，我们采用 CCK-8、Transwell 和流式细胞术评估细胞增殖、侵袭和凋亡。通过荧光素酶报告基因实验证实了 LncRNA TUG1、miR-144-3p 和 RRAGD 之间的关系。此外，通过挽救实验和异种移植动物实验验证了 HCC 细胞中的 LncRNA TUG1-miR-144-3p-RRAGD-mTOR 信号通路。通过 qRT-PCR 在 HCC 细胞（Huh7、Hep3B）中进一步验证了来自三个数据库的 HCC 组织中的 LncTUG1。敲低 LncTUG1 可增加 HCC 细胞的凋亡并抑制侵袭和增殖。利用 mTOR/S6K 通路的抑制剂和激活剂，通过 mTOR/S6K 通路证实 LncTUG1 调节 HCC 进展。荧光素酶报告基因实验表明 TUG1 负调控 miR-144-3p。此外，通过荧光素酶报告基因实验证实 miR-144-3p 通过与 RRAGD mRNA 的 3'UTR 相互作用负调控 RRAGD。在体内，我们还发现，皮下异种移植裸鼠模型中，来源于 sh-LncTUG1 表达 Huh7 细胞的肿瘤重量和肿瘤体积显著降低。并且，在皮下异种移植裸鼠模型中，p-mTOR、p-S6K 和 RRAGD 的表达明显降低，而 miR144-3p 增加。总之，该研究表明 LncTUG1 靶向 miR-144-3p，而 miR-144-3p 结合 RRAGD mRNA，从而诱导 mTOR/S6K 通路激活并促进 HCC 的进展。
