The Third Central Hospital of Tianjin, Tianjin, China; Tianjin Key Laboratory of Extracorporeal Life Support for Critical Diseases, Tianjin, China; Artificial Cell Engineering Technology Research Center, Tianjin, China; Tianjin Institute of Hepatobiliary Disease, Tianjin, China.
Respiratory and Critical Care Medicine of Tianjin Chest Hospital, Tianjin, China.
Bosn J Basic Med Sci. 2021 Apr 1;21(2):187-197. doi: 10.17305/bjbms.2020.4641.
Hepatocellular carcinoma (HCC) is the third most common malignant tumor of the digestive system. Plasma cell tumor heterotopic gene 1 (PVT1) is an intergenic long non-coding RNA that is aberrantly expressed in different cancers. Myocardin-related transcription factor A or megakaryoblastic leukemia 1 (MKL1) is a transcriptional coactivator of serum response factor that has been shown to promote cancer cell migration and invasion. In this study, we investigated the relationship between PVT1 and MKL1 as a novel regulatory mechanism underlying HCC progression. We used HepG2 and Cos‑7 cell lines. Transfection experiments with miR-3619-5p mimics/inhibitor, PVT1, siRNA-PVT1, MKL1, or siRNA-MKL1 were performed. RNA and protein levels were analyzed by quantitative reverse transcription PCR and Western blot, respectively. Cell migration was assessed by transwell assay. Luciferase assays, RNA-FISH, RNA immunoprecipitation, and chromatin immunoprecipitation assays were performed to confirm the interaction between PVT1, miR-3619-5p, and MKL1 in HCC cells. Overexpression of PVT1 was positively correlated with MKL1 upregulation, which promoted HepG2 cell migration. miR-3619-5p inhibited MKL1 expression in HCC cells by acting on its 3'-UTR. Furthermore, PVT1 promoted MKL1 expression and migration in HCC cells by directly binding to miR-3619-5p. In a positive feedback loop, MKL1 could activate PVT1 transcription by binding to the CArG box in the promoter region. Our findings may provide a basis for the development of novel targeted therapies in HCC.
肝细胞癌（HCC）是消化系统中第三大常见的恶性肿瘤。浆细胞瘤异位基因 1（PVT1）是一种基因间的长非编码 RNA，在不同的癌症中异常表达。肌球蛋白相关转录因子 A 或巨核细胞白血病 1（MKL1）是血清反应因子的转录共激活因子，已被证明可促进癌细胞迁移和侵袭。在这项研究中，我们研究了 PVT1 和 MKL1 之间的关系，作为 HCC 进展的新的调节机制。我们使用了 HepG2 和 Cos-7 细胞系。进行了 miR-3619-5p 模拟物/抑制剂、PVT1、siRNA-PVT1、MKL1 或 siRNA-MKL1 的转染实验。通过定量逆转录 PCR 和 Western blot 分别分析 RNA 和蛋白质水平。通过 Transwell 测定评估细胞迁移。进行了荧光素酶测定、RNA-FISH、RNA 免疫沉淀和染色质免疫沉淀测定，以确认 PVT1、miR-3619-5p 和 MKL1 在 HCC 细胞中的相互作用。PVT1 的过表达与 MKL1 的上调呈正相关，这促进了 HepG2 细胞的迁移。miR-3619-5p 通过作用于其 3'-UTR 抑制 HCC 细胞中的 MKL1 表达。此外，PVT1 通过直接结合 miR-3619-5p 促进 HCC 细胞中 MKL1 的表达和迁移。在正反馈回路中，MKL1 可以通过结合启动子区域的 CArG 盒来激活 PVT1 转录。我们的研究结果可能为 HCC 的新型靶向治疗的发展提供基础。
