Department of Laboratory Medicine Center, Nanfang Hospital, Southern Medical University/The First School of Clinical Medicine, Southern Medical University, Guangzhou, Guangdong, 510515, China.
Department of Clinical Laboratory, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital, Qingyuan, Guangdong, China.
J Exp Clin Cancer Res. 2018 Jun 4;37(1):113. doi: 10.1186/s13046-018-0727-1.
Angiogenesis is considered as an important process in the development of malignancies and is associated with cancer progression and metastasis. Hepatocellular carcinoma (HCC) is the most common primary tumor of the liver and is recognized as a typical angiogenic tumor. Thus, it is of great importance to study the underlying mechanism of angiogenesis in HCC. The long non-coding RNA (lncRNA) ubiquitin conjugating enzyme E2C pseudogene 3 (UBE2CP3) has been reported as an oncogene that promotes tumor metastasis in HCC. However, the role and underlying mechanisms of UBE2CP3 in HCC angiogenesis are still unclear.
We measured the expression levels of UBE2CP3 by in situ hybridization (ISH) and quantitative real-time polymerase chain reaction (qRT-PCR) in HCC patient samples. We also concomitantly used CD31/PAS double-staining to measure endothelial vessel (EV) density and used qRT-PCR to measure the CD31 mRNA level. HepG2 and SMMC-7721 cells were transfected with Lv-UBE2CP3 or Sh-UBE2CP3 virus to obtain stably over-expressing or knocking-down UBE2CP3 cell lines. The indirect effects of UBE2CP3 on ECs were studied by establishing a co-culture system using Transwell chambers with a 0.4-μm pore size. HCC cells and ECs in the co-culture system were separated, but the cytokines and growth factors were able to communicate with each other. Following exposed to HCC cells, ECs were collected for functional studies. Finally, we studied the function of UBE2CP3 in vivo by chick embryo chorioallantoic membrane (CAM) angiogenesis assays and nude mouse tumorigenicity assays.
In this study, we found that UBE2CP3 expression was higher in HCC tissues than in para-tumor tissues and was up-regulated in tissues with high EV density. Functionally, we found that in the co-culture systems, HCC cells overexpressing UBE2CP3 promoted HUVEC proliferation, migration and tube formation via the activation of ERK/HIF-1α/p70S6K/VEGFA signalling, increasing the level of VEGFA in HCC cell supernatant. In addition, the opposite results appeared when the expression of UBE2CP3 in HCC cells was knocked down. Consistent with these results, CAM angiogenesis assays and nude mouse tumorigenicity assays showed that UBE2CP3 expression up-regulated EV density in vivo.
Our study suggests that UBE2CP3 can enhance the interaction between HCC tumor cells and HUVECs and promote HCC tumorigenicity by facilitating angiogenesis.
血管生成被认为是恶性肿瘤发展过程中的一个重要过程，与癌症的进展和转移有关。肝细胞癌（HCC）是最常见的原发性肝癌，被认为是一种典型的血管生成肿瘤。因此，研究 HCC 中血管生成的潜在机制具有重要意义。长链非编码 RNA（lncRNA）泛素缀合酶 E2C 假基因 3（UBE2CP3）已被报道为一种促进 HCC 肿瘤转移的癌基因。然而，UBE2CP3 在 HCC 血管生成中的作用及其潜在机制尚不清楚。
我们通过原位杂交（ISH）和实时定量聚合酶链反应（qRT-PCR）测量 HCC 患者样本中 UBE2CP3 的表达水平。我们还同时使用 CD31/PAS 双重染色来测量内皮血管（EV）密度，并使用 qRT-PCR 来测量 CD31 mRNA 水平。HepG2 和 SMMC-7721 细胞用 Lv-UBE2CP3 或 Sh-UBE2CP3 病毒转染以获得稳定过表达或敲低 UBE2CP3 的细胞系。通过 Transwell 小室建立 0.4μm 孔径的共培养系统，研究 UBE2CP3 对 ECs 的间接影响。该共培养系统中分离了 HCC 细胞和 ECs，但细胞因子和生长因子可以相互交流。在暴露于 HCC 细胞后，收集 ECs 进行功能研究。最后，我们通过鸡胚绒毛尿囊膜（CAM）血管生成试验和裸鼠肿瘤发生试验研究了 UBE2CP3 在体内的功能。
在这项研究中，我们发现 UBE2CP3 在 HCC 组织中的表达高于癌旁组织，并且在 EV 密度较高的组织中上调。功能上，我们发现共培养系统中，过表达 UBE2CP3 的 HCC 细胞通过激活 ERK/HIF-1α/p70S6K/VEGFA 信号通路促进 HUVEC 增殖、迁移和管形成，增加 HCC 细胞上清液中 VEGFA 的水平。当 HCC 细胞中 UBE2CP3 的表达被敲低时，出现了相反的结果。这些结果与 CAM 血管生成试验和裸鼠肿瘤发生试验一致，表明 UBE2CP3 表达在体内上调了 EV 密度。
我们的研究表明，UBE2CP3 可以通过促进血管生成增强 HCC 肿瘤细胞与 HUVECs 的相互作用，促进 HCC 肿瘤发生。
