Guo Kai, Yao Jie, Yu Qiang, Li Zijian, Huang Hu, Cheng Jianguo, Wang Zhigang, Zhu Yunfeng
1 Department of Gastroenterology, The 161th Hospital of PLA, Wuhan, China.
2 Department of Oncology, The 161th Hospital of PLA, Wuhan, China.
Tumour Biol. 2017 Apr;39(4):1010428317699122. doi: 10.1177/1010428317699122.
The plasmacytoma variant translocation 1 gene (PVT1) is a large non-coding locus at adjacent of c-Myc, and long non-coding RNA PVT1 is now recognized as a cancerous gene co-amplified with c-Myc in various cancers. But the expression and functional role of PVT1 in colorectal cancer are still unelucidated. In addition, all the reported long non-coding RNAs so far are discovered in either cells or tissues, but no research about long non-coding RNAs detection in extracellular vesicles has been reported yet. In the present study, we firstly investigated the expression of PVT1 in colorectal cancer specimens and its correlation with the expression of c-Myc and other related genes by real-time polymerase chain reaction. Then, we isolated the extracellular vesicles from colorectal cancer cells culturing medium by differential centrifugation and detected the PVT1 expression in extracellular vesicles by using real-time polymerase chain reaction. The PVT1 targeting siRNA was transfected into SW480 and SW620 cells, and 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and flow cytometry were used to evaluate the cell proliferation and apoptosis. The results showed that the PVT1 expression in tumor tissues was higher than that in normal tissues, which was significantly correlated with the expression of c-Myc and three c-Myc regulating genes FUBP1, EZH2, and NPM1 and also correlated with the expression of two other PVT1-associated transcript factors nuclear factor-κB and myocyte-specific enhancer factor 2A. Here, we reported for the first time that PVT1 as a long non-coding RNA was successfully detected in extracellular vesicles excluded from SW620 and SW480 cells, and the expression level of PVT1 was higher in extracellular vesicles from the more aggressive cell SW620 than from SW480. The results also showed that by down-regulating the PVT1 expression, the c-Myc expression was suppressed, the cell proliferation was inhibited, and cell apoptosis was increased. Taken together, these findings implicated that PVT1 may be a new oncogene co-amplified with c-Myc in colorectal cancer tissues and extracellular vesicles and functionally correlated with the proliferation and apoptosis of colorectal cancer cells.
浆细胞瘤变异易位1基因(PVT1)是位于c-Myc附近的一个大型非编码基因座,长链非编码RNA PVT1现在被认为是一种在多种癌症中与c-Myc共同扩增的癌基因。但PVT1在结直肠癌中的表达及功能作用仍不明确。此外,目前所有已报道的长链非编码RNA都是在细胞或组织中发现的,尚未有关于细胞外囊泡中长链非编码RNA检测的研究报道。在本研究中,我们首先通过实时聚合酶链反应研究了PVT1在结直肠癌标本中的表达及其与c-Myc和其他相关基因表达的相关性。然后,我们通过差速离心从结直肠癌细胞培养基中分离出细胞外囊泡,并使用实时聚合酶链反应检测细胞外囊泡中的PVT1表达。将靶向PVT1的小干扰RNA转染到SW480和SW620细胞中,并用3-(4,5-二甲基噻唑-2-基)-5-(3-羧甲氧基苯基)-2-(4-磺基苯基)-2H-四唑盐比色法和流式细胞术评估细胞增殖和凋亡。结果显示,肿瘤组织中PVT1的表达高于正常组织,这与c-Myc以及三个c-Myc调节基因FUBP1、EZH2和NPM1的表达显著相关,也与另外两个与PVT1相关转录因子核因子κB和肌细胞特异性增强因子2A的表达相关。在此,我们首次报道在SW620和SW480细胞释放的细胞外囊泡中成功检测到作为长链非编码RNA的PVT1,且在侵袭性更强的SW620细胞的细胞外囊泡中PVT1的表达水平高于SW480细胞。结果还表明,下调PVT1表达可抑制c-Myc表达,抑制细胞增殖并增加细胞凋亡。综上所述,这些发现表明PVT1可能是结直肠癌组织和细胞外囊泡中与c-Myc共同扩增的一个新的癌基因,并且在功能上与结直肠癌细胞的增殖和凋亡相关。