外泌体环状锌指蛋白652通过miR-29a-3p/葡糖糖代谢蛋白1轴促进肝细胞癌的细胞增殖、迁移、侵袭和糖酵解
Exosomal Circ-ZNF652 Promotes Cell Proliferation, Migration, Invasion and Glycolysis in Hepatocellular Carcinoma via miR-29a-3p/GUCD1 Axis.
作者信息
Li Yuhui, Zang Hongliang, Zhang Xue, Huang Guomin
机构信息
Department of General Surgery, China-Japan Union Hospital of Jilin University, Changchun 130012, People's Republic of China.
出版信息
Cancer Manag Res. 2020 Aug 24;12:7739-7751. doi: 10.2147/CMAR.S259424. eCollection 2020.
BACKGROUND
Circular RNAs (circRNAs) play a crucial role in hepatocellular carcinoma (HCC) progression. However, the role of exosomal circRNAs in HCC is still largely unknown. We aimed to explore the function of exosomal circ-ZNF652 in HCC.
METHODS
The morphology and size of exosomes were examined by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The expression of circ-ZNF652, ZNF652 mRNA, microRNA-29a-3p (miR-29a-3p) and guanylyl cyclase domain containing 1 (GUCD1) mRNA was determined by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of CD63, CD81, hexokinase 2 (HK2) and GUCD1 were examined via Western blot assay. The stability of circ-ZNF652 was examined by RNase R digestion assay. Cell proliferation was analyzed by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Cell migration and invasion were assessed by transwell assay. The glycolysis level was detected via specific kits. The association between miR-29a-3p and circ-ZNF652 or GUCD1 was analyzed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. A murine xenograft model was constructed to explore the effect of circ-ZNF652 in vivo.
RESULTS
Exosomal circ-ZNF652 was upregulated in HCC patients' serums and HCC cells. Exosomal circ-ZNF652 could transfer to HCC cells, and circ-ZNF652 silencing suppressed HCC cell proliferation, migration, invasion and glycolysis. Circ-ZNF652 was a sponge of miR-29a-3p, and the inhibitory effect of circ-ZNF652 silencing on HCC cell progression was weakened by miR-29a-3p inhibitor. GUCD1 was a target gene of miR-29a-3p, and GUCD1 overexpression restored the effect of miR-29a-3p on HCC cell development. Moreover, circ-ZNF652 knockdown repressed tumor growth in vivo.
CONCLUSION
Exosomal circ-ZNF652 contributes to HCC cell proliferation, migration, invasion and glycolysis by miR-29a-3p/GUCD1 axis.
背景
环状RNA（circRNAs）在肝细胞癌（HCC）进展中起关键作用。然而，外泌体circRNAs在HCC中的作用仍 largely未知。我们旨在探讨外泌体circ-ZNF652在HCC中的功能。
方法
通过透射电子显微镜（TEM）和纳米颗粒跟踪分析（NTA）检测外泌体的形态和大小。通过定量实时聚合酶链反应（qRT-PCR）测定circ-ZNF652、ZNF652 mRNA、微小RNA-29a-3p（miR-29a-3p）和含鸟苷酸环化酶结构域1（GUCD1）mRNA的表达。通过蛋白质印迹法检测CD63、CD81、己糖激酶2（HK2）和GUCD1的蛋白质水平。通过RNase R消化试验检测circ-ZNF652的稳定性。通过3-（4,5-二甲基-2-噻唑基）-2,5-二苯基-2-H-四氮唑溴盐（MTT）试验分析细胞增殖。通过transwell试验评估细胞迁移和侵袭。通过特定试剂盒检测糖酵解水平。通过双荧光素酶报告基因试验和RNA免疫沉淀（RIP）试验分析miR-29a-3p与circ-ZNF652或GUCD1之间的关联。构建小鼠异种移植模型以探讨circ-ZNF652在体内的作用。
结果
外泌体circ-ZNF652在HCC患者血清和HCC细胞中上调。外泌体circ-ZNF652可转移至HCC细胞，circ-ZNF652沉默抑制HCC细胞增殖、迁移、侵袭和糖酵解。circ-ZNF652是miR-29a-3p的海绵，miR-29a-3p抑制剂减弱了circ-ZNF652沉默对HCC细胞进展的抑制作用。GUCD1是miR-29a-3p的靶基因，GUCD1过表达恢复了miR-29a-3p对HCC细胞发育的影响。此外，circ-ZNF652敲低在体内抑制肿瘤生长。
结论
外泌体circ-ZNF652通过miR-29a-3p/GUCD1轴促进HCC细胞增殖、迁移、侵袭和糖酵解。
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