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Circ_0046599 Promotes the Development of Hepatocellular Carcinoma by Regulating the miR-1258/RPN2 Network
Quangang Fang1, Haiyun Liu1, Aiqun Zhou1
1Department of Laboratory, Jiangxi Provincial People's Hospital Affiliated to Nanchang University, Nanchang 330006, Jiangxi, People's Republic of China.
Background:
Many studies have confirmed that circular RNAs (circRNAs) play a key role in the biological progression of cancers. However, the function of a novel circRNA, circ_0046599, in hepatocellular carcinoma (HCC) progression has not been explored.
Methods:
Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure the expression of circ_0046599, microRNA (miR)-1258 and Ribophorin II (RPN2). Subcellular fractionation location assay was used to localize circ_0046599 in HCC cells. The circular characteristic of circ_0046599 was verified using Ribonuclease R (RNase R) digestion assay. Besides, cell counting kit 8 (CCK8) assay, colony formation assay, wound healing assay and transwell assay were used to detect cell proliferation, migration and invasion, respectively. The lactate production and glucose level were determined by Lactate and Glucose Assay Kits. Furthermore, the protein levels of glycolysis, metastasis and proliferation-related marker proteins, as well as RPN2 were tested by Western blot (WB) analysis. Moreover, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were employed to confirm the interactions among circ_0046599, miR-1258 and RPN2. In addition, mice xenograft models were applied to observe the effect of circ_0046599 silencing on HCC tumor growth in vivo.
Results:
Circ_0046599 was highly expressed in HCC tissues and cells, and its knockdown could suppress HCC cell proliferation, migration, invasion and glycolysis process. MiR-1258 could be targeted by circ_0046599, and its inhibitor could invert the suppressing effect of circ_0046599 knockdown on HCC progression. Further, RPN2 was a target of miR-1258. Overexpressed RPN2 could reverse the inhibiting effect of miR-1258 overexpression on HCC progression. Also, knockdown of circ_0046599 could restrain HCC tumor growth in vivo.
Conclusion:
Our results provided new evidence that circ_0046599 could promote the progression of HCC by increasing RPN2 expression via sponging miR-1258.
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