The circular RNA circ2176, targeting on NFATC3, acts as a cancer-suppressor of hepatocellular carcinoma via JNK, c-Jun, AKT and mTOR pathways

Objective: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-induced death worldwide. Circular RNAs (circRNAs) were reported as regulators involved in HCC, but the mechanism of the circRNAs in regulating HCC is not clearly clarified.Methods: Expression of the circ2176 in the hepatocellular carcinoma tissues from HCC patients was determined by RT-PCR. The circ2176 overexpression vector was packaged in lentivirus and transfected into HepG2 and Huh7 cells. The viability of the cells was determined by the MTT assay, and the cell apoptosis was evaluated by flow cytometer. Transwell assay was conducted for measuring the capacity of cell migration and cell invasion of both cell lines. In addition, tumor formation assay was performed in nude mice to determine the relationship between circ2176 and tumor growth. RNA-seq and KEGG functional analysis were done to screen the candidate target genes of circ2176. The target gene, NFATC3 was further verified with the expression level in HCC patients. The correlation between NFATC3 and staging and prognosis of HCC was calculated as well. The correlation between circ2176, NFATC3 and the molecules involved in JNK/c-Jun/AKT/mTOR pathway was determined by western blotting.Results: circ2176 was proven to down-regulated express in HCC patients. When over-expressed circ2176, the cell viability was accordingly inhibited whereas the apoptosis was enhanced. Moreover, the amount of the migrated and invasive cancerous cells was also decreased for both cell lines. The tumor generated in the nude mice became smaller in size and weight under the regulation of the overexpressed circ2176. The RNA-seq results shown that NFATC3 was the most significant differentially expressed gene when circ2176 was overexpressed. It also proved that the level of NFATC3 was down-regulated expressed in HCC patients, but was not correlated to the staging and prognosis of HCC. Both circ2176 and NFATC3 was proven to perform functions in inhibiting the phosphorylation of JNK, c-Jun, AKT and mTOR.Conclusions: circ2176 is positively correlated to the expression of NFATC3 and thereby regulate HCC via JNK, c-Jun, AKT and mTOR pathways.