Circular RNA ACVR2A promotes the progression of hepatocellular carcinoma through mir-511-5p targeting PI3K-Akt signaling pathway

Abstract

Circular RNA (circRNA) is thought to mediate the occurrence and development of human cancer and usually acts as a tiny RNA (miRNA) sponge to regulate downstream gene expression. However, it is not clear whether and how circACVR2A (hsa_circ_0001073) is involved in the progression of HCC. The purpose of this study is to clarify the potential role and molecular mechanism of circACVR2A in regulating the progression of hepatocellular carcinoma cells (HCC). The abundance of related proteins in circACVR2A, microRNA (miR511-5p) and PI3K-Akt signaling pathway was determined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) or Western blotting. Cell viability, invasion and apoptosis were analyzed by CCK-8, Transwell analysis and Tunel staining, respectively. The interaction between circACVR2A and microRNA was evaluated by double luciferase reporter gene assay. The results showed that circACVR2A was highly expressed in hepatocellular carcinoma cell lines. Our in vivo and in vitro data showed that circACVR2A promoted the proliferation, migration and invasion of HCC. In terms of mechanism, we found that circACVR2A can directly interact with miR511-5p and act as a miRNA sponge to regulate the expression of related proteins in PI3K-Akt signaling pathway.In HCC, circACVR2A can mediate miR-511-5p/mRNA network to activate PI3K signal pathway. This shows that the molecular regulatory network with circACVR2A as the core is a new potential target for diagnosis and treatment of hepatocellular carcinoma.

Keywords: HCC; PI3K; circACVR2A; mRNA; miRNA.

Fig. 1

circACVR2A was highly expressed in hepatoma cells and was used as a sponge for miR-511-5p (A) RT-qPCR to detect the expression of circACVR2A in normal liver cells and liver cancer cells. (B) Volcano map of miRNAs differential expression. (C) Intersection the differentiated miRNAs with the circACVR2A-miRNAs predicted in the circbank database. (D) RT-qPCR assay to verify the efficiency of overexpression and knockdown of circACVR2A in hepatocellular carcinoma cells. (E) RT-qPCR assay verified the expression of miR-511-5p after overexpression and knockdown of circACVR2A in hepatoma cells. (F) HEK293T cotransfected with miRNA mimics, Psi-Check2-wild-type circACVR2A (circACVR2A-WT) or Psi-Check2-mutant circACVR2A (circACVR2A-MUT) plasmid Luciferase reporter gene was detected in cells. All experiments were repeated three times, expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. In Fig. 1A * LO2 serves as the control group; # MIHA serves as the control group compared with pcDNA3.1 and PCDNA3.1-U6 in Fig. 1D and E, compared with mimics NC in Fig. 1F

Fig. 2

circACVR2A can promote the proliferation of hepatoma cells in vitro. (A) The effect of circACVR2A on the proliferation of hepatoma cell line Huh-7 was detected by CCK8 assay. (B) The effect of circACVR2A on the proliferation of hepatoma cell line Hep3B cells was detected by CCK8 assay. (C) The effect of circACVR2A on the clonal formation of hepatoma cells Huh 7 cells were detected by cell cloning assay. (D) The effect of circACVR2A on the clonal formation of hepatoma cells Hep3B cells was detected by cell cloning assay. All experiments were repeated three times, expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, compared with pCDNA3.1 or pCDNA3.1-U6 in Fig. 2

Fig. 3

circACVR2A can promote the migration and invasion of hepatoma cells. (A, C) Cell migration and invasion test: to detect the migration and invasion ability of hepatoma cell Huh-7 in vitro; (B, D) Cell migration and invasion test: to detect the migration and invasion ability of hepatoma cell Hep3B in vitro. All the experiments were repeated three times, expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, compared with pCDNA3.1 or pCDNA3.1-U6

Fig. 4

circACVR2A can inhibit the apoptosis of hepatoma cells in vitro. (A) In Huh-7 and Hep3B cells overexpressing circACVR2A, the climbing slices fixed with 4% paraformaldehyde were stained with TdT enzyme and DAPI enzyme respectively. (B) In the silent circACVR2A hepatoma cells Huh-7 and Hep3B, the climbing slices fixed with 4% paraformaldehyde were stained with TdT enzyme and DAPI enzyme, respectively

Fig. 5

circACVR2A can regulate the abundance of mRNAs related to PI3K signal pathway through miR-511-5p. (A) Predict hsa-miR511-5p/mRNAs intersection through three databases (TargetScan, mirDIP, miRWalk); (B) KEGG enrichment analysis of overlapping mRNAs; (C) Difference analysis of 9 mRNAs enriched to PI3K signal pathway; (D-K) RT-qPCR verification of 8 mRNAs with differences. All the experiments were repeated three times, expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, and compared with pCDNA3.1 or pCDNA3.1-U6

Fig. 6

circACVR2A can regulate the PI3K/AKT/FOXO1 signaling pathway. (A) After overexpression and silencing of circACVR2A in Huh-7 and Hep3B, the abundance of PI3K, Akt, FOXO1 and their phosphorylated proteins, with GAPDH as the internal reference protein; (B, C) After overexpression and silencing of circ ACVR2A in Huh-7 and Hep3B, the abundance of Bax and BCL2, with β-Actin as the internal reference protein. *P < 0.05, **P < 0.01, ***P < 0.001, and compared with pCDNA3.1 or pCDNA3.1-U6

Fig. 7

Overexpression or silencing of circACVR2A promoted or inhibited the growth of hepatoma cells in vivo. (A, B) Hep3B cells stably expressing circACVR2 or control vector and Hep3B cells stably silencing circ-ACVR2 or control vector were inoculated into the right axilla of BALB/c nude mice. (C, D) The tumor volume and weight of circACVR2A overexpression group were significantly increased, while those of silent group were significantly decreased. (E) The representative images of HE staining analysis of tumors in each group. (F) The schematic diagram shows that circACVR2A inhibits the proliferation, metastasis and apoptosis of hepatocellular carcinoma cells through the miR-511/PI3K axis. *P < 0.05, **P < 0.01, ***P < 0.001, and compared with pCDNA3.1 or pCDNA3.1-U6