5-Azacytidine Downregulates the Proliferation and Migration of Hepatocellular Carcinoma Cells In Vitro and In Vivo by Targeting miR-139-5p/ROCK2 Pathway

Abstract

Background: For hepatocellular carcinoma (HCC), effective therapeutic approaches are lacking. As aberrant gene methylation is a major contributor to HCC development, demethylating drugs such as 5-azacytidine (5-Aza) have been proposed. As most 5-Aza mechanisms of action are unknown, we investigated its phenotypic/molecular effects. Methods: 5-Aza effects were examined in the human HCC cell lines JHH-6/HuH-7 and in the rat cell-line N1-S1. We also employed a xenograft mouse model (HuH-7), a zebrafish model (JHH-6), and an orthotopic syngeneic rat model (N1-S1) of HCC. Results: 5-Aza downregulated cell viability/growth/migration/adhesion by upregulating miR-139-5p, which in turn downregulated ROCK2/cyclin D1/E2F1 and increased p27kip1, resulting in G1/G0 cell accumulation. Moreover, a decrease in cyclin B1 and an increase in p27kip1 led to G2/M accumulation. Finally, we observed a decrease in MMP-2 levels, a stimulator of HCC cell migration. Aza effects were confirmed in the mouse model; in the zebrafish model, we also demonstrated the downregulation of tumor neo-angiogenesis, and in the orthotopic rat model, we observed impaired N1-S1 grafting in a healthy liver. Conclusion: We demonstrate for the first time that 5-Aza can impair HCC development via upregulation of miR-139-5p, which in turn impairs the ROCK2/cyclin D1/E2F1/cyclin B1 pro-proliferative pathway and the ROCK2/MMP-2 pro-migratory pathway. Thus, we provide novel information about 5-Aza mechanisms of action and deepen the knowledge about the crosstalk among ROCK2/cyclin D1/E2F1/cyclin B1/p27kip1/MMP-2 in HCC.

5-azacytidine inhibits the tumorigenesis of esophageal cancer cells through the suppression of NOTCH1 signaling

“…Therefore, according to this research and our results, 5-azaC may promote EC cell death by inhibiting and demethylating NICD. Studies also showed that 5-azaC could suppress tumor cell growth by demethylating cell cycle-associated genes such as p16INK4b and p14ARF [24], regulating miRNA-200C and miR-124 [25], or incorporating into RNA in hepatocellular carcinoma cells [11]. The molecular mechanism of how 5-azaC downregulated the NOTCH1 pathway was beyond our study and more studies are required to solve this question.…”

“…5-azacytidine (5-azaC) is an FDAapproved methyltransferase inhibitor that has been widely used to treat hematological malignancies and some malignant solid tumors. Apart from the demethylation function, 5-azaC could also alter the Wnt/β-catenin signaling pathway [10] or miR-139-5p/ROCK2 pathway expression [11]. 5-azaC is an effective drug in Myelodysplastic Syndromes (MDS) and NOTCH1 is one of its main targets [12].…”

Targeting Non-Coding RNAs for the Development of Novel Hepatocellular Carcinoma Therapeutic Approaches

“…This results in gene transcription repression, as transcription factors do not have access to the gene promoter. Recently, we studied [47] the effects of 5-azacytidine (5-Aza), a drug with the ability to revert pathological promoter methylation, on HCC cell lines. Among many other effects, we observed the reactivation of the expression of miR-139-5p.…”

Advances in the Biological Functions and Mechanisms of miRNAs in the Development of Osteosarcoma

“…For example, demethylating drugs such as 5-azacytidine (5-aza) can inhibit the proliferation and migration of hepatocellular carcinoma cells by upregulating the expression of miRNA-139-5p. 182 Therefore, the accuracy of circulating miRNAs will be interfered by diseases complicated with osteosarcoma or the use of some drugs. In addition, the low expression level of miRNAs in cells makes them exist less in biological fluids.…”