Background
Hepatocellular carcinoma (HCC) is one of the most common tumors with high mortality. MicroRNAs (miRNAs) were reported as crucial markers for the diagnosis of HCC. Paeonol exerted many pharmacological effects on tumor progression. This study aimed to elucidate the underlying molecular mechanism of paeonol in HCC progression.
Conclusion
Paeonol impeded cell viability, migration and invasion and triggered apoptosis by regulating miR-21-5p/KLF6 axis in HCC cells. Xenograft assay confirmed that paeonol inhibited tumor growth through miR-21-5p/KLF6 axis in HCC in vivo.
Methods
Cell viability was determined by Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was examined by flow cytometry. The levels of Cyclin D1, cyclin-dependent kinase 4 (CDK4), B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) were detected by Western blot assay. Cell migration and invasion were assessed by transwell assay. The levels of matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) were measured by Western blot. The expression of miR-21-5p and kruppel-like factor 6 (KLF6) was detected by quantitative real-time PCR (qRT-PCR) or Western blot assay, respectively. Dual-luciferase reporter assay was performed to analyze the interaction between miR-21-5p and KLF6. The enrichment of miR-21-5p was determined by RNA pull-down assay. Xenograft assay was conducted to analyze tumor growth in vivo.
Results
The results demonstrated that cell viability of Hep3B and Huh-7 cells was inhibited, while cell apoptosis was promoted after treatment with paeonol. Transwell assay indicated that cell migration and invasion were blocked in paeonol-treated cells. Moreover, miR-21-5p expression was markedly decreased in paeonol-treated cells and its knockdown suppressed cell viability, migration and invasion, but contributed to cell apoptosis. MiR-21-5p targeted KLF6 and its silencing prominently elevated KLF6 level. Furthermore, the restoration experiment determined that miR-21-5p and KLF6 were antagonisms on cell viability, apoptosis, migration and invasion. Also, paeonol abated the decrease in KLF6 level caused by miR-21-5p up-regulation. Besides, paeonol suppressed tumor growth in vivo.