ABSTRACT
We studied the effects of berberine on the proliferation, apoptosis, and migration of skin melanoma A375 cells, as well as cell cycle-related miRNAs and their target genes, CDK1, CDK2, and cyclins D1 and A. The inhibitory effect of berberine on the growth of A375 cells was evaluated by MTT assay. Cell apoptosis was detected by trypan blue staining. Cell migration was assessed by the scratch test. Cell cycle phases were determined by flow cytometry. The levels of miRNA-582-5p and miRNA-188-5, and mRNA of their target genes encoding CDK1, CDK2, and cyclins D1 and A were measured by qRT-PCR. The expression of cell cycle-related proteins (CDK1, CDK2, and cyclins D1 and A) was determined by Western blotting. Berberine inhibited the proliferation of A375 cells in a time- and dose-dependent manner and significantly and dose-dependently enhanced cell apoptosis. Scratch assay showed an inhibitory effect of berberine on migration of A375 cells. Berberine in low concentrations (20 and 40 µM) caused cell cycle arrest in the S and G2/M phases, while treatment with high concentrations of berberine (60 and 80 µM) arrested cell-cycle in the G2/M phase. The increase in berberine concentration led to an increase in miRNA-582-5p and miRNA-188-5p expression and a decrease in the expression of mRNA for the corresponding target genes encoding CDK1, CDK2, and cyclins D1 and A. Western blotting also revealed reduced expression of CDK1, CDK2, and cyclins D1 and A. Thus, berberine suppressed the growth and migration of human melanoma cells and promoted their apoptosis. Berberine can increase the expression of cell cycle-related miRNAs and cause degradation of the corresponding target genes, thereby blocking the cell cycle progression and inhibiting the melanoma A375 cells.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Berberine/pharmacology , Cell Cycle Checkpoints/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Melanocytes/drug effects , Apoptosis/drug effects , Apoptosis/genetics , CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/metabolism , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cyclin A/genetics , Cyclin A/metabolism , Cyclin D1/genetics , Cyclin D1/metabolism , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 2/metabolism , Dose-Response Relationship, Drug , Humans , Melanocytes/metabolism , Melanocytes/pathology , MicroRNAs/genetics , MicroRNAs/metabolism , Signal TransductionABSTRACT
BACKGROUND AND AIMS: To clarify the role of miR-23a in the onset and development of hepatocarcinoma on the cellular, genetic and molecular levels. PATIENTS AND METHODS: Seventy-eight patients were included after hepatectomy. Relationships between the clinical pathological factors of tumor and paracancerous tissues were analyzed. Risk factors of overall and recurrence-free survival rates were subject to multi-variable analysis. Tissues were sequenced by digital miRNA expression profiling, and new miRNA was subject to target gene prediction. RESULTS: miR-23a expression was correlated with the stage of the TNM Classification of Malignant Tumours most significantly, followed by tumor size (P=0.041 and 0.047). High miR-23a, vascular invasion, tumor size≥7cm, tumor capsule and late pathological stage were the risk factors of overall survival rate, and those of recurrence-free survival rate also included alpha-fetoprotein level≥200µg/L and multiple tumors. Compared with normal hepatic cell line L-02, the miR-23a expression levels in tumor cell lines SMMC-7721 and HepG2 were up-regulated and down-regulated respectively. Transfecting miR-23a inhibitor suppressed cell growth. Apoptotic rates of the control and those transfected with inhibitor-NC and miR-23a inhibitor for 48h were similar. CONCLUSION: High miR-23a expression is the independent prognostic factor of overall and recurrence-free survival rates, and miR-23a may be involved in the onset of hepatocarcinoma as an oncogene.
