ABSTRACT
Matrine, one of the main alkaloid components extracted from the traditional Chinese herb, Sophora flavescens Ait, has various pharmacological effects, and has been reported to exert antitumor activity in melanoma. In the current study, the molecular mechanisms underlying the inhibitory effects of matrine were investigated in melanoma cell line. It was initially confirmed that matrine inhibited proliferation, invasion and induced apoptosis in human A375 and SK-MEL-2 melanoma cell lines in vitro. Subsequently, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis demonstrated that the expression of microRNA (miR)-19b-3p was significantly increased in melanoma cells and was downregulated by treatment with matrine. Furthermore, downregulated miR-19b-3p exerted effects similar to 500 µg/ml matrine on cell proliferation, invasion and apoptosis. Phosphatase and tensin homolog (PTEN) mRNA was identified as a direct target of miR-19b-3p through bioinformatics analysis and a dual-luciferase reporter assay. Additionally, western blotting and RT-qPCR analysis demonstrated that the expression of PTEN protein and mRNA were increased by the treatment with matrine. Furthermore, silencing of PTEN expression reversed the effects of matrine and miR-19b-3p downregulation in A375 and SK-MEL-2 cells. Taken together, the results indicated that matrine may suppress cell proliferation and invasion and induce cell apoptosis partially via miR-19b-3p targeting of PTEN.
Subject(s)
Alkaloids/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Melanoma/genetics , MicroRNAs/genetics , PTEN Phosphohydrolase/genetics , Quinolizines/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Melanoma/metabolism , Neoplasm Invasiveness , PTEN Phosphohydrolase/metabolism , MatrinesABSTRACT
The template-independent teminal transferase activity of Taq DNA polymerase results in an overhanging dA at the 3′end of its PCR products. The pGEMX vector constructed in this experiment forms a single overhanging dT at its 3′end as the result of cleavage with Xcm I restriction enzyme. This vector is very efficient for direct cloning of PCR product obtained by using Taq DNA polymerase.Recombinant colonies can be selected by Blue/white screening. Moreover,insertion fragment can be easily released from the vector simply with either BamH I or Hind III digestion.
