MicroRNA hsa-miR-29b potentiates etoposide toxicity in HeLa cells via downregulation of Mcl-1.

Introduction

Etoposide is a semisynthetic derivate of podophyllotoxin. It is in use for over 30 years in various treatment combinations for cancer treatment. Primary mechanism of its action is topoisomerase II inhibition. miRNAs are 18 - 25 nucleotide short RNA chains which modulate protein expression. Aim of this project was to evaluate the effect of miR-29 family members on etoposide cytotoxicity.

Materials/methods

We selected HeLa cells for the experiment. xCELLigence system was utilized for cell viability monitoring through the experiment. Western blot technique was used for detecting expression changes in selected proteins. Lipofectamine 2000 was used for transfection of miRNA precursors into the cells. Specific siRNAs were used to verify miR-29 down-regulation effect.

Results and conclusions

We observed statistically significant enhancement of etoposide toxicity in cells treated with miR-29b precursor. Based on literature data and miRNA database we selected Mcl-1, Bak and Bax proteins as known/possible miR-29 targets. In accordance with data published by others we found downregulation of Mcl-1 protein expression and negligible influence on Bak and Bax proteins by miR-29 family members. We investigated influence of Mcl-1 protein on toxicity of etoposide via siRNA interference, the protein was knocked out using specific siRNAs and the effect of etoposide was re-evaluated. Our results from xCELLigence system showed that modulation of Mcl-1 protein is important for enhancing the etoposide cytotoxicity. In conclusion, our results showed that miRNA 29b significantly enhances cytotoxicity of etoposide in HeLa cells by modulating Mcl-1 protein expression. Enhancing cytotoxicity of etoposide through miRNA 29b precursor is probably connected to the nuclear shuttling of mature miR-29b.

Acknowledgements: This work was supported by grants IGA_ LF_2015_007 and LO1304.