Functional analysis of down-regulated miRNA-221-5p in HeLa cell treated with polyphenol-rich Polyalthia longifolia as regulators of apoptotic HeLa cell death.

Abstract

MicroRNAs are endogenous small non-coding-RNAs that control gene expression and cancer development. Previous studies reported that Polyalthia longifolia treatment induced apoptotic cell death in HeLa cells by down-regulation of miR-221-5p. Hence, the current study was conducted to validate the down-regulated miR-221-5p in HeLa cells. Functional analysis of miR-221-5p was conducted through the gain-of-function, and loss-of-function approach and the miRNA expression was quantified by a real-time polymerase chain reaction. The P. longifolia treatment significantly (p < 0.05) reduced miR-221-5p expression when compared to the untreated HeLa cells with a double delta Ct value of 6.32 and the expression fold change value was reduced up to 0.013. The transfection of miR-221-5p mimic significantly increased the expression of miR-221-5p with an expression fold change as high as 0.53 while anti-miR-221-5p transfected HeLa cells show the most significant decrease in miR-221-5p expression with an expression fold change of 0.011. The MTT assay results revealed that the over-expression of miR-221-5p increased the cell proliferation and viability of polyphenol-rich P. longifolia-treated HeLa cells and confirmed the role of downregulated miRNA 221-5p in HeLa cell death. The flow-cytometry analysis showed that the miR-221-5p over-expressed cells decreased the apoptosis of cells induced by polyphenol-rich P. longifolia treatment in HeLa cells, which proved the oncogenic role of miR-221-5p to inhibit apoptosis. Moreover, the depletion of caspase-3 in miR-221-5p-overexpressed HeLa cells showed the roles of downregulated miR-221-5p in the induction of apoptosis. In conclusion, these results suggest that the down-regulated miR-221-5p was involved in regulating apoptosis in HeLa cancer cells.