Effects of Moringa oleifera extracellular vesicles on human microRNAs involved in the HeLa cell tumorigenesis

The effects of extracellular vesicles from Moringa oleifera on the modulation of tumour associated human-microRNAs profile in cervical cancer-derived HeLa cells were investigated. Plant extracellular vesicles have been shown to carry a variety of bioactive compounds, such as proteins, lipids, phytochemicals (e.g., phenols and flavonoids), and RNAs, including nucleic acids (e.g., miRs), mediating the transfer of information to recipient cells and thus modulating their function and gene expression. To define the microRNA profile in EVs-treated and untreated HeLa, the expression of 84 human miRNAs involved in human cancer was investigated. Total RNA was isolated from the pellets of MOES-EVs treated and untreated HeLa cells by TRIzol extraction method, according to the manufacturer’s instructions (Invitrogen, CA, USA). RNA was quantified using a NanoDropTM light spectrophotometer (Thermo Fisher Scientific, MA; USA) and stored at -20 °C until use. cDNA was obtained from total RNA by cDNA Reverse Transcription Kit (Thermo Fisher Scientific, MA; USA) and the expression analyses using a miScriptTM miRNA PCR Array Human Cancer Pathway array (cat # MIHS-102Z, Qiagen, Hilden, Germany). The assays were performed in a Bio-rad instrument (CFX96 Real-Time System, Biorad, Hercules, California, USA) using SYBR Green chemistry (Fast QPCR Master Mix, SmoBio, Taiwan), following the manufacturer’s instructions. The basal miRs expression detected in untreated HeLa cells was categorized in three groups with respect to Ct values: group n.1 from 15 to 20 Ct; group n.2 from 21 to 30 Ct; group n.3 >30 Ct. Data were obtained by the application of the 2−ΔCt method, normalising for the housekeeping controls SNORD 61 and SNORD 96A. The fold change was calculated between 2−ΔCt of treated cells/untreated cells. The miRs with >1.5-fold change were considered over expressed and those with a value between 0.5 and 1.5 equally expressed, while the miRs with a fold change <0.5 as under expressed.