Sequence-specific inhibition of microRNA-130audgene by CRISPR/Cas9 system in breast cancerudcell line

摘要

MicroRNAs (miRNAs) are short stranded noncoding RNA that play important roles inudapoptosis, cell survival, development and cell proliferation. However, gene expression control viaudsmall regulatory RNA, particularly miRNA in breast cancer is still less explored. Therefore, thisudproject aims to develop an approach to target microRNA-130a using the Clustered RegularlyudInterspaced Short Palindromic Repeat (CRISPR)/Cas9 system in MCF7, breast cancer cell line. Theud20 bp sequences target at stem loop, 3' and 5' end of miR130a were cloned into pSpCas9(BB)-2AGFPud(PX458) plasmid, and the positive clones were confirmed by sequencing. A total of 5 μg ofudPX458-miR130a was transfected to MCF7 using Lipofectamine® 3000 according to manufacturer’sudprotocol. The transfected cells were maintained in the incubator at 37 ⁰C under humidified 5% CO2.udAfter 48 hours, cells were harvested and total RNA was extracted using miRNeasy Mini Kitud(Qiagen). cDNAs were synthesised specific to miR-130a using TaqMan MicroRNA ReverseudTranscription Kit (Applied Biosystems). Then, qRT-PCR was carried out using TaqMan UniversaludMaster Mix (Applied Biosystems) to quantify the knockdown level of mature miRNAs in the cells.udResult showed that miR-130a-5p was significantly downregulated in MCF7 cell line. However, noudsignificant changes were observed for sequences targeting miR-130a-3p and stem loop. Thus, thisudstudy showed that the expression of miR-130a-5p was successfully down-regulated using CRISPRudsilencing system. This technique may be useful to manipulate the level of miRNA in various celludtypes to answer clinical questions at the molecular level.