| Summary: | MicroRNAs (miRNAs) are short stranded noncoding RNA that play important roles in
apoptosis, cell survival, development and cell proliferation. However, gene expression control via
small regulatory RNA, particularly miRNA in breast cancer is still less explored. Therefore, this
project aims to develop an approach to target microRNA-130a using the Clustered Regularly
Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system in MCF7, breast cancer cell line. The
20 bp sequences target at stem loop, 3' and 5' end of miR130a were cloned into pSpCas9(BB)-2AGFP
(PX458) plasmid, and the positive clones were confirmed by sequencing. A total of 5 μg of
PX458-miR130a was transfected to MCF7 using Lipofectamine® 3000 according to manufacturer’s
protocol. The transfected cells were maintained in the incubator at 37 ⁰C under humidified 5% CO2.
After 48 hours, cells were harvested and total RNA was extracted using miRNeasy Mini Kit
(Qiagen). cDNAs were synthesised specific to miR-130a using TaqMan MicroRNA Reverse
Transcription Kit (Applied Biosystems). Then, qRT-PCR was carried out using TaqMan Universal
Master Mix (Applied Biosystems) to quantify the knockdown level of mature miRNAs in the cells.
Result showed that miR-130a-5p was significantly downregulated in MCF7 cell line. However, no
significant changes were observed for sequences targeting miR-130a-3p and stem loop. Thus, this
study showed that the expression of miR-130a-5p was successfully down-regulated using CRISPR
silencing system. This technique may be useful to manipulate the level of miRNA in various cell
types to answer clinical questions at the molecular level.
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