Sequence-specific inhibition of microRNA-130a gene by CRISPR/Cas9 system in breast cancer cell line

10th International Seminar on Medical Physics; 1st AMDI International Oncology Symposium

物理学;医药卫生

Abdollah, Nur Ainina^1 ; Kumitaa, Theva Das^2 ; Narazah, Mohd Yusof^3 ; Abdul Razak, Siti Razila^1

Oncology and Radiological Sciences Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, Kepala Batas, Pulau Pinang

13200, Malaysia^1

Infectomics Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, Kepala Batas, Pulau Pinang

13200, Malaysia^2

Regenerative Medicine Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, Kepala Batas, Pulau Pinang

13200, Malaysia^3

关键词: Applied Biosystems;    Breast cancer cells;    Expression controls;    Molecular levels;    Non-coding RNAs;    Reverse transcription;    Silencing systems;    Transfected cells;   

Others  :  https://iopscience.iop.org/article/10.1088/1742-6596/851/1/012037/pdf
DOI  :  10.1088/1742-6596/851/1/012037

学科分类：卫生学

来源: IOP

【 摘 要 】

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)-2A-GFP (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 °C 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.