Lipofection of Single Guide RNA Targeting MMP8 Decreases Proliferation and Migration in Lung Adenocarcinoma Cells
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Published on: July 21, 2018
Oscar David Hernandez Maradiaga1, Pooi Ling Mok2,3,4, Gothai Sivapragasam5
1Department of Medical Microbiology and Parasitology, Universiti Putra Malaysia, Serdang 43400, Malaysia.
Abstract:
Background and Objectives: Matrix metalloproteinases (MMP) have been implicated as major determinants of tumour growth and metastasis, which are considered two of the main hallmarks of cancer. The interaction of MMP8 and other signalling molecules within and adjacent tumoral tissues, including immune cells, are rather elusive, particularly of adenocarcinoma cell type. In this study, we aimed to investigate the role of MMP8 in non-small cell lung cancer proliferation and invasiveness potential. Materials and Methods: We individually lipofected with two different single guide RNA (sgRNAs) that specifically targeted on MMP8, with CRISPR-Cas 9 protein into the cells. Results: Our results clearly indicated that the lipofection of these complexes could lead to reduced ability of A549 cells to survive and proliferate to form colonies. In addition, when compared to non-transfected cells, the experimental cell groups receiving sgRNAs demonstrated relatively decreased migration rate, hence, wider wound gaps in scratch assay. The quantitative real time-polymerase chain reaction (qRT-PCR) demonstrated significant reduction in the MAP-K, survivin and PI3-K gene expression. MMP8 might have protective roles over tumour growth and spread in our body. Conclusions: The delivery of sgRNAs targeting on the MMP8 gene could induce tumour cell death and arrest cell migratory activity.
Insights
Targeting matrix metalloproteinase-8 (MMP8) with CRISPR-Cas9 inhibited non-small cell lung cancer cell survival and migration. This suggests MMP8 may play a protective role in tumor progression.
Area of Science:
- Oncology
- Molecular Biology
- Gene Editing
Background:
- Matrix metalloproteinases (MMPs), including MMP8, are implicated in tumor growth and metastasis.
- The specific role of MMP8 in adenocarcinoma, particularly non-small cell lung cancer (NSCLC), remains unclear.
- Understanding MMP8's function is crucial for developing targeted cancer therapies.
Purpose of the Study:
- To investigate the role of MMP8 in the proliferation and invasiveness of non-small cell lung cancer (NSCLC) cells.
- To evaluate the potential of targeting MMP8 using CRISPR-Cas9 gene editing technology.
- To assess the impact of MMP8 inhibition on key cancer-related signaling pathways.
Main Methods:
- Utilized CRISPR-Cas9 gene editing with single guide RNAs (sgRNAs) to specifically target and inhibit the MMP8 gene in A549 NSCLC cells.
- Assessed cell viability and colony formation post-transfection.
- Performed scratch assays to evaluate cell migration rates.
- Quantified gene expression of MAP-K, survivin, and PI3-K using quantitative real-time PCR (qRT-PCR).
Main Results:
- CRISPR-Cas9 mediated MMP8 inhibition significantly reduced A549 cell survival and proliferation.
- MMP8-targeted cells exhibited decreased migration rates in scratch assays.
- Quantitative real-time PCR revealed significant downregulation of MAP-K, survivin, and PI3-K gene expression following MMP8 inhibition.
- These findings suggest MMP8 may possess tumor-protective functions.
Conclusions:
- Targeting the MMP8 gene with sgRNAs effectively induces cell death and inhibits migratory activity in NSCLC cells.
- MMP8 inhibition presents a potential therapeutic strategy for controlling NSCLC progression.
- Further research into MMP8's role could uncover novel therapeutic targets for lung cancer.
