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The Effect of Gene Editing by CRISPR-Cas9 of miR-21 and the Indirect Target MMP9 in Metastatic Prostate Cancer
Juliana A Camargo1, Nayara I Viana1,2, Ruan Pimenta1,3
1Laboratory of Medical Investigation (LIM 55), Urology Department, Medicine School, University of Sao Paulo (FMUSP), São Paulo 01246-903, SP, Brazil.
Abstract:
Prostate cancer (PCa) has a high prevalence and represents an important health problem, with an increased risk of metastasis. With the advance of CRISPR-Cas9 genome editing, new possibilities have been created for investigating PCa. The technique is effective in knockout oncogenes, reducing tumor resistance. MMP9 and miR-21 target genes are associated with PCa progression; therefore, we evaluated the MMP-9 and miR-21 targets in PCa using the CRISPR-Cas9 system. Single guide RNAs (sgRNAs) of MMP9 and miR-21 sequences were inserted into a PX-330 plasmid, and transfected in DU145 and PC-3 PCa cell lines. MMP9 and RECK expression was assessed by qPCR, WB, and IF. The miR-21 targets, integrins, BAX and mTOR, were evaluated by qPCR. Flow cytometry was performed with Annexin5, 7-AAD and Ki67 markers. Invasion assays were performed with Matrigel. The miR-21 CRISPR-Cas9-edited cells upregulated RECK, MARCKS, BTG2, and PDCD4. CDH1, ITGB3 and ITGB1 were increased in MMP9 and miR-21 CRISPR-Cas9-edited cells. Increased BAX and decreased mTOR were observed in MMP9 and miR-21 CRISPR-Cas9-edited cells. Reduced cell proliferation, increased apoptosis and low invasion in MMP9 and miR-21 edited cells was observed, compared to Scramble. CRISPR-Cas9-edited cells of miR-21 and MMP9 attenuate cell proliferation, invasion and stimulate apoptosis, impeding PCa evolution.
Insights
CRISPR-Cas9 gene editing effectively targeted MMP9 and miR-21 in prostate cancer (PCa) cells. This approach reduced PCa cell proliferation and invasion while increasing apoptosis, offering a promising strategy to impede cancer progression.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Prostate cancer (PCa) is a prevalent disease with a high risk of metastasis.
- CRISPR-Cas9 genome editing offers novel avenues for PCa research and therapeutic development.
- MMP9 and miR-21 are implicated in PCa progression and metastasis.
Purpose of the Study:
- To investigate the role of MMP9 and miR-21 targets in prostate cancer using the CRISPR-Cas9 system.
- To evaluate the impact of CRISPR-Cas9-mediated gene editing of MMP9 and miR-21 on PCa cell behavior.
Main Methods:
- CRISPR-Cas9 technology was employed to edit MMP9 and miR-21 in DU145 and PC-3 PCa cell lines.
- Gene expression (MMP9, RECK, integrins, BAX, mTOR) was assessed using qPCR, Western Blot (WB), and Immunofluorescence (IF).
- Cell proliferation, apoptosis, and invasion were evaluated using flow cytometry (Annexin V, 7-AAD, Ki67) and Matrigel invasion assays.
Main Results:
- CRISPR-Cas9 editing of miR-21 upregulated RECK, MARCKS, BTG2, and PDCD4.
- MMP9 and miR-21 editing increased CDH1, ITGB3, and ITGB1 expression.
- Edited cells showed increased BAX, decreased mTOR, reduced proliferation, enhanced apoptosis, and diminished invasion compared to controls.
Conclusions:
- CRISPR-Cas9-mediated editing of MMP9 and miR-21 effectively attenuates prostate cancer cell proliferation and invasion.
- This gene-editing strategy stimulates apoptosis, presenting a potential therapeutic approach to impede PCa evolution.
- Targeting MMP9 and miR-21 with CRISPR-Cas9 holds promise for controlling prostate cancer progression.
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