CRISPR/Cas9 genomic editing to evaluate the role of MMP9 and microRNA-21 in metastatic prostate cancer: In vitro and in vivo study

Author(s)

Juliana Alves de Camargo

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2022-05-03

Examining board members

Sabrina Thalita dos Reis Faria; Katia Ramos Moreira Leite; André Almeida Schenka

• Advisor: Sabrina Thalita dos Reis Faria

Abstract

INTRODUCTION: Prostate Cancer (PCa) has a high prevalence and represents an important health problem, with a strong economic impact if we consider screening, diagnosis, treatment, and mortality from the disease. With the advance of CRISPR methodology, new possibilities have opened for the understanding and control of cellular resistance mechanisms. Gene editing by the CRISPR-Cas9 technique is effective in correcting cellular mutations that promote resistance. The microRNA-21, which acts on the tumor suppressor gene RECK and the oncogene MMP-9, has an important role in the process of migration and invasion of tumor cells into other tissues, generating metastases. Due to the importance of these molecules in carcinogenesis, further investigations of their role in PCa are needed. OBJECTIVE: To evaluate the role of MMP-9 and the indirect regulator miR-21 in PCa, with gene editing by CRISPR-Cas9 technique. METHODS: Initially, insertion of the MMP9 and miR-21 guide RNAs (sgRNA) sequences into the PX-330 plasmid was performed. Then, the plasmids with the inserts for MMP-9 were transfected into PCa DU145 and PC-3M-luc-C6 cell lines. MMP9 gene and protein expression analysis was performed by qPCR, Western Blotting, and immunofluorescence. The target genes of miR-21, including RECK, MARKS, BTG2, PDCD4, the integrins ITGB1 and ITGB3, CDH1, BAX and mTOR, were evaluated by qPCR. Flow cytometry was performed to validate protein expression related to apoptosis and cell proliferation, in cells edited for MMP9 and miR-21, using the markers Annexin5, 7-AAD andKi67 respectively. Matrigel invasion assays were performed, and cells were analyzed after 48 hours, with light microscopy. The control group consisted of cells transfected with plasmid PX-330 without the sgRNA insertions(Scramble). Tumor growth was assessed by an in vivo bioluminescence system in mice Balb/c Nude. After injection of 300.000 cells, divided into groups, Scramble, edited for MMP9 and miR-21 with CRISPR-Cas9 These animals were followed up for 14 days. RESULTS: We standardized the techniques of digestion and insertion of the sgRNAs of MMP9 and miR-21 into the plasmid PX-330, validated by sequencing. After this step, we observed that the cell lines transfected with the plasmid with sgRNA1 and 2 for MMP9 showed positive GFP, which shows the efficiency of the transfection. Cells edited for miR-21 with CRISPR-Cas9 showed increased gene expression of RECK, MARKS, BTG2, and PDCD4 compared to the Scramble group. CDH1 and integrins ITGB3 and ITGB1 were increased in cells edited for MMP9 and miR-21 with CRISPR-Cas9 when compared to Scramble. Increased BAX and decreased mTOR gene expression were observed in cells edited for MMP9 and miR-21 with CRISPR-Cas9 compared to the Scramble group. These results were validated by flow cytometry, showing reduced proliferation and increased apoptosis in cells edited for MMP9 and miR-21 with CRISPR-Cas9 compared to Scramble. In the cell invasion assay, we observed that cells edited for MMP9 and miR-21 have lower invasion rates than the Scramble group. In the in vivo experiments, tumor growth was significantly reduced in animals that received cells edited with CRISPR-Cas9 for MMP9 compared to Scramble. We found no difference in animals that received cells edited for miR-21. CONCLUSION: Gene editing by CRISPR-Cas9 for MMP9 and miR-21 in metastatic PCa cell lines modulates molecular factors that attenuate cell proliferation and invasion and stimulate cell apoptosis, possibly preventing PCa tumor evolution (AU)

• FAPESP's process: 18/19906-3 - Genomic edition with CRISPR/Cas9 to evaluate the role of MMP9 and the regulator miR21 in Prostate Cancer
• Grantee: Juliana Alves de Camargo
• Support Opportunities: Scholarships in Brazil - Doctorate