مقالات پذیرفته شده در ششمین کنگره بین المللی زیست پزشکی
Targeting miRNAs with CRISPR/Cas System in Cancer
Targeting miRNAs with CRISPR/Cas System in Cancer
Negar Sadeghi,1,*
1. Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran
Introduction: Cancer is a fatal disease that requires multiple gene mutations and alters epigenetics throughout the genome. In a cancer cell, oncogenes are activated and tumor suppressors are inactivated, as well as dysregulation of the epigenome, which regulates normal gene expression, happens. MicroRNAs (miRNA) are short, small, and non-coding protein RNA molecules with a length of 20–24 nucleotides. These tiny RNAs can function as oncogenes or tumor suppressors in cancerous cells. CRISPR (clustered regularly interspaced short palindromic repeats) and Cas (CRISPR-associated) proteins are crucial elements of an ancient bacterial adaptive immune system. The Cas proteins are divided into two main classes. Mostly the class 2 systems (type II Cas9 and Cas12a) have been adopted for gene editing applications. The main difference between these two Cas is the ability of Cas12a to process the pre-crRNA (CRISPR RNA) itself so this Cas protein can be used for multiplex editing. Furthermore, Cas12a has only one nuclease domain (RuvC domain) and induces breaks in double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). Cas12 can be guided by its processed-crRNA to recognize complementary ssDNA or dsDNA with protospacer-adjacent motif (PAM) sequences and cleaves target DNA. The CRISPR-Cas system with the target-specific binding and cleavage has been applied in various fields such as genetics, gene therapy, and molecular diagnosis. Glioblastoma is the most widespread and lethal primary tumor of the central nervous system. Several studies have shown aberrant expression of miR-21 in many cancers including glioblastoma with a high expression of miR-21. In a survey, the knocking out of this important miRNA in glioma caused decreased proliferation capacity of these cells.
Methods: For studying the knocking-out function of CRISPR-Cas12a on miR-21 in glioma cells, animal models, and cell cultures were used. A combination of encoding plasmids with different sgRNAs for miR-21 and a plasmid expressing Cas12a were transfected to glioma cultured cells. Cell sorting was done using different fluorescence dyes, and cells with both Cas12a and sgRNA plasmid were selected. miRNA levels were analyzed using the TaqMan qRT-PCR. In the following steps, DNA and RNA sequencing, and western blotting were carried out to validate the CRISPR activity and to verify the miR-21 knock-out in mouse and human glioma lines. Then for imaging, immunofluorescence staining using anti-GFP was accomplished. Moreover, different assays were applied for evaluating the amount of cell proliferation, migration, and invasion.
Results: Following a comparison of expression levels of miR-21 in wild-type (WT) and CRISPR-edited human and mouse cells, it was shown that miR-21 levels of the miR-21 knockout (KO) of mouse and human glioma cells were significantly lower than the WT cells. Next, the CRISPR-edited clones were aligned to the WT sequences to analyze the CRISPR-induced insertion-deletion (INDEL). There were no changes outside of the intended target. NGS results represented that miR-21 KO cell lines showed INDELs around the cut site compared with no INDELs in the WT sequence. Moreover, RNA-seq analysis of downstream miR-21 targets showed that the lack of miR-21 resulted in enhanced levels of several miR-21-regulated anti-proliferation mRNAs. Proliferation was significantly decreased in miR 21 KO mouse and human glioma cells, as compared with miR-21 WT cell lines. Also, animal experiments showed that the mice injected with miR-21 KO cells indicated a reduction in tumor growth and an increase in survival compared with the mice injected with miR-21 WT cells.
Conclusion: CRISPR-Cas system is a reliable and powerful gene modifier technology by taking advantage of the sequence-specific target binding and cleavage. It provides an incredible method for genome analysis, regulation, and editing which impacts the treatment and prognoses of patients with cancer. Disruption in the regulation of miRNAs expression in cancer cells can act as either a tumor suppressor or oncogene, resulting in inhibition or promotion of tumor development, respectively. In many cancers, including glioma, miR-21 is highly expressed and functions as an oncogene regulating many various downstream mRNA targets. Knocking out of miR-21 in glioma using the CRISPR technique was confirmed by non-detectable miR-21 expression levels and NGS. This deletion resulted in reduced proliferation, migration, and invasion of these cells in vitro, as well as increased survival of tumor-bearing mice in vivo.