Exploring miR-21 Knock-Out Using CRISPR/Cas as a Treatment for Lung Cancer

Abstract

Background: Lung cancer is a leading cause of cancer-related deaths worldwide. Its high incidence and poor prognosis demonstrate the need to investigate new therapies. The PI3K/AKT pathway is activated in carcinogenic processes such as invasion, proliferation, and drug resistance. MiR-21 is a microRNA overexpressed in numerous types of cancer and which activates PI3K/AKT pathway by down-regulating its main targets, PTEN and PDCD4. CRISPR is a revolutionary gene-editing technology that allows genes to be deleted. The aim of this study was to use CRISPR/Cas9 technology as an option to reduce carcinogenic and drug resistance processes by eliminating miR-21.

Methods: CRISPR/Cas9 was used to knock out miR-21 (miR-21 KO) in A549 lung cancer cells and thus reverse the carcinogenic processes activated by miR-21 overexpression. Furthermore, the effect of miR-21 KO on drug resistance was studied, choosing the main chemotherapeutic agents used for the treatment of lung cancer: gemcitabine, carboplatin, paclitaxel, and oxaliplatin.

Results: miR-21 KO A549 cells exhibited a reduction in proliferation, migration, and colony formation compared to A549 cells. In contrast, the expression of PTEN and PDCD4 increased in miR-21 KO A549 cells. Furthermore, miR-21 KO A549 cells showed a decrease in the IC₅₀ of the drugs used for the treatment of lung cancer: gemcitabine, carboplatin, paclitaxel, and oxaliplatin.

Conclusions: Based on these results, miR-21 knock-out using CRISPR/Cas could be a promising strategy for the treatment of lung cancer.

Keywords: A549 cells; CRISPR/Cas9; lung cancer; miR-21; miRNA.

Figure 1

Strategy to knock out miR-21 in A549 cells. (A) Schematic of the strategy designed to generate miR-21 KO A549 cells. (B). Representative scheme of the nucleofection process with RNP and analysis of the edited cells. Image created with https://www.biorender.com/ (23 September 2023). (C) Editing efficiencies of CRISPR/Cas9 targeted in miR-21, determined by the ICE algorithm (see M&M). (D) Graph showing the profile of indels generated in miR-21 KO A549 cells nucleofected using the ICE algorithm. The coordinate zero represents the cutite, negative values represent deletions of different lengths, and positive values represent insertions.

Figure 2

Proliferation, migration, and colony formation analysis of miR-21 KO A549 cells. (A) Graphical representation of cell growth of A549 cells and miR-21 KO A549 cells. Statistical analysis performed by ANOVA test. Values represent means ± SD (n = 3). * p < 0.1 ** p < 0.01, and *** p < 0.001. (B) Graphical representation of the percentage of cell migration in A549 cells and miR-21 KO A549 cells (area of the scratch). Statistical analysis performed by ANOVA test. Values represent means ± D (n = 3). ** p < 0.01. Representative microscopic images (right) of wound-healing assays. Scale bar: 200 µm. (C) Graphical representation of the number of colonies formed by A549 cells and miR-21 KO A549 cells. Statistical analysis performed by ANOVA test. Values represent means ± D (n = 3). ** p < 0.01. Representative image (right) of colony formation.

Figure 3

Graphical representation of the IC₅₀ results of gemcitabine, carboplatin, paclitaxel, and oxaliplatin. Statistical analysis performed by ANOVA test. Values represent means ± D (n = 3). *** p < 0.001.

Figure 4

Analysis of PTEN and PDCD4 expression. Relative expression levels were calculated upon GADPH (housekeeping gene) normalization, respectively. Statistical analysis performed by ANOVA test. Values represent means ± D (n = 3). * p < 0.1 ** p < 0.01.