106P - Knockout of lncRNA-CCAT1 with the use of CRISPR-Cas9 system and G7 PAMAM dendrimers influences apoptosis and proliferations of NSCLC cells

Background

Colon cancer-associated transcript 1 (CCAT-1) is an oncogenic lncRNA that has been emerged as a vital biomarker for diagnosis, prognosis and therapeutic interventions in multiple malignancies. The previous studies showed that lncRNA-CCAT1 was upregulated in NSCLC cells and its expression was related to tumor growth and reduced survival rate. The aim of our study was to evaluate influence of the knockout of lncRNA-CCAT1 with the use of CRISPR-Cas9 system and G7 PAMAM dendrimers on apoptosis and proliferations of NSCLC cells.

Methods

We used two human lung adenocarcinoma cell lines: A549, H1975 and H1703 squamous cell carcinoma cell line. The knockout of the lncCCAT expression was performed using the CRISPR-Cas9 system and G7 PAMAM dendrimers. We used 4 combinations of gRNAs. The apoptosis of NSCLC after lncRNA-CCAT1 knockout was estimated with the use of flow cytometry and Annexin V staining, evaluation of caspase-3/7 and measurement of mitochondrial membrane potential changes. Expression of Ki67 was measured by flow cytometry to evaluate NSCLC cells proliferation. All mentioned above parameters were evaluated 24, 48 and 72 hours after transfection. Nonparametric ANOVA tests was used for statistical analysis.

Results

We found that transfection with conjugates of G7 PAMAM dendrimers and px459 v2.0, the appropriate gRNAs (for lncCDH5-3:3 knockout), and pcDNA3.1 plasmid are downregulating expression of lncRNA-CCAT1. We confirmed that apoptosis of NSCLC was increased after transfection and cells proliferation was reduced. We also found differences in timing and intensity of biological effects when different combination of gRNAs are used in particular NSCLC cell lines.

Conclusions

The conjugates of G7 PAMAM dendrimers and px459 v2.0, the appropriate gRNAs (for lncCDH5-3:3 knockout), and pcDNA3.1 plasmid can be used for knockout of the expression of lncRNA-CCAT1. On the other hand the gRNAs shall be individually chosen for particular NSCLC cells according to their genetic mutation status.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

University of Rzeszow.

Funding

Polish Ministry for Higher Education and Science.

Disclosure

All authors have declared no conflicts of interest.