Modulation of gene expression after inducing expression of 14q32 miRNAs by CRISPR activation technology in lung adenocarcinoma

Most lung adenocarcinoma deaths are related to metastases, indicating the necessity of detecting and inhibiting tumor cell dissemination. We have identified that overexpression of miRNAs located on 14q32 was associated with metastasis in lung adenocarcinoma patients. For functional analysis, we utilized CRISPR activation technology to increase levels of miRNAs clustered on 14q32 in a coordinated manner, and the results showed that 14q32 miRNA overexpression promoted tumor cell migratory and invasive properties. Whole transcriptome microarray analysis of the miRNA-overexpressing cells was performed to define the underlying molecular mechanisms. Microarray analysis was used to investigate the gene expression changes underlying the tumor cell migratory and invasive properties induced by 14q32 miRNAs in lung adenocarcinoma. CRISPR-Cas9-based gene activation technology (CRISPRa) using a mutant Cas9 lacking endonuclease activity (dCas9) fused with the activating domains VP64, p65, and Rta (VPR) was used to generate cell lines overexpressing the miRNAs located on 14q32. Two different miRNA-overexpressing cell clones were generated from H2009 lung adenocarcinoma cells by using two distinct single guide RNAs (sgRNA-A and sgRNA-B). Cells transfected with the empty sgRNA vector were used as control. Increased expression of 14q32 miRNA levels in CRISPRa-sgRNA-A and CRISPRa-sgRNA-B cells in comparison with CRISPRa-control cells was confirmed by RT-qPCR before microarray expression analysis. Three biological replicates from each cell clone were used for hybridization on Affymetrix microarrays.