Implication of leucyl-tRNA synthetase 1 (LARS1) over-expression in growth and migration of lung cancer cells detected by siRNA targeted knock-down analysis

Abstract

Molecular mechanism of lung carcinogenesis and its aggressive nature is still largely elusive. To uncover the biomarkers related with tumorigenesis and behavior of lung cancer, we screened novel differentially expressed genes (DEG) in A549 lung cancer cell line by comparison with CCD-25Lu, normal pulmonary epithelial cell line, using annealing control primer(ACP)-based GeneFishing system. Of the DEGs, over-expression of leucyl-tRNA synthetase 1 (LARS1) was prominent and this up-regulation was confirmed by immunoblotting and real-time quantitative RT-PCR analysis. In addition to A549 cell line, primary lung cancer tissues also expressed higher level of LARS1 mRNA than their normal counter tissues. To explore the oncogenic potential of LARS1 over-expression in lung cancer, we knocked-down LARS1 by treating siRNA and observed the tumor behavior. LARS1 knock-down cells showed reduced ability to migrate through transwell membrane and to form colonies in both soft agar and culture plate. Taken together, these findings suggest that LARS1 may play roles in migration and growth of lung cancer cells, which suggest its potential implication in lung tumorigenesis.

Figure 1

Over-expression of LARS1 gene in lung cancer cells. (A) Total RNA of CCD-25Lu and A549 cells were used for ACP-based GeneFishing analysis as described in Materials and Methods. The amplicon bands showing different intensities between two cell lines were re-amplified and sequenced for gene annotation. Arrow indicates a DEG band turned out to be LARS1. (B) Expression level of LARS1 in each cell was determined by qRT-PCR as described in Materials and Methods. As an internal control, GAPDH mRNA was used. (C) Cell lysates of CCD-25Lu and A549 cells were used for immunoblot analysis with anti-LARS1 antibody. β-actin was probed for equal protein loading. (D) Expression level of LARS1 in 17 primary lung cancer samples was determined by qRT-PCR as described in Materials and Methods. Ten of the 17 primary cancers showed over two fold up-regulation of LARS1 (criteria of over-expression in this study) compared to normal control lung tissues.

Figure 2

Down-regulation of LARS1 expression in A549 cells by treating siRNA against LARS1 (siLARS). (A) A549 cells were transfected with 50 nM of siLARS or siCon and incubated for 14 hours. Then LARS1 expression was compared between siLARS and siCon treated cells by qRT-PCR as described in Materials and Methods. As an internal control, GAPDH mRNA was used. (B) LARS1 protein expression was compared between the same cells by immunoblot analysis. β-actin was probed for equal protein loading.

Figure 3

Effect of siLARS on colony formation and migration of A549 cells. (A) Colony formation assay. A549 cells were transfected with siCon and siLARS, respectively, and incubated in 6-well plates for 6 days. Colonies larger than 1 mm in diameter were counted. Bar chart in the right box represents the percentage of colony numbers in siLARS-treated cells to those in siCon-treated cells. Data represents mean of percentages ± SD of three independent experiments. (B) Soft agar assay. Same cells were incubated in low-melting agarose as described in Materials and Methods. Two weeks later, colonies were photographed and numbers of colonies were counted. (C) Transwell assay. Same cells were incubated in upper chamber of 24-well transwell chambers for migration assay. After 24 h, migrated cells were counted as described in Materials and Methods. The migrated cells were photographed and percentages of migrated siLARStreated cells to migrated siCon-treated cells were calculated. Data represents mean of percentages ± SD of three dependent experiments.