PGC-1α Regulates Cell Proliferation, Migration, and Invasion by Modulating Leucyl-tRNA Synthetase 1 Expression in Human Colorectal Cancer Cells

Abstract

Although mounting evidence has demonstrated that peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) can promote tumorigenesis, its role in cancer remains controversial. To find potential target molecules of PGC-1α, GeneFishing^TM DEG (differentially expressed genes) screening was performed using stable HEK293 cell lines expressing PGC-1α (PGC-1α-HEK293). As results, leucyl-tRNA synthetase 1 (LARS1) was upregulated. Western blot analysis showed that LARS1 was increased in PGC-1α overexpressed SW480 cells but decreased in PGC-1α shRNA knockdown SW620 cells. Several studies have suggested that LARS1 can be a potential target of anticancer agents. However, the molecular network of PGC-1α and LARS1 in human colorectal cancer cells remains unclear. LARS1 overexpression enhanced cell proliferation, migration, and invasion, whereas LARS1 knockdown reduced them. We also observed that expression levels of cyclin D1, c-Myc, and vimentin were regulated by LARS1 expression. We aimed to investigate whether effects of PGC-1α on cell proliferation and invasion were mediated by LARS1. Our results showed that PGC-1α might modulate cell proliferation and invasion by regulating LARS1 expression. These results suggest that LARS1 inhibitors might be used as anticancer agents in PGC-1α-overexpressing colorectal cancer. Further studies are needed in the future to clarify the detailed molecular mechanism by which PGC-1α regulates LARS1 expression.

Keywords: AKT; PGC-1α; colorectal cancer; invasion; leucyl-tRNA synthetase 1 (LARS1).

Figure 1

PGC-1α overexpression enhances expression of LARS1 in HEK293 cells. (A) Total RNAs isolated from PGC-1α- or pcDNA-expressing HEK293 cells were used for ACP-based GeneFishing analysis as described in Materials and Methods. Amplified bands showing different densities between two cell lines were re-amplified and sequenced for gene annotation. Arrow indicates DEG band identified as LARS1. (B) Left: The mRNA levels of PGC-1α and LARS1 were examined by qRT-PCR. β-actin is used as an endogenous control. Middle and Right: The protein levels of PGC-1α and LARS1 in control pcDNA-HEK293 and PGC-1α-HEK293 cells. Protein lysates were prepared and subjected to Western blot analysis as described in Materials and Methods. Equal protein loading was ensured by showing uniform β-actin expression. Densitometry results are indicated above the bands. Representative data of three independent experiments are shown. Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, pcDNA-HEK293 cells. (C,D) Left panel: Immunofluorescence staining was performed as described in Materials and Methods using anti-PGC-1α ((C), green) and anti-LARS1 ((D), green) antibodies. Right panel: Mean Fluorescence Intensity (MFI) of PGC-1α (C) and LARS1 (D) of immunofluorescence images were quantified using Image J software (v.1.53t). GP, arbitrary general primer; 1, control HEK293 cells; 2, PGC-1α-HEK293 cells. Molecular weights for proteins are indicated in the full, uncropped, annotated Western blot images (Figure S3).

Figure 2

LARS1 overexpression enhances cell proliferation, migration, and invasion of SW480 cells. (A) Left panel: Expression levels of PGC-1α and LARS1 mRNA in pcDNA-, PGC-1α-1-, or -2-SW480 cells. Middle panel: Expression of PGC-1α and LARS1 in SW620 and SW480 cells. Right panel: SW480 cells were transfected with LARS1 or pCMV6 expression vector and screened based on their resistance to G418 (800 μg/mL). Western blot was used to detect PGC-1α and LARS1. β-actin was used as an internal control. Densitometry results are indicated above bands. Representative data of three independent experiments are shown. Data are expressed as the mean ± SD of three independent experiments. *** p < 0.001 vs. pcDNA-SW480 cells. (B,C) pCMV6-, LARS1-2, or -4-SW480 cells were seeded and cultured for the indicated times and cell proliferation was determined by cell counting (B) and MTT assay (C). Data are presented as the mean ± SD of three separate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. pCMV6-SW480 cells. (D,E) Left panel: Representative figures of pCMV6-, LARS1-2-, and -4-SW480 cells in the transwell migration assay (D) and transwell invasion assay (E) are shown (×200 magnification). Right panel: The numbers of transmembrane migrated cells (D) and transmembrane invaded cells (E) were counted for five randomly chosen visual fields. Data are expressed as the mean ± SD of three independent experiments. *** p < 0.001 vs. pCMV6-SW480 cells. Molecular weights for proteins are indicated in the full, uncropped, annotated Western blot images (Figure S4).

Figure 3

LARS1 knockdown reduces cell proliferation, migration, and invasion of SW620 cells. (A) Left panel: Expression levels of PGC-1α and LARS1 mRNA in NC shRNA-, PGC-1α shRNA-1-, or -2-SW620 cells. Data are expressed as the mean ± SD of three independent experiments. ** p < 0.01, *** p < 0.001, NC shRNA-SW620 cells. Middle panel: SW620 cells were transfected with NC shRNA or LARS1 shRNA expression vector and screened based on their resistance to G418 (800 μg/mL). Western blot was used to detect PGC-1α and LARS1. β-actin was used as an internal control. Densitometry results are indicated above bands. Representative data of three independent experiments are shown. Right panel: Data are expressed as the mean ± SD of three independent experiments. * p < 0.05 vs. NC shRNA-SW620 cells. NS, not significant. (B,C) NC shRNA-, LARS1 shRNA-4-, or -5-SW620 cells were seeded and cultured for the indicated time and cell proliferation was determined by cell counting (B) and MTT assay (C). Data are presented as the mean ± SD of three separate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. NC shRNA-SW620 cells. (D,E) Left panel: Representative figures of NC shRNA- and LARS1 shRNA-5-SW620 cells in the transwell migration assay (D) and transwell invasion assay (E) are shown (×200 magnification). Right panel: The numbers of transmembrane migrated cells (D) and transmembrane invaded cells (E) are shown. Data are expressed as the mean ± SD of three independent experiments. *** p < 0.001 vs. NC shRNA-SW620 cells. Molecular weights for proteins are indicated in the full, uncropped, annotated Western blot images (Figure S5).

Figure 4

PGC-1α regulates cell proliferation, migration, and invasion of SW480 and SW620 cells by regulating LARS1 expression. (A–C) PGC-1α-1-SW480 cells were transiently transfected with NC shRNA vector or LARS1 shRNA expression vector, respectively. (A) After transfection, the cell counts of each type of cell are presented as the mean ± SD of three separate experiments. (B,C) Left panel: Representative figures of pcDNA-, PGC-1α-1/NC shRNA-, and PGC-1α-1/LARS1 shRNA-SW480 cells in the transwell migration assay (B) and transwell invasion assay (C) are shown (×200 magnification). Right panel: The numbers of transmembrane migrated cells (B) and transmembrane invaded cells (C) are shown. Data are expressed as the mean ± SD of three independent experiments. ** p < 0.01, *** p < 0.001 vs. pcDNA-SW480 cells. ^## p < 0.01, ^### p < 0.001 vs. PGC-1α-1/NC shRNA-SW480 cells. (D–F) PGC-1α shRNA-1-SW620 cells were transiently transfected with pCMV6 expression vector or LARS1 expression vector, respectively. (D) After transfection, the cell counts of each type of cell are presented as the mean ± SD of three separate experiments. *** p < 0.001 vs. NC shRNA-SW620 cells. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. PGC-1α shRNA-1/pCMV6-SW620 cells. (E,F) Left panel: Representative figures of NC shRNA-, PGC-1α shRNA-1/pCMV6-, and PGC-1α shRNA-1/LARS1-SW620 cells in the transwell migration assay (E) and transwell invasion assay (F) are shown (×200 magnification). Right panel: The numbers of transmembrane migrated cells (E) and transmembrane invaded cells (F) are shown. Data are expressed as the mean ± SD of three independent experiments. *** p < 0.001 vs. NC shRNA-SW620 cells. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. PGC-1α shRNA-1/pCMV6-SW620 cells. (G–I) PGC-1α shRNA-1-SW620 cells were transiently transfected with NC shRNA expression vector or LARS1 shRNA expression vector, respectively. (G) After transfection, the cell counts of each type of cell are presented as the mean ± SD of three separate experiments. (H,I) Left panel: Representative figures of NC shRNA-, PGC-1α shRNA-1/NC shRNA-, and PGC-1α shRNA-1/LARS1 shRNA-SW620 cells in the transwell migration assay (H) and the transwell invasion assays (I) are shown (×200 magnification). Right panel: The numbers of transmembrane migrated cells (H) and transwell-membrane invaded cells (I) are shown. Data are expressed as the mean ± SD of three independent experiments. *** p < 0.001 vs. NC shRNA-SW620 cells. NS, not significant.

Figure 5

Expression levels of PGC-1α, LARS1, and several signaling molecules in pcDNA-, PGC-1α-, pCMV6-, LARS1-SW480, NC shRNA-, PGC-1α shRNA-, LARS1 shRNA-SW620 cells. (A) (Left panel) Representative three independent Western blot analysis results of pcDNA-, PGC-1α-1-, PGC-1α-2-SW480 cells are shown. β-actin was used as a loading control. Densitometry results are expressed above bands. (Right panel) Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. pcDNA-SW480 cells. (B) (Left panel) Representative three independent Western blot analysis results of pCMV6-, LARS1-2-, LARS1-4-SW480 cells are shown. β-actin was used as a loading control. Densitometry results are expressed above the bands. (Right panel) Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. pCMV6-SW480 cells. (C) (Left panel) Representative three independent Western blot analysis results of NC shRNA-, PGC-1α shRNA-1-, PGC-1α shRNA-2-SW620 cells are shown. β-actin was used as a loading control. Densitometry results are expressed above bands. (Right panel) Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. NC shRNA-SW620 cells. (D) (Left panel) Representative three independent Western blot analysis results of NC shRNA-, LARS1 shRNA-4-, LARS1 shRNA-5-SW620 cells are shown. β-actin was used as a loading control. Densitometry results are expressed above bands. (Right panel) Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. NC shRNA-SW620 cells. Molecular weights for proteins are indicated in the full, uncropped, annotated Western blot images (Figures S6–S9).

Figure 5

Expression levels of PGC-1α, LARS1, and several signaling molecules in pcDNA-, PGC-1α-, pCMV6-, LARS1-SW480, NC shRNA-, PGC-1α shRNA-, LARS1 shRNA-SW620 cells. (A) (Left panel) Representative three independent Western blot analysis results of pcDNA-, PGC-1α-1-, PGC-1α-2-SW480 cells are shown. β-actin was used as a loading control. Densitometry results are expressed above bands. (Right panel) Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. pcDNA-SW480 cells. (B) (Left panel) Representative three independent Western blot analysis results of pCMV6-, LARS1-2-, LARS1-4-SW480 cells are shown. β-actin was used as a loading control. Densitometry results are expressed above the bands. (Right panel) Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. pCMV6-SW480 cells. (C) (Left panel) Representative three independent Western blot analysis results of NC shRNA-, PGC-1α shRNA-1-, PGC-1α shRNA-2-SW620 cells are shown. β-actin was used as a loading control. Densitometry results are expressed above bands. (Right panel) Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. NC shRNA-SW620 cells. (D) (Left panel) Representative three independent Western blot analysis results of NC shRNA-, LARS1 shRNA-4-, LARS1 shRNA-5-SW620 cells are shown. β-actin was used as a loading control. Densitometry results are expressed above bands. (Right panel) Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. NC shRNA-SW620 cells. Molecular weights for proteins are indicated in the full, uncropped, annotated Western blot images (Figures S6–S9).

Figure 6

LARS1 enhances cell proliferation, migration, and invasion through AKT activation in SW480 cells. (A,B) LARS1-2-SW480 cells were treated with/without 0.5 μM AKT inhibitor IV for indicated time (A) or for 48 h (B) and cell proliferation was measured by cell counting (A) and MTT assay (B), respectively. (C,D) LARS1-2-SW480 cells were treated with/without 0.5 μM AKT inhibitor IV for 48 h. Transwell migration assays (C) and invasion assays (D) were performed as described in Materials and Methods. (C) Left panel: Representative figures of pCMV6-, LARS1-2-SW480 cells treated without/with 0.5 μM AKT inhibitor IV in the transwell migration assay are shown (×200 magnification). Right panel: The number of transmembrane migrated cells was counted for five randomly chosen visual fields. Data are expressed as the mean ± SD of three independent experiments. *** p < 0.001 vs. pCMV-6-SW480 cells; ^### p < 0.001 vs. LARS1-2-SW480 cells. (D) Left panel: Representative figures of pCMV6-, LARS1-2-SW480 cells treated without/with 0.5 μM AKT inhibitor IV in the transwell invasion assay are shown (×200 magnification). Right panel: The number of transmembrane migrated cells was counted for five randomly chosen visual fields. Data are expressed as the mean ± SD of three independent experiments. *** p < 0.001 vs. pCMV-6-SW480 cells; ^### p < 0.001 vs. LARS1-2-SW480 cells. (E) After treatment with AKT inhibitor IV, protein lysates were prepared and used for Western blot analysis with corresponding antibodies. β-actin was used as a loading control. (Left panel) The blot is representative of three separate experiments. Densitometry results are expressed above bands. (Right panel) Data are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. pCMV6-SW480 cells. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. LARS1-2-SW480 cells. Molecular weights for proteins are indicated in the full, uncropped, annotated Western blot images (Figure S10).

Figure 7

Potential molecular mechanism by which PGC-1α regulates cell proliferation, migration, and invasion of human colorectal cancer cells. In summary, PGC-1α regulates cell proliferation, migration, and invasion via regulation of LARS1/AKT/GSK-3β/β-catenin axis.