Cancer-testis gene PIWIL1 promotes cell proliferation, migration, and invasion in lung adenocarcinoma

Abstract

Piwi-like RNA-mediated gene silencing 1 (PIWIL1) has been identified as a novel extremely highly expressed cancer-testis (CT) gene in lung adenocarcinoma. However, the exact function and mechanism of PIWIL1 in lung adenocarcinoma remains unclear. Herein, we sought to investigate the role of PIWIL1 in the occurrence and development of lung adenocarcinoma. We examined the expression pattern of PIWIL1 in The Cancer Genome Atlas (TCGA) lung adenocarcinoma samples, and validated it by Real-Time PCR (RT-PCR) in additional 21 paired lung adenocarcinoma tissues and 16 normal tissues. Subsequently, we explored the biological function of PIWIL1 in A549 and H1299 cell lines by gain and loss-of-function analyses. Using TCGA lung adenocarcinoma data, we further performed coexpression and Gene Ontology (GO) analyses, and analyzed the association of DNA methylation levels in PIWIL1 promoter region with its expression. Finally, we evaluated its expression in different mutation status of significantly mutated genes (SMGs) in TCGA lung adenocarcinoma data. We observed that PIWIL1 was expressed in testis and lung adenocarcinoma but not in other normal tissues, and its high expression was associated with shortened survival of lung cancer patients. Overexpression of PIWIL1 could facilitate the proliferation, invasion and migration of lung adenocarcinoma cells and vice versa. GO analysis revealed that PIWIL1 upregulated genes were enriched in embryonic development, cell proliferation and regulation of transcription. Moreover, promoter DNA hypomethylation of PIWIL1 could contribute to its aberrant expression in tumors. Interestingly, PIWIL1 expression was significantly higher in patients without hepatocyte growth factor (HGF) or serine/threonine kinase 11 (STK11) mutation (P = 0.006 and 0.005, respectively). PIWIL1 is an epidriver gene in lung adenocarcinoma, indicating a potential target for further therapy.

Keywords: PIWIL1; Cancer-testis genes; DNA methylation; Epi-driver genes; Lung adenocarcinoma.

Figure 1

PIWIL 1 was overexpressed in lung adenocarcinoma tissues and the testis tissue. (A) PIWIL1 mRNA level was significantly higher in lung adenocarcinoma compared with paired normal tissues (n = 57) based on the TCGA lung adenocarcinoma dataset. (B) PIWIL1 expression was significantly increased in tumor tissues compared with matched adjacent normal tissues (n = 21) from Nantong Cancer hospital. (C) RT‐PCR verified the testis‐enriched expression of PIWIL1 from human MTC panels.

Figure 2

PIWIL 1 facilitated the proliferation, migration and invasion of lung adenocarcinoma cell. (A) RT‐PCR (left panel) and western blot (right panel) demonstrated expression levels of PIWIL1 in A549 and H1299 cells. β‐actin was used as a positive control for RT‐PCR. Tubulin was detected as the internal control for western blot. (B) According to western blot analysis, PIWIL1 expression in A549 and H1299 cells was significantly increased or inhibited after lentiviral transduction (PIWIL1+) or siRNA transfection (siPIWIL1), respectively. (C) The effects of PIWIL1 on the cell growth in A549^{ex PIWIL 1} cells (upper panel) and H1299^{si RNA} cells (lower panel) measured by CCK8 assay. (D) Cell proliferation was assessed by EdU (Images, 100 × ). The histogram shows the EdU‐positive cells in A549^{ex PIWIL 1} cells and H1299^{si RNA} cells (right panel). (E) Colonies were counted in A549^{ex PIWIL 1} cells and H1299^{si RNA} cells. (F) Representative images of Migration and (G) Matrigel invasion assays in A549^{ex PIWIL 1} cells and H1299^{si RNA} cells (Images, 100×). Quantification of cells was shown at the right. We conducted the experiments in triplicate. Data are presented as means ± SD (SD) (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3

PIWIL 1 expression is regulated by promoter hypomethylation in lung adenocarcinoma. (A) Schematic diagrams showed a CpG island located in the promoter and first exon of PIWIL1 gene. CpG sites were shown as black bars. The region of CpG sites analyzed by BSP was indicated by a horizontal line marked with BSP, spanning 410 bp. (B) A negative correlation between DNA methylation and PIWIL1 mRNA level from the TCGA lung adenocarcinoma dataset (n = 426). ρ: Spearman's rank correlation coefficient. (C) The methylation status of 46 CpG sites was analyzed by BSP in A549 and H1299 cells and (D) the second paired lung adenocarcinoma tissues. Filled and open circles represented methylated and unmethylated CpG sites, respectively. BSP, bisulfite sequencing PCR.