Analysis of lung cancer-related genetic changes in long-term and low-dose polyhexamethylene guanidine phosphate (PHMG-p) treated human pulmonary alveolar epithelial cells

Abstract

Background: Lung injury elicited by respiratory exposure to humidifier disinfectants (HDs) is known as HD-associated lung injury (HDLI). Current elucidation of the molecular mechanisms related to HDLI is mostly restricted to fibrotic and inflammatory lung diseases. In our previous report, we found that lung tumors were caused by intratracheal instillation of polyhexamethylene guanidine phosphate (PHMG-p) in a rat model. However, the lung cancer-related genetic changes concomitant with the development of these lung tumors have not yet been fully defined. We aimed to discover the effect of long-term exposure of PHMG-p on normal human lung alveolar cells.

Methods: We investigated whether PHMG-p could increase distorted homeostasis of oncogenes and tumor-suppressor genes, with long-term and low-dose treatment, in human pulmonary alveolar epithelial cells (HPAEpiCs). Total RNA sequencing was performed with cells continuously treated with PHMG-p and harvested after 35 days.

Results: After PHMG-p treatment, genes with transcriptional expression changes of more than 2.0-fold or less than 0.5-fold were identified. Within 10 days of exposure, 2 protein-coding and 5 non-coding genes were selected, whereas in the group treated for 27-35 days, 24 protein-coding and 5 non-coding genes were identified. Furthermore, in the long-term treatment group, 11 of the 15 upregulated genes and 9 of the 14 downregulated genes were reported as oncogenes and tumor suppressor genes in lung cancer, respectively. We also found that 10 genes of the selected 24 protein-coding genes were clinically significant in lung adenocarcinoma patients.

Conclusions: Our findings demonstrate that long-term exposure of human pulmonary normal alveolar cells to low-dose PHMG-p caused genetic changes, mainly in lung cancer-associated genes, in a time-dependent manner.

Keywords: Human pulmonary alveolar epithelial cells; Humidifier disinfectant; Lung cancer related genes; Polyhexamethylene guanidine phosphate; Total RNA sequencing.

Fig. 1

Cell viability evaluation of human pulmonary alveolar epithelial cells (HPAEpiCs) after polyhexamethylene guanidine phosphate (PHMG-p) treatment. HPAEpiCs were exposed to PHMG-p in a 10-point dose for 24 h, 48 h, and 72 h. All experiments were performed 3 times with biological replicates. Statistically significant differences were analyzed using one-way analysis of variance (ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus 0 µg/mL 24 h; ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001, ^^^^ p < 0.0001 versus 0 µg/mL 48 h; + p < 0.05, +  + p < 0.01, +  +  + p < 0.001, +  +  +  + p < 0.0001 versus 0 µg/mL 72 h.)

Fig. 2

Schematic of PHMG-p low-dose exposure with long-term subculture and cell harvesting. Odd numbers indicate control samples and even numbers indicate PHMG-p-treated samples. The detailed description is present in the main text

Fig. 3

Effects of PHMG-p on the gene expression of HPAEpiCs. (a) Heatmap of selected genes (more than twofold; P < 0.05) in HPAEpiCs after exposure to 1 ug/mL of PHMG-p. Left panel indicates altered genes in each group of short-term treatment and right panel indicates altered genes in long-term treatment group. (b) Gene ontology enrichment analysis of the top 10 biological processes for selected genes

Fig. 4

Changes in transcriptional expression levels (more than twofold or less than 0.5-fold) in PHMG-p treated HPAEpiCs. (a) Numbers of commonly changed genes are shown in a Venn diagram. Upregulated gene counts are shown in bold and downregulated gene counts are underlined. (b) Upregulated and downregulated genes at short-term treatment (within 10 days) and long-term treatment (within 35 days), respectively

Fig. 5

RT-qPCR validation for upregulated protein coding genes in the long-term treatment group. To validate the upregulated candidate genes on total RNA sequencing, the expression of MX1, KRT19, HMGA2, ISG15, IL33, MMP1, TRPA1, IFI6, PLAU, CDKN1A, PLAT, AK5 and NT5E was determined by RT-qPCR. All selected genes were verified to be increased in the long-term treatment group (within 35 days from 27 days). All experiments were performed 3 times with technical replicates

Fig. 6

RT-qPCR validation for downregulated protein coding genes in the long-term treatment group. To validate the upregulated candidate genes on total RNA sequencing, the expression of NDUFA4L2, SLITRK6, TIMP3, FMO3, HBG1, MGP, HBG2, TBX4, GPX3, and FMO2 was determined by RT-qPCR. All selected genes were verified to be decreased in the long-term treatment group. All experiments were performed 3 times with technical replicates

Fig. 7

Clinical significance of selected genes in patients with lung adenocarcinoma. Kaplan–Meier survival analysis generated for groups of patients based on the expression levels of upregulated genes (ISG15, MMP1, TRPA1 and KRT19) (a) and downregulated genes (FMO3, COL14A1, FMO2 and TIMP3) (b) in the database