Downregulation of MGAT3 Promotes Benzo[ a]pyrene-Mediated Lung Carcinogenesis by Regulating Cell Invasion and Migration Activity

Abstract

Environmental chemical carcinogens are major factors in the induction of lung cancer, with benzo[a]pyrene (B[a]P) being one of the most widespread and highly carcinogenic among them. Although studies have reported that B[a]P exerts its carcinogenic effects by causing mutations, inducing cytotoxicity, and inhibiting DNA synthesis, the early molecular regulatory events and mechanisms involved in B[a]P-induced tumor initiation remain unclear. This study found that the MGAT3 gene was significantly downregulated in B[a]P-induced mouse lung tumorigenesis, suggesting its important tumor-suppressive function. Further investigation revealed that suppression of MGAT3 expression promoted the invasion and migration abilities of lung cancer cells, while overexpression of MGAT3 in these cells inhibited these effects. Western blot analysis also showed that MGAT3 regulated the expression of epithelial-mesenchymal transition markers, thereby affecting the motility of lung cancer cells. Xenograft assay also confirmed the inhibitory effect of MGAT3 overexpression on tumor proliferation. Analysis of lung cancer tissue expression further validated that MGAT3 is significantly downregulated in lung cancer tissues, and this decrease in expression is associated with a poor prognosis in lung cancer patients. Our research indicates that the suppression of MGAT3 expression and its downstream regulatory molecules plays a crucial role in lung cancer development induced by environmental chemical carcinogens.

Figure 1

MGAT3 was downregulated in B[a]P-induced lung carcinogenesis. (a) Schematic map of B[a]P-induced lung carcinogenesis mice model. (b) Left, representative images of lung primary tumor in mice with or without B[a]P treatment; right, representative images of HE staining. (c) Heatmap of the mRNA expression of different genes by RNA-sequencing in different lung tissues from mice treated with or without carcinogens. Control: normal lung tissue samples from the mice without B[a]P treatment; adjacent: paraneoplastic tissue samples from the mice with B[a]P treatment; cancer: neoplastic tissue samples from the mice with B[a]P treatment. (d) Expressions of mRNA levels of different genes selected by RNA-sequencing were analyzed by RT-PCR and normalized with normal lung tissues mRNA expression in paraneoplastic and neoplastic lung tissue samples from the mice with B[a]P treatment. The analyses were repeated three times, and the results were expressed as mean ± SD.

Figure 2

MGAT3 was downregulated in BPDE-induced malignant transformed lung cells. (a–f) Expressions of mRNA levels of different genes selected by RNA-sequencing were analyzed by RT-PCR in BPDE-induced malignant transformed lung cells. The cell without BPDE treatment was used as control. (g) mRNA expression of MGAT3 at the different time points during the B[a]P-induced carcinogenesis. Control: the sample without B[a]P treatment. 1,2,3,4: the samples after B[a]P injection for 8 weeks and raised in different months. (h) MGAT3 gene expression after chemical carcinogen N-nitroso compounds exposure. The analyses were repeated three times, and the results were expressed as mean ± SD *p < 0.05.

Figure 3

MGAT3 was downregulated in lung cancer cells. (a–f) Expression of mRNA levels of different genes selected by RNA-sequencing was analyzed by RT-PCR in lung cancer cells (H1975 and A549) and normal lung epithelial cells (HBE). (g) MGAT3 mRNA expression in different cancer cell lines by CCLE database. (h) Expression of mRNA of MGAT3 was analyzed by RT-PCR in different lung cancer cells. Normal lung epithelial cells HBE and BEAS-2B were used as control. The analyses were repeated three times, and the results were expressed as mean ± SD *p < 0.05, **p < 0.01.

Figure 4

MGAT3 inhibited the invasion and migration of lung cancer cells. (a–d) Left, representative images of wound healing assays; right, relative percentage wound closure after treatment. (e–h) Left, representative images of transwell invasion; right, relative percentage of numbers of transwell invasion after treatment. The cell transfected with empty vector was used as the NC. The analyses were repeated three times, and the results were expressed as mean ± SD *p < 0.05.

Figure 5

MGAT3 regulated the expression of EMT marker genes. (a) Protein levels of E-cadherin and β-catenin in lung cancer cells and normal lung epithelial cells after transfection with MGAT3 siRNA and MGAT3 overexpressed plasmid by Western blot. (b,c) E-cadherin and β-catenin mRNA expression in lung cancer cells and normal lung epithelial cells after transfection with MGAT3 siRNA and MGAT3 overexpressed plasmid. The cell transfected with empty vector or scramble siRNA was used as NC. The analyses were repeated three times, and the results were expressed as mean ± SD *p < 0.05. (d) Gene set enrichment plots of differentially expressed genes belonging to the EMT pathway in MGAT3 downregulated cells. (e) MGAT3 and the proteins included in the GSEA analysis were explored for protein–protein interaction network analysis with STRING database. Node colors represents the correlation values and line colors represents the strength of protein–protein interaction action, except for MGAT3.

Figure 6

MGAT3 has an onco-suppressive effect in lung cancer. (a–c) Xenograft tumor growth assay of lung cancer cells after empty vector and MGAT3 overexpression. Photos of tumors, tumor growth curves, and tumor weight are shown. Empty vector was used as NC. (d,e) Qualification of MGAT3 mRNA and protein expression in lung cancer and normal tissues by TCGA and PDC database. (f,g) Expression of MGAT3 mRNA and protein levels in clinical lung adenocarcinoma tissue samples of different stages by TCGA and CPTAC database. (h) Representative image of IHC staining of MGAT3, E-cadherin, and β-catenin in human lung normal and tumor tissues. (i,j) Correlation analysis of MGAT3 expression and the clinical prognostic indicators of lung cancer patients. (k) Correlation of MGAT3 and E-cadherin expression in lung cancer tissues. The analyses were repeated three times, and the results were expressed as mean ± SD *p < 0.05, **p < 0.01.