Let-7a Downregulation Accompanied by KRAS Mutation Is Predictive of Lung Cancer Onset in Cigarette Smoke-Exposed Mice

Abstract

Background: Let-7 is a tumor suppressor microRNA targeting the KRAS lung oncogene. Let-7a downregulation is reversible during the early stages of lung carcinogenesis but is irreversible in cancer cells. The aim of this study is to shed light on the relationship between oncogene (KRAS) mutation and let-7a downregulation in cigarette smoke (CS)-induced lung carcinogenesis.

Methods: A total of 184 strain H Swiss albino mice were either unexposed (control) or exposed to CS for 2 weeks (short CS) or 8 months (long CS). After 8 months, the lungs were individually collected. The following end points have been evaluated: (a) DNA methylation of the let-7a gene promoter by bisulphite-PCR and pyrosequencing; (b) let-7a expression by qPCR; (c) KRAS mutation by DNA pyrosequencing; (d) cancer incidence by histopathological examination.

Results: let-7a expression decreased by 8.3% in the mice exposed to CS for two weeks (CS short) and by 33.4% (p ≤ 0.01) in the mice exposed to CS for 8 months (CS long). No significant difference was detected in the rate of let-7a-promoter methylation between the Sham-exposed mice (55.1%) and the CS short-(53%) or CS long (51%)-exposed mice. The percentage of G/T transversions in KRAS codons 12 and 13 increased from 2.3% (Sham) to 6.4% in CS short- and to 11.5% in CS long-exposed mice. Cancer incidence increased significantly in the CS long-exposed mice (11%) as compared to both the Sham (4%) and the CS short-exposed (2%) mice. In the CS long-exposed mice, the correlation between let-7a expression and the number of KRAS mutations was positive (R = +0.5506) in the cancer-free mice and negative (R = -0.5568) in the cancer-bearing mice.

Conclusions: The effects of CS-induced mutations in KRAS are neutralized by the high expression of let-7a in cancer-free mice (positive correlation) but not in cancer-bearing mice where an irreversible let-7a downregulation occurs (negative correlation). This result provides evidence that both genetic (high load of KRAS mutation) and epigenetic alterations (let-7a irreversible downregulation) are required to produce lung cancer in CS-exposed organisms.

Keywords: KRAS mutation; cigarette smoke; let-7a expression; lung cancer; microRNA.

Figure 1

Quantification of let-7a expression intensity calculated by referring to RNU38B reference standard (vertical axis, copies per microliter) as valuated by qPCR in mice either unexposed to CS (Sham) or exposed for 2 weeks (CS short) and for 8 months (CS long). * p < 0.01 as evaluated by t-Student test by unpaired data CS long exposure vs. Sham and CS long vs. CS short exposure.

Figure 2

(A) example of pyrogram trace illustrating the analysis of mouse let-7 promoter. (B) example of pyrogram trace illustrating the percentages of CpG methylation for the two analyzed CpG positions of mouse let-7 promoter (C) Methylation percentage of let-7a promoter gene (vertical axis) in mice either unexposed to CS (Sham) or exposed for 2 weeks (CS short) and for 8 months (CS long).

Figure 2

(A) example of pyrogram trace illustrating the analysis of mouse let-7 promoter. (B) example of pyrogram trace illustrating the percentages of CpG methylation for the two analyzed CpG positions of mouse let-7 promoter (C) Methylation percentage of let-7a promoter gene (vertical axis) in mice either unexposed to CS (Sham) or exposed for 2 weeks (CS short) and for 8 months (CS long).

Figure 3

Pyrosequencing analysis of KRAS gene mutation (percentage of G/T transversions in codons 12 and 13) in DNA isolated from lungs of mice either unexposed (Sham) or exposed to CS for 2 weeks (short CS) or 8 months (long CS). * p < 0.05 vs. Sham, ** p ≤ 0.01 vs. Sham.

Figure 4

(A) cancer incidence in mice belonging to the 3 experimental groups. (B) upper panel, microscope morphology (A: Sham magnification 25×; B: adenoma 100×; C: adenocarcinoma 25×; D: acinar 25×; E: papillary 25×; F: solid 200×); (C) number of cancers distributed according to their histotypes as detected in the lungs of mice exposed to cigarette smoke for 8 months.

Figure 4

(A) cancer incidence in mice belonging to the 3 experimental groups. (B) upper panel, microscope morphology (A: Sham magnification 25×; B: adenoma 100×; C: adenocarcinoma 25×; D: acinar 25×; E: papillary 25×; F: solid 200×); (C) number of cancers distributed according to their histotypes as detected in the lungs of mice exposed to cigarette smoke for 8 months.

Figure 5

Regression analysis of the relationship between KRAS mutation amount (vertical axis) and let-7a expression (horizontal axis) in cancer-free (upper panel) and cancer-bearing mice (lower panel), both exposed to CS for 8 months.