The association between genetic polymorphism and the processing efficiency of miR-149 affects the prognosis of patients with head and neck squamous cell carcinoma

Abstract

MicroRNAs (miRNAs) play important roles in modulating the neoplastic process of cancers including head and neck squamous cell carcinoma (HNSCC). A genetic polymorphism (rs2292832, C>T) has been recently identified in the precursor of miR-149; nevertheless its clinicopathological implications remain obscure. In this study, we showed that miR-149 is down-regulated in HNSCC compared to normal mucosa and this is associated with a poorer patient survival. In addition, HNSCC patients with the T/T genotype have more advanced tumors and a worse prognosis. Multivariate analysis indicated that patients carried the T/T genotype have a 2.81-fold (95% CI: 1.58-4.97) increased risk of nodal metastasis and 1.66-fold (95% CI: 1.05-2.60) increased risk of mortality compared to other groups. T/T genotype also predicted the worse prognosis of buccal mucosa carcinoma subset of HNSCC. In vitro analysis indicated that exogenous miR-149 expression reduces the migration of HNSCC cells. Moreover, HNSCC cell subclones carrying the pri-mir-149 sequence containing the T variant show a low processing efficacy when converting the pre-mir-149 to mature miR-149. These findings suggest that miR-149 suppresses tumor cell mobility, and that the pre-mir-149 polymorphism may affect the processing of miR-149, resulting in a change in the abundance of the mature form miRNA, which, in turn, modulates tumor progression and patient survival.

Figure 1. miR-149 expression in HNSCC tissues.

(A) Before-and-after plots of the −ΔCt values from paired NCMT tissue, primary HNSCC and neck lymph node metastasis (LN). Paired t-test. (B) ROC analysis across the –ΔΔCt values from survived patients and dead patients. (C) Kaplan-Meier analysis of overall survival as related to miR-149 expression using −ΔΔCt = −1.43 as a cutoff. (D) ISH. Upper, NCMT, lower HNSCC. Note the miR-149 signals in nucleus and cytosol. NCMT exhibited miR-149 signals that were more intense than HNSCC.

Figure 2. miR-149 expression and HNSCC cell migration.

SAS and OECM-1 cells were transfected with miR-149 mimic and miR-149 inhibitor. (A) qRT-PCR analysis. This detected increased miR-149 expression and decreased miR-149 expression following transfecting with miR-149 mimic and miR-149 inhibitor, respectively, relative to the controls. (B) Transwell migration assay. This indicated that transient miR-149 expression decreased cell migration, and knockdown of miR-149 expression increased cell migration. The results are means ± SE from at least triplicate analysis; un-paired t-test.

Figure 3. Analysis of the pre-mir-149 polymorphism.

(A) Sequencing analysis. Representative sequencing results showing the genotypes of pre-mir-149 polymorphisms using blood DNA. (B) Scatter spot charts to show the levels of miR-149 expression in HNSCC patients with various genotypes. The comparison was made against the C/C genotype. Un-paired t-test. (C, D) Kaplan-Meier analysis of overall survival in all HNSCC (C) and HNSCC occurring on buccal mucosa (D).

Figure 4. Analysis of miR-149 expression in HNSCC subclones.

(A) miR-149 expression in SAS, Fadu and OECM-1 cells carrying the C/T, T/T and T/T phenotypes, respectively. (B) miR-149 expression in the SAS-GFP, SAS-miR-149(C) and SAS-miR-149(T) subclones (Lt); and OECM-1-GFP, OECM-1-miR-149(C) and OECM-1-miR-149(T) subclones (Rt). T variant seemed to be associated with decreased miR-149 expression. Data shown are the means ± SE from triplicate analysis. (C) Schematic diagram to illustrate the analytical strategies used to quantify the expression of the pri-mir-149, pre-mir-149 and miR-149 in subclones. (D) Processing efficiency at different time points from 48 h to 120 h. Lt, processing of pri-mir-149 (normalized against viral integration copy revealed by Late-RT qPCR); Middle, pri-mir-149 to pre-mir-149 processing; Rt, pre-mir-149 to mature miR-149 processing. Both SAS and OECM-1 unequivocally display a decrease in pre-mir-149 to miR-149 processing in the T variant subclones. All ratios (Y axis) were normalized against the OECM-1-miR-149(C) ratio at the same time point. Data shown are the means ± SE from duplicate analysis.