The <i>MKK7</i> p.Glu116Lys Rare Variant Serves as a Predictor for Lung Cancer Risk and Prognosis in Chinese

<div><p>Accumulated evidence indicates that rare variants exert a vital role on predisposition and progression of human diseases, which provides neoteric insights into disease etiology. In the current study, based on three independently retrospective studies of 5,016 lung cancer patients and 5,181 controls, we analyzed the associations between five rare polymorphisms (i.e., p.Glu116Lys, p.Asn118Ser, p.Arg138Cys, p.Ala195Thr and p.Leu259Phe) in <i>MKK7</i> and lung cancer risk and prognosis. To decipher the precise mechanisms of <i>MKK7</i> rare variants on lung cancer, a series of biological experiments was further performed. We found that the <i>MKK7</i> p.Glu116Lys rare polymorphism was significantly associated with lung cancer risk, progression and prognosis. Compared with Glu/Glu common genotype, the 116Lys rare variants (Lys/Glu/+ Lys/Lys) presented an adverse effect on lung cancer susceptibility (odds ratio [OR] = 3.29, 95% confidence interval [CI] = 2.70–4.01). These rare variants strengthened patients’ clinical progression that patients with 116Lys variants had a significantly higher metastasis rate and advanced N, M stages at diagnosis. In addition, the patients with 116Lys variants also contributed to worse cancer prognosis than those carriers with Glu/Glu genotype (hazard ratio [HR] = 1.53, 95% CI = 1.32–1.78). Functional experiments further verified that the <i>MKK7</i> p.116Lys variants altered the expression of several cancer-related genes and thus affected lung cancer cells proliferation, tumor growth and metastasis <i>in vivo</i> and <i>in vitro</i>. Taken together, our findings proposed that the <i>MKK7</i> p.Glu116Lys rare polymorphism incurred a pernicious impact on lung cancer risk and prognosis through modulating expressions of a serial of cancer-related genes.</p></div

Stratification analysis of association between the <i>MKK7</i> p.Glu116Lys genotypes and lung cancer prognosis by selected variables.

<p>Stratification analysis of association between the <i>MKK7</i> p.Glu116Lys genotypes and lung cancer prognosis by selected variables.</p

Effects of <i>MKK7</i> p.Glu116Lys on cellular proliferation, apoptosis, migration, and invasion.

<p>(A). A549 and L78 cells were seeded into 96-well plates after transfected with <i>MKK7</i>-116Glu or <i>MKK7</i>-116Lys lentivirus, and cell proliferation was evaluated every other day for a week using the MTT assay. OD values from four independent experiments were assessed (* indicated that a statistical significance with <i>P</i> < 0.05 between the groups). (B). Representative colony formation assay in 6-well plates both for A549 and L78 cells. The comparison of different colonies level between <i>MKK7</i>-116Glu-cells and <i>MKK7</i>-116Lys-cells was assessed by student’s <i>t</i>-test. (C). In soft agar assay, colonies number in cells with over-expressing <i>MKK7</i>-116Lys were much higher than cells transfected with <i>MKK7</i>-116Glu. Colonies were stained with crystal violet, and were counted in four randomly selected points in each well under the microscopy (original magnification: ×100). (D). Cell cycle analysis of A549 and L78 cells after transfection with lentiviruses containing different p.Glu116Lys allele. (E). Annexin V-FITC/PI apoptosis assay of A549 and L78 cells after transfection with <i>MKK7</i>-116Glu or <i>MKK7</i>-116Lys lentivirus. (F, G). Cell migration and invasion assays were performed. The upper chambers were seeded with various cell lines. The membranes of the chambers were stained with crystal violet. All data were representative of at least three separate experiments.</p

Frequency distributions of selected variables in lung cancer patients and cancer-free controls.

<p>Frequency distributions of selected variables in lung cancer patients and cancer-free controls.</p

Associations between <i>MKK7</i> rare polymorphisms and lung cancer risk.

<p>Associations between <i>MKK7</i> rare polymorphisms and lung cancer risk.</p

The effects of <i>MKK7</i> p.Glu116Lys on tumor growth and metastasis.

<p>(A, B). Subcutaneously implanted <i>MKK7</i>-116Glu (A549<i>-MKK7</i>-116Glu and L78<i>-MKK7</i>-116Glu cells) and <i>MKK7</i>-116Lys (A549<i>-MKK7</i>-116Lys and L78<i>-MKK7</i>-116Lys cells) cells xenografted tumors were established and were observed for a total of 4 weeks. Tumor volumes represented the mean ± SD of 6 mice per group. Columns, mean; bars, SD. The symbol “^★” indicated a statistical significance with <i>P</i> < 0.05 between the cells transfected with the two different transfectants. (C-E). A549<i>-MKK7</i>-116Glu or A549<i>-MKK7</i>-116Lys cells were separately injected into the tail vein of each mouse. After proximately 10 weeks, lung metastases were evaluated using magnetic resonance imaging, macroscopic observation and histomorphology under microscopy. The red loops and arrows indicate the metastases.</p

Kaplan-Meier (KM) survival curves for lung cancer patients carrying the different <i>MKK7</i> p.Glu116Lys genotypes.

<p>The <i>P</i> value was calculated by the Log-rank test. (A). KM curves of patients from the discovery set; (B). KM curves of patients from validation set I; (C). KM curves of patients from validation set II.</p

Associations between <i>MKK7</i> rare polymorphisms and lung cancer survival in the three populations.

<p>Associations between <i>MKK7</i> rare polymorphisms and lung cancer survival in the three populations.</p

Additional file 1: Table S1. of Overexpression of lncRNA IGFBP4–1 reprograms energy metabolism to promote lung cancer progression

Sequence of primers using for qRT-PCR analysis (DOCX 12 kb