Identification of Blood Let-7e-5p as a Biomarker for Ischemic Stroke

<div><p>Circulating microRNAs (miRNAs) are emerging as novel disease biomarkers. Using a miRNA microarray, we previously showed that the whole blood level of let-7e-5p was significantly higher in ischemic stroke patients than in control subjects. However, the association between let-7e-5p expression and the occurrence of ischemic stroke remains unknown. In this study, we validated the expression levels of let-7e-5p in two case-control populations using miRNA TaqMan assays and further investigated the potential targets of let-7e-5p. The results suggest that the blood level of let-7e-5p was significantly higher in patients with ischemic stroke than in controls (p<0.05). Higher levels of let-7e-5p were associated with increased occurrence of ischemic stroke (adjusted OR, 1.89; 95% CI, 1.61~2.21, p<0.001) in the combined population. The addition of let-7e-5p to traditional risk factors led to an improvement in the area under the curve, which increased from 0.74 (95% CI, 0.70~0.78) to 0.82 (95% CI, 0.78~0.85), with a net reclassification improvement of 16.76% (p<0.0001) and an integrated discrimination improvement of 0.10 (p<0.0001) for patients with ischemic stroke. Bioinformatics prediction and cell experiments suggested that the expression levels of four genes enriched in the MAPK signaling pathway were down-regulated by let-7e-5p transfection. Specifically, the expression levels of the genes CASP3 and NLK were significantly lower in ischemic stroke patients than in controls and were negatively correlated with let-7e-5p expression. In summary, our study suggests the potential use of blood let-7e-5p as a biomarker for ischemic stroke and indicates its involvement in the related pathomechanism.</p></div

AUC, NRI, and IDI calculations for let-7e-5p in the combined population.

<p>AUC, NRI, and IDI calculations for let-7e-5p in the combined population.</p

Association of the expression level of let-7e-5p with ischemic stroke.

<p>Association of the expression level of let-7e-5p with ischemic stroke.</p

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

The mRNA levels of the target genes in different cell groups.

<p>U937 cells were transfected with 50 nM control mimics (negative control) or 50 nM let-7e-5p mimics. The normal control is normal cultured cells. *p<0.05 compared to the negative control; **p<0.01 compared to the negative control.</p

Correlations between let-7e-5p expression and platelet parameters.

<p>The correlation was evaluated by the Spearman test in the combined patient population.</p

The whole blood mRNA levels of the target genes in control subjects (n = 20) and ischemic stroke patients (n = 20).

<p>The relative expression levels were normalized to beta-actin. *p<0.05; **p<0.01.</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

The expression levels of let-7e-5p in stage I and II populations.

<p>Let-7e-5p expression was detected in (A) stage I (n = 44 patients and controls) and (B) stage II (n = 302 patients and controls) population. The relative expression levels were normalized to U6 and then log-transformed. The whiskers of the plots represent the 2.5 to 97.5 percentiles.</p

General characteristics of the study population.

<p>General characteristics of the study population.</p