Effect of let-7b on tumor growth.

<p>MDA-MB-435 cells were injected subcutaneously into the right flank of nude mice to generate the mouse model of breast cancer. Two weeks later, mice received the let-7b treatment randomly. The let-7b expression vector (pSilencer-let-7b plasmid) was injected into mice through a tail vein at a dose of 4 mg/kg body weight every 3 weeks. <b>A</b>, the x-axis was labeled as the days of pSilencer-let-7b treatment. Tumor volume was measured weekly and calculated as TV (mm³)  =  length×width²×0.5236. <b>B</b>, the measurement of 14,15-DHET level in nude mice urine was performed by ELISA according to the manufacturer’s instructions. Points, mean of three experiments; bars, SD. *, <i>P</i><0.05. <b>C</b>, average tumor weight and body weight of control and let-7b treatment groups after growth for 8 weeks. Columns, mean; bars, SD. *, <i>P</i><0.05 versus control. <b>D–E</b>, the expression of the mature let-7b in the tumors and primary organs was validated by real-time RT-PCR. Columns, mean of three experiments; bars, SD. *, <i>P</i><0.05. <b>F</b>, Western blot analysis showed alteration of the expression level of CYP2J2 protein and tumor-related genes of tumor samples.</p

Effect of exogenous let-7b on CYP2J2 expression and its enzymatic activity.

<p><b>A</b>, protein level of CYP2J2. HeLa, Tca-8113, MDA-MB-435, and SK-MES-1 cells were treated with let-7b or random let-7b (100 nM) for 48 h. The protein level of CYP2J2 was examined by western blot analysis. <b>B</b>, protein level of CYP2J2 was quantified by densitometry. Columns, mean of three experiments; bars, SD. *, <i>P</i><0.05. <b>C</b>, stable metabolite 14, 15-DHET in HeLa, Tca-8113, and MDA-MB-435 cells were determined as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039197#s4" target="_blank">Materials and Methods</a>. Cells treated with exogenous hsa-let-7b produced fewer EETs than those treated with random let-7b. Points, mean of three experiments; bars, SD. *, <i>P</i><0.05.</p

Relationship between CYP2J2 protein and let-7b expression in lung cancer and adjacent nontumor tissues.

<p><b>A</b>, expression levels of CYP2J2 in lung cancerous (C) and adjacent nontumor tissues (N) was measured by Western blot analysis. <b>B</b>, comparison between the expression levels of let-7b in lung cancerous and adjacent nontumor tissues (n = 18). Although changes of −ΔΔCT values [−(ΔCT_{non-tumor tissue}−ΔCT_{cancer tissue})] are relatively mild (from -1.85 to 5.4, 2.6±1.27), the fold changes of let-7b expression between 18 paired human lung squamous cancer and adjacent nontumor tissues are significant (from 0.277 to 42.22, 6.06±2.43). <b>C</b>, relationship between CYP2J2 protein and let-7b expression in lung cancer and paired adjacent nontumor tissues. Expression level of CYP2J2 protein was increased in 13 of 18 sets of lung cancer compared with the adjacent normal tissues (the fold change>1). As expected, let-7b levels were commonly reduced in these tumors compared with the adjacent normal tissues (the fold change<1). <b>D–E</b>, expression levels of CYP2J2 and let-7b in breast cancerous and adjacent nontumor tissues (n = 4). Real-time RT-PCR was used to determine mature let-7b levels. The U6 snRNA expression level was used to normalize the relative let-7b level. Columns, mean of three experiments; bars, SD. *, <i>P</i><0.05.</p

Circulating miR-30a, miR-195 and let-7b Associated with Acute Myocardial Infarction

<div><h3>Background</h3><p>MicroRNAs (miRNAs) play key roles in diverse biological and pathological processes, including the regulation of proliferation, apoptosis, angiogenesis and cellular differentiation. Recently, circulating miRNAs have been reported as potential biomarkers for various pathologic conditions. This study investigated miR-30a, miR-195 and let-7b as potential of biomarker for acute myocardial infarction (AMI).</p> <h3>Methods and Results</h3><p>Plasma samples from 18 patients with AMI and 30 healthy adults were collected. Total RNA was extracted from plasma with TRIzol LS Reagent. MiRNA levels and plasma cardiac troponin I (cTnI) concentrations were measured by quantitative real-time PCR and ELISA assay, respectively. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050926#s3">Results</a> showed that circulating miR-30a in AMI patients was highly expressed at 4 h, 8 h and 12 h after onset of AMI, and miR-195 was highly expressed at 8 h and 12 h. However, let-7b was lower in AMI patients than in controls throughout the whole time points. Interestingly, in these patients, circulating miR-30a, miR-195 and let-7b all reached their expression peak at 8 h. By the receiver operating characteristic (ROC) curve analyses, these plasma miRNAs were of significant diagnostic value for AMI. The combined ROC analysis revealed the an AUC value of 0.93 with 94% sensitivity and 90% specificity at 8 h after onset, and an AUC value of 0.92 with 90% sensitivity and 90% specificity at 12 h after onset, in discriminating the AMI patients from healthy controls.</p> <h3>Conclusions</h3><p>Our results imply that the plasma concentration of miR-30a, miR-195 and let-7b can be potential indicators for AMI.</p> </div

Circulating let-7b expressions in AMI group and control group at all different time points.

<p>(A–G) The let-7b-score was shown as median values in different groups.</p

Circulating miR-195 expressions in AMI group and control group at 8 h and 12 h.

<p>(A and B) The miR-195-score was shown as median values in different groups; (C and D) ROC curve analyzed the diagnosis value of circulating miR-195.</p

Discrimination between AMI and control group at 8 h and 12 h using the composite miRNA score (miRNA-score).

<p>(A and B) The composite miRNA-score was shown as median values in different groups; (C and D) ROC curve analyzed the diagnosis value of the composite miRNA-score.</p

miR-30a, miR-195 and let-7b in human AMI patients and healthy adults.

<p>Δct value of miR-195, miR-30a and let-7b in AMI groups and healthy adult is presented as an average group Δct±SD. Corresponding p values were calculated using the Independent-samples T test. And missing p values represent non significant Δct changes. AUC indicates the area under the receiver operating characteristic (ROC) curve for the discrimination between MI and control groups.</p

MiRNAs plasma levels in patients with AMI detected by real-time PCR assays.

<p>Plasma samples were collected at 4 h, 8 h, 12 h, 24 h, 48 h, 72 h and 1 w after the onset of symptoms. (A) The expression levels of miR-30a at different time points; (B) The expression levels of miR-195 at different time points; (C) The expression levels of let-7b at different time points; (D) Concentrations of cTnI at different time points. The results were reported as mean±SD. Values indicated fold changes of miRNAs vs. its level in the control group, arbitrarily set at 1 as indicated by CTRL. (E) Time courses of circulating miR-30a and cTnI from same plasma samples in patients with AMI and healthy control people; (F) Time courses of circulating miR-195 and cTnI from same plasma samples in patients with AMI and healthy control people; (G) Time courses of circulating let-7b and cTnI from same plasma samples in patients with AMI and healthy control people; The data were normalized to the peak level that each miRNA and cTnI achieved in each patient, and the time of the peak-fold increase vs. miRNAs and cTnI time courses were analyzed by repeated-measures ANOVA. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050926#s3" target="_blank">Results</a> were reported as mean+SD (*, p<0.05; **, p≤0.01).</p

Circulating let-7b expressions in AMI group and control group at all different time points.

<p>(A–G) ROC curve analyzed the diagnosis value of circulating let-7b.</p