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Background: Because of limited stability and sensitivity, circulating miRNAs as noninvasive biomarkers have not so far been used for early diagnosis and prognosis of non–small cell lung cancer (NSCLC) in clinic. Therefore, it is imperative to find more reliable biomarker(s). Methods: We performed one of most sensitive qRT-PCR assays, S-Poly(T) Plus, to select differently expressed miRNAs from genome-wide miRNA profiling. miRNA candidates were validated through a three-phase selection and two validation processes with 437 NSCLC cases and 415 controls. Results: A unique set of 7 and 9 miRNAs differed significantly in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) samples compared with those in controls, of which, there were 5 universal biomarkers for NSCLC (ADC or SCC). Ten of 11 miRNAs could discriminate early stage (stage I) of NSCLC from healthy individuals. Risk score was obtained from the validation set-1 and was tested using the ROC curves with a high area under ROC curve of 0.89 in ADC and 0.96 in SCC. Ultimately, potential biomarkers and the risk score were verified by the validation set-2 with a sensitivity of 94% and a specificity of 91.6% in ADC, and a sensitivity of 98.5% and a specificity of 51.5% in SCC, respectively. Conclusions: Taken together, 7 miRNAs and 9 miRNAs may provide noninvasive biomarkers for diagnosis and prognosis in ADC and SCC, respectively. Impact: On the basis of our sensitive and accurate method, we hope that these candidate miRNAs may have strong impact on the early lung cancer diagnosis.

Quantitative real-time PCR (qPCR) is the most frequently used method for measuring expression levels of microRNAs (miRNAs), which is based on normalization to endogenous references. Although circulating miRNAs have been regarded as potential non-invasive biomarker of disease, no study has been performed so far on reference miRNAs for normalization in colorectal cancer. In this study we tried to identify optimal reference miRNAs for qPCR analysis across colorectal cancer patients and healthy individuals. 485 blood-derived miRNAs were profiled in serum sample pools of both colorectal cancer and healthy control. Seven candidate miRNAs chosen from profiling results as well as three previous reported reference miRNAs were validated using qPCR in 30 colorectal cancer patients and 30 healthy individuals, and thereafter analyzed by statistical algorithms BestKeeper, geNorm and NormFinder. Taken together, hsa-miR-93-5p, hsa-miR-25-3p and hsa-miR-106b-5p were recommended as a set of suitable reference genes. More interestingly, the three miRNAs validated from 485 miRNAs are derived from a single primary transcript, indicting the cluster may be highly conserved in colorectal cancer. However, all three miRNAs differed significantly between healthy individuals and non-small cell lung cancer or breast cancer patients and could not be used as reference genes in the two types of cancer.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a kind of primary cardiomyopathy characterized by the fibro-fatty replacement of right ventricular myocardium. Currently, myocardial microRNAs have been reported to play critical role in the pathophysiology of cardiovascular pathophysiology. So far, the profiling of microRNAs in ARVC has not been described. In this study, we applied S-Poly (T) Plus method to investigate the expression profile of microRNAs in 24 ARVC patients heart samples. The tissue levels of 1078 human microRNAs were assessed and were compared with levels in a group of 24 healthy controls. Analysis of the area under the receiver operating characteristic curve (ROC) supported the 21 validated microRNAs to be miRNA signatures of ARVC, eleven microRNAs were significantly increased in ARVC heart tissues and ten microRNAs were significantly decreased. After functional enrichment analysis, miR-21-5p and miR-135b were correlated with Wnt and Hippo pathway, which might involve in the molecular pathophysiology of ARVC. Overall, our data suggested that myocardial microRNAs were involved in the pathophysiology of ARVC, miR-21-5p and miR-135b were significantly associated with both the myocardium adipose and fibrosis, which was a potential disease pathway for ARVC and might to be useful as therapeutic targets for ARVC.

We herein describe a simple, sensitive and specific method for analysis of circulating microRNAs (miRNA), termed S-Poly(T) Plus real-time PCR assay. This new method is based on our previously developed S-Poly(T) method, in which a unique S-Poly(T) primer is used during reverse-transcription to increase sensitivity and specificity. Further increased sensitivity and simplicity of S-Poly(T) Plus, in comparison with the S-Poly(T) method, were achieved by a single-step, multiple-stage reaction, where RNAs were polyadenylated and reverse-transcribed at the same time. The sensitivity of circulating miRNA detection was further improved by a modified method of total RNA isolation from serum/plasma, S/P miRsol, in which glycogen was used to increase the RNA yield. We validated our methods by quantifying miRNA expression profiles in the sera of the patients with pulmonary arterial hypertension associated with congenital heart disease. In conclusion, we developed a simple, sensitive and specific method for detecting circulating miRNAs that allows the measurement of 266 miRNAs from 100 μl of serum or plasma. This method presents a promising tool for basic miRNA research and clinical diagnosis of human diseases based on miRNA biomarkers.

Chronic hypoxia triggers pulmonary vascular remodeling, which is associated with de-differentiation of pulmonary artery smooth muscle cells (PASMC). Here, we show that miR-20a expression is up-regulated in response to hypoxia in both mouse and human PASMC. We also observed that miR-20a represses the protein kinase, cGMP-dependent, type I (PRKG1) gene and we identified two crucial miR-20a binding sites within the coding region of PRKG1. Functional studies showed that miR-20a promotes the proliferation and migration of human PASMC, whereas it inhibits their differentiation. In summary, we provided a possible mechanism by which hypoxia results in decreased PRKG1 expression and in the phenotypic switching of PASMC.