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.

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.

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.

Coronary heart disease (CHD) is one of the leading causes of heart‐associated deaths worldwide. Conventional diagnostic techniques are ineffective and insufficient to diagnose CHD with higher accuracy. To use the circulating microRNAs (miRNAs) as non‐invasive, specific and sensitive biomarkers for diagnosing of CHD, 203 patients with CHD and 144 age‐matched controls (126 high‐risk controls and 18 healthy volunteers) were enrolled in this study. The direct S‐Poly(T)Plus method was used to identify novel miRNAs expression profile of CHD patients and to evaluate their clinical diagnostic value. This method is an RNA extraction‐free and robust quantification method, which simplifies procedures, reduces variations, in particular increases the accuracy. Twelve differentially expressed miRNAs between CHD patients and high‐risk controls were selected, and their performances were evaluated in validation set‐1 with 96 plasma samples. Finally, six (miR‐15b‐5p, miR‐29c‐3p, miR‐199a‐3p, miR‐320e, miR‐361‐5p and miR‐378b) of these 12 miRNAs were verified in validation set‐2 with a sensitivity of 92.8% and a specificity of 89.5%, and the AUC was 0.971 (95% confidence interval, 0.948‐0.993, P < .001) in a large cohort for CHD patients diagnosis. Plasma fractionation indicated that only a small amount of miRNAs were assembled into EVs. Direct S‐Poly(T)Plus method could be used for disease diagnosis and 12 unique miRNAs could be used for diagnosis of CHD.

See editorial comment on page 1650Objective: Pulmonary hypertension is a lethal disease characterized by pulmonary vascular remodeling and is mediated by abnormal proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs). Plateletderived growth factor BB (PDGF-BB) is the most potent mitogen for PASMCs and is involved in vascular remodeling in pulmonary hypertension development. Therefore, the objective of our study is to identify novel mechanisms underlying vascular remodeling in pulmonary hypertension. Methods:We explored the effects and mechanisms of PTPRD downregulation in PASMCs and PTPRD knockdown rats in pulmonary hypertension induced by hypoxia. Results:We demonstrated that PTPRD is dramatically downregulated in PDGF-BB-treated PASMCs, pulmonary arteries from pulmonary hypertension rats, and blood and pulmonary arteries from lung specimens of patients with hypoxic pulmonary arterial hypertension (HPAH) and idiopathic PAH (iPAH). Subsequently, we found that PTPRD was downregulated by promoter methylation via DNMT1. Moreover, we found that PTPRD knockdown altered cell morphology and migration in PASMCs via modulating focal adhesion and cell cytoskeleton. We have demonstrated that the increase in cell migration is mediated by the PDGFRB/PLCg1 pathway. Furthermore, under hypoxic condition, we observed significant pulmonary arterial remodeling and exacerbation of pulmonary hypertension in heterozygous PTPRD knock-out rats compared with the wild-type group. We also demonstrated that HET group treated with chronic hypoxia have higher expression and activity of PLCg1 in the pulmonary arteries compared with wild-type group. Conclusion:We propose that PTPRD likely plays an important role in the process of pulmonary vascular remodeling and development of pulmonary hypertension in vivo. Video abstract http://links.lww.com/HJH/B989.