N6-methyladenosine-driven miR-143/145-KLF4 circuit orchestrates the phenotypic switch of pulmonary artery smooth muscle cells.

Pulmonary hypertension (PH) is characterized by vascular remodeling predominantly driven by a phenotypic switching in pulmonary artery smooth muscle cells (PASMCs). However, the underlying mechanisms for this phenotypic alteration remain incompletely understood. Here, we identified that RNA methyltransferase METTL3 is significantly elevated in the lungs of hypoxic PH (HPH) mice and rats, as well as in the pulmonary arteries (PAs) of HPH rats. Targeted deletion of Mettl3 in smooth muscle cells exacerbated hemodynamic consequences of hypoxia-induced PH and accelerated pulmonary vascular remodeling in vivo. Additionally, the absence of METTL3 markedly induced phenotypic switching in PASMCs in vitro. Mechanistically, METTL3 depletion attenuated m6A modification and hindered the processing of pri-miR-143/145, leading to a downregulation of miR-143-3p and miR-145-5p. Inhibition of hnRNPA2B1, an m6A mediator involved in miRNA maturation, similarly resulted in a significant reduction of miR-143-3p and miR-145-5p. We demonstrated that miR-145-5p targets Krüppel-like factor 4 (KLF4) and miR-143-3p targets fascin actin-bundling protein 1 (FSCN1) in PASMCs. The decrease of miR-145-5p subsequently induced an upregulation of KLF4, which in turn suppressed miR-143/145 transcription, establishing a positive feedback circuit between KLF4 and miR-143/145. This regulatory circuit facilitates the persistent suppression of contractile marker genes, thereby sustaining PASMC phenotypic switch. Collectively, hypoxia-induced upregulation of METTL3, along with m6A mediated regulation of miR-143/145, might serve as a protective mechanism against phenotypic switch of PASMCs. Our results highlight a potential therapeutic strategy targeting m6A modified miR-143/145-KLF4 loop in the treatment of PH.

N6-methyladenosine modification of KLF2 may contribute to endothelial-to-mesenchymal transition in pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a progressive disease characterized by pulmonary vascular remodeling. Increasing evidence indicates that endothelial-to-mesenchymal transition (EndMT) in pulmonary artery endothelial cells (PAECs) is a pivotal trigger initiating this remodeling. However, the regulatory mechanisms underlying EndMT in PH are still not fully understood. METHODS: Cytokine-induced hPAECs were assessed using RNA methylation quantification, qRT-PCR, and western blotting to determine the involvement of N6-methyladenosine (m6A) methylation in EndMT. Lentivirus-mediated silencing, overexpression, tube formation, and wound healing assays were utilized to investigate the function of METTL3 in EndMT. Endothelial-specific gene knockout, hemodynamic measurement, and immunostaining were performed to explore the roles of METTL3 in pulmonary vascular remodeling and PH. RNA-seq, RNA Immunoprecipitation-based qPCR, mRNA stability assay, m6A mutation, and dual-luciferase assays were employed to elucidate the mechanisms of RNA methylation in EndMT. RESULTS: The global levels of m6A and METTL3 expression were found to decrease in TNF-α- and TGF-ß1-induced EndMT in human PAECs (hPAECs). METTL3 inhibition led to reduced endothelial markers (CD31 and VE-cadherin) and increased mesenchymal markers (SM22 and N-cadherin) as well as EndMT-related transcription factors (Snail, Zeb1, Zeb2, and Slug). The endothelial-specific knockout of Mettl3 promoted EndMT and exacerbated pulmonary vascular remodeling and hypoxia-induced PH (HPH) in mice. Mechanistically, METTL3-mediated m6A modification of kruppel-like factor 2 (KLF2) plays a crucial role in the EndMT process. KLF2 overexpression increased CD31 and VE-cadherin levels while decreasing SM22, N-cadherin, and EndMT-related transcription factors, thereby mitigating EndMT in PH. Mutations in the m6A site of KLF2 mRNA compromise KLF2 expression, subsequently diminishing its protective effect against EndMT. Furthermore, KLF2 modulates SM22 expression through direct binding to its promoter. CONCLUSIONS: Our findings unveil a novel METTL3/KLF2 pathway critical for protecting hPAECs against EndMT, highlighting a promising avenue for therapeutic investigation in PH.

Comprehensive characterization of small noncoding RNA profiles in hypoxia-induced pulmonary hypertension (HPH) rat tissues.

Hypoxia-induced pulmonary hypertension (HPH) is a fatal cardiovascular disease characterized by an elevation in pulmonary artery pressure, resulting in right ventricular dysfunction and eventual heart failure. Exploring the pathogenesis of HPH is crucial, and small noncoding RNAs (sncRNAs) are gaining recognition as potential regulators of cellular responses to hypoxia. In this study, we conducted a comprehensive analysis of sncRNA profiles in eight tissues of male HPH rats using high-throughput sequencing. Our study unveiled several sncRNAs, with the brain, kidney, and spleen exhibiting the highest abundance of microRNA (miRNA), tRNA-derived small RNA (tDR), and small nucleolar RNA (snoRNA), respectively. Moreover, we identified numerous tissue-specific and hypoxia-responsive sncRNAs, particularly miRNAs and tDRs. Interestingly, we observed arm switching in miRNAs under hypoxic conditions and a significant increase in the abundance of 5' tRNA-halves among the total tDRs during hypoxia. Overall, our study provides a comprehensive characterization of the sncRNA profiles in HPH rats.

Endothelium-specific SIRT7 targeting ameliorates pulmonary hypertension through Krüpple-like factor 4 deacetylation.

AIMS: Pulmonary hypertension (PH) is a pulmonary vascular disease characterized by a high mortality rate. Pulmonary arterial endothelium cells (PAECs) serve as a primary sensor of various environmental cues, such as shear stress and hypoxia, but PAEC dysfunction may trigger vascular remodelling during the onset of PH. This study aimed to illustrate the role of Sirtuin 7 (SIRT7) in endothelial dysfunction during PH and explore the potential therapeutic strategy for PH. METHODS AND RESULTS: SIRT7 levels were measured in human and murine experimental PH samples. Bioinformatic analysis, immunoprecipitation, and deacetylation assay were used to identify the association between SIRT7 and Krüpple-like factor 4 (KLF4), a key transcription factor essential for endothelial cell (EC) homeostasis. Sugen5416 + hypoxia (SuHx)-induced PH mouse models and cell cultures were used for the study of the therapeutic effect of SIRT7 for PH. SIRT7 level was significantly reduced in lung tissues and PAECs from PH patients and the SuHx-induced PH mouse model as compared with healthy controls. Pulmonary endothelium-specific depletion of Sirt7 increased right ventricular systolic pressure and exacerbated right ventricular hypertrophy in the SuHx-induced PH model. At the molecular level, we identified KLF4 as a downstream target of SIRT7, which deacetylated KLF4 at K228 and inhibited the ubiquitination-proteasome degradation. Thus, the SIRT7/KLF4 axis maintained PAEC homeostasis by regulating proliferation, migration, and tube formation. PAEC dysfunction was reversed by adeno-associated virus type 1 vector-mediated endothelial overexpression of Sirt7 or supplementation with nicotinamide adenine dinucleotide (NAD)+ intermediate nicotinamide riboside which activated Sirt7; both approaches successfully reversed PH phenotypes. CONCLUSION: The SIRT7/KLF4 axis ensures PAEC homeostasis, and pulmonary endothelium-specific SIRT7 targeting might constitute a PH therapeutic strategy.

Terminal modifications independent cell-free RNA sequencing enables sensitive early cancer detection and classification.

Cell-free RNAs (cfRNAs) offer an opportunity to detect diseases from a transcriptomic perspective, however, existing techniques have fallen short in generating a comprehensive cell-free transcriptome profile. We develop a sensitive library preparation method that is robust down to 100 µl input plasma to analyze cfRNAs independent of their 5'-end modifications. We show that it outperforms adapter ligation-based method in detecting a greater number of cfRNA species. We perform transcriptome-wide characterizations in 165 lung cancer, 30 breast cancer, 37 colorectal cancer, 55 gastric cancer, 15 liver cancer, and 133 cancer-free participants and demonstrate its ability to identify transcriptomic changes occurring in early-stage tumors. We also leverage machine learning analyses on the differentially expressed cfRNA signatures and reveal their robust performance in cancer detection and classification. Our work sets the stage for in-depth study of the cfRNA repertoire and highlights the value of cfRNAs as cancer biomarkers in clinical applications.

Critical view on oligo(dT)-based RNA-seq: bias arising, modeling, and mitigating.

The precise biological interpretation of oligo(dT)-based RNA sequencing (RNA-seq) datasets, particularly in single-cell RNA-seq (scRNA-seq), is invaluable for understanding complex biological systems. However, the presence of biases can lead to misleading results in downstream analysis. This study has now identified two additional biases that are not accounted for in established bias models: poly(A)-tail length bias and fixed-position GC-content bias. These biases have a significant negative impact on the overall quality of oligo(dT)-based RNA-seq data. To address these biases, we have developed a universal bias-mitigating method based on the lower-affinity binding of short and nonanchored oligo(dT) primers to poly(A) tails. This method significantly reduces poly(A) length bias and completely eliminates fixed-position GC bias. Furthermore, the use of short oligo(dT) with impartial binding behavior toward the diverse poly(A) tails renders RNA-seq with more reliable measurements. The findings of this study are particularly beneficial for scRNA-seq datasets, where accurate benchmarking is critical.

Altered cfDNA fragmentation profile in hypomethylated regions as diagnostic markers in breast cancer.

BACKGROUND: Breast cancer, the most common malignancy in women worldwide, has been proven to have both altered plasma cell-free DNA (cfDNA) methylation and fragmentation profiles. Nevertheless, simultaneously detecting both of them for breast cancer diagnosis has never been reported. Moreover, although fragmentation pattern of cfDNA is determined by nuclease digestion of chromatin, structure of which may be affected by DNA methylation, whether cfDNA methylation and fragmentation are biologically related or not still remains unclear. METHODS: Improved cfMeDIP-seq were utilized to characterize both cfDNA methylation and fragmentation profiles in 49 plasma samples from both healthy individuals and patients with breast cancer. The feasibility of using cfDNA fragmentation profile in hypo- and hypermethylated regions as diagnostic markers for breast cancer was evaluated. RESULTS: Mean size of cfDNA fragments (100-220 bp) mapped to hypomethylated regions decreased more in patients with breast cancer (4.60 bp, 172.33 to 167.73 bp) than in healthy individuals (2.87 bp, 174.54 to 171.67 bp). Furthermore, proportion of short cfDNA fragments (100-150 bp) in hypomethylated regions when compared with it in hypermethylated regions was found to increase more in patients with breast cancer in two independent discovery cohort. The feasibility of using abnormality of short cfDNA fragments ratio in hypomethylated genomic regions for breast cancer diagnosis in validation cohort was evaluated. 7 out of 11 patients were detected as having breast cancer (63.6% sensitivity), whereas no healthy individuals were mis-detected (100% specificity). CONCLUSION: We identified enriched short cfDNA fragments after 5mC-immunoprecipitation (IP) in patients with breast cancer, and demonstrated the enriched short cfDNA fragments might originated from hypomethylated genomic regions. Furthermore, we proved the feasibility of using differentially methylated regions (DMRs)-dependent cfDNA fragmentation profile for breast cancer diagnosis.

The protective role of EP300 in monocrotaline-induced pulmonary hypertension.

Background: Pulmonary hypertension (PH) is a lethal disease characterized by pulmonary vascular remodeling, which is mediated by the abnormal proliferation/migration of pulmonary arterial smooth muscle cells (PASMCs). Recent reports suggest the involvement of histone acetylation in PAH development and that histone deacetylase (HDAC) inhibitors have therapeutic potential for the treatment of PAH. EP300 is an acetyltransferase that plays diverse roles in cell proliferation, differentiation, and apoptosis. However, the functions of EP3000 in PH are rarely studied. Results: In this work, we found that the expression of EP300 was increased in the pulmonary arteries of monocrotaline (MCT)-induced PH rats. Knockdown of EP300 by AAV-mediated shRNA exacerbated the PH, with a higher right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and wall thickness in the pulmonary artery of MCT-induced PH rat. On the cellular level, the proliferation of PASMCs was promoted by EP300 knockdown. In addition, the expression of EP300 was increased in PASMCs by the overexpression of EGR1, while the deletion of EGR1 binding sites in the EP300 promoter region decreased the activity of EP300 promoter. Moreover, deleting the EP300 promoter region containing EGR1 binding sites using CRISPR/Cas9 abolished the upregulation of EP300 in MCT-induced rats and exacerbated MCT-induced PH. To summarize, our data indicate that EP300 upregulation mediated by EGR1 has a protective effect on MCT-induced PH. Conclusion: These findings showed EP300 expression was increased in the MCT-induced PH model in rats, which could be mediated by EGR1; the EP300 also displayed the potential to provide protection from PH.

Methylation-mediated silencing of PTPRD induces pulmonary hypertension by promoting pulmonary arterial smooth muscle cell migration via the PDGFRB/PLCγ1 axis.

OBJECTIVE: 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). Platelet-derived 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/PLCγ1 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 PLCγ1 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 .

Decoding ceRNA regulatory network in the pulmonary artery of hypoxia-induced pulmonary hypertension (HPH) rat model.

BACKGROUND: Hypoxia-induced pulmonary hypertension (HPH) is a lethal cardiovascular disease with the characteristic of severe remodeling of pulmonary vascular. Although a large number of dysregulated mRNAs, lncRNAs, circRNAs, and miRNAs related to HPH have been identified from extensive studies, the competitive endogenous RNA (ceRNA) regulatory network in the pulmonary artery that responds to hypoxia remains largely unknown. RESULTS: Transcriptomic profiles in the pulmonary arteries of HPH rats were characterized through high-throughput RNA sequencing in this study. Through relatively strict screening, a set of differentially expressed RNAs (DERNAs) including 19 DEmRNAs, 8 DElncRNAs, 19 DEcircRNAs, and 23 DEmiRNAs were identified between HPH and normal rats. The DEmRNAs were further found to be involved in cell adhesion, axon guidance, PPAR signaling pathway, and calcium signaling pathway, suggesting their crucial role in HPH. Moreover, a hypoxia-induced ceRNA regulatory network in the pulmonary arteries of HPH rats was constructed according to the ceRNA hypothesis. More specifically, the ceRNA network was composed of 10 miRNAs as hub nodes, which might be sponged by 6 circRNAs and 7 lncRNAs, and directed the expression of 18 downstream target genes that might play important role in the progression of HPH. The expression patterns of selected DERNAs in the ceRNA network were then validated to be consistent with sequencing results in another three independent batches of HPH and normal control rats. The diagnostic effectiveness of several hub mRNAs in ceRNA network was further evaluated through investigating their expression profiles in patients with pulmonary artery hypertension (PAH) recorded in the Gene Expression Omnibus (GEO) dataset GSE117261. Dysregulated POSTN, LTBP2, SPP1, and LSAMP were observed in both the pulmonary arteries of HPH rats and lung tissues of PAH patients. CONCLUSIONS: A ceRNA regulatory network in the pulmonary arteries of HPH rats was constructed, 10 hub miRNAs and their corresponding interacting lncRNAs, circRNAs, and mRNAs were identified. The expression patterns of selected DERNAs were further validated to be consistent with the sequencing result. POSTN, LTBP2, SPP1, and LSAMP were suggested to be potential diagnostic biomarkers and therapeutic targets for PAH.

LncPTSR Triggers Vascular Remodeling in Pulmonary Hypertension by Regulating [Ca2+]i in Pulmonary Arterial Smooth Muscle Cells.

Pulmonary hypertension (PH) is characterized by vascular remodeling and sustained increase in right ventricular systolic pressure. The molecular mechanisms behind PH development remain unclear. Here, a long noncoding RNA (lncRNA) attenuated by platelet-derived growth factor BB (PDGF-BB) was identified, and its functional roles were investigated in vitro and in vivo. Using RNA-sequencing data and rapid amplification of cDNA ends, an lncRNA neighboring the locus of ATPase plasma membrane Ca2+ transporting 4 (PMCA4) was identified and named lncPTSR. It is a highly conserved nuclear lncRNA and was downregulated in pulmonary arterial smooth muscle cells (PASMCs) with PDGF-BB stimulation or hypoxia induction. Gene interruption or overexpression assays revealed that lncPTSR negatively regulates rat PASMC proliferation, apoptosis, and migration. LncPTSR interruption in Sprague Dawley rats using adeno-associated virus type 9-mediated shRNA resulted in a significant increase in right ventricular systolic pressure and vascular remodeling in normoxic condition. LncPTSR knockdown also suppressed PMCA4 expression and attenuated the intracellular Ca2 + efflux of PASMCs in vitro and in vivo. Further studies suggest a complex crosstalk between lncPTSR and mitogen-activated protein kinase pathway: inhibition of mitogen-activated protein kinase kinase and extracellular signal-regulated kinase abolishes the PDGF-BB-mediated lncPTSR downregulation, and lncPTSR plays a feedback regulation for mitogen-activated protein kinase-signaling molecules. The present study suggests that lncPTSR participates in pulmonary artery remodeling via modulating the expression of PMCA4 and intracellular Ca2 + homeostasis downstream of PDGF-BB-driven mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling. These results suggest that lncPTSR may be a promising therapeutic target in PH treatment.

A novel tonicity-responsive microRNA miR-23a-5p modulates renal cell survival under osmotic stress through targeting heat shock protein 70 HSPA1B.

There is growing evidence that microRNAs (miRNAs) are implicated in cellular adaptation to osmotic stress, but the underlying osmosignaling pathways are still not completely understood. In this study, we found that a passenger strand miRNA, miR-23a-5p, was significantly downregulated in response to high NaCl treatment in mouse inner medullary collecting duct cells (mIMCD3) through an miRNA profiling assay. The decrease of miR-23a-5p is hypertonicity-dependent and osmotolerant cell type-specific. Knockdown of miR-23a-5p increased cellular survival and proliferation in mIMCD3. In contrast, miR-23a-5p overexpression repressed cell viability and proliferation under hypertonic stress. RNA deep-sequencing revealed that a heat shock protein 70 (HSP70) isoform, HSP70 member 1B (HSPA1B), was significantly increased under hypertonic treatment. Based on the prediction analysis by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and TargetScan, and a further validation via a dual-luciferase assay, HSPA1B was identified as a potential target of miR-23a-5p. Overexpressed miR-23a-5p suppressed HSPA1B, whereas downregulated miR-23a-5p promoted HSPA1B expression in mIMCD3. In addition, an in vivo study demonstrated that there is a reverse correlation between the levels of miR-23a-5p and HSPA1B in mouse renal inner medulla (papilla) that is exposed to extremely high osmolality. In summary, this study elucidates that passenger strand miR-23a-5p is a novel tonicity-responsive miRNA. The downregulation of miR-23a-5p facilitates cellular adaptation to hypertonic stress in mammalian renal cells through modulating HSPA1B.

Circulating microRNA profiles based on direct S-Poly(T)Plus assay for detection of coronary heart disease.

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.

Direct S-Poly(T) Plus assay in quantification of microRNAs without RNA extraction and its implications in colorectal cancer biomarker studies.

BACKGROUND: Advances in microRNAs (miRNAs) biomarkers have generated disease markers with potential clinical values. However, none of these published results have been applied in clinic until today. The main reason could be the lack of simple but robust miRNA measurements. METHODS: We built up a simple but ultrasensitive RT-qPCR protocol, Direct S-Poly(T) Plus assay, for detecting miRNAs without RNA purification. In this study, the method was optimized and compared with other RNA purification-based miRNA assays, and the sensitivity was tested. Using Direct S-Poly(T) Plus method, seven potential miRNA biomarkers of colorectal cancer were validated. RESULTS: It is possible to detect approximately 100 miRNAs with minimal plasma inputs (20 µl) and time (~ 140 min) with this approach. The sensitivity of this method was 2.7-343-fold higher than that of the stem-loop method, and comparable with S-Poly(T) plus method. 7 validated miRNA biomarkers of colorectal cancer by Direct S-Poly(T) plus assay could discriminate colorectal cancer stage I from healthy individuals, and promised satisfactory discrimination with the area under receiver operating characteristic (ROC) curve ranging from 0.79 to 0.94 (p value < 0.001). CONCLUSIONS: This simple and robust protocol may have strong impact on the development of specific miRNAs as biomarkers in clinic.

Circulating Plasma miRNAs as Potential Biomarkers of Non-Small Cell Lung Cancer Obtained by High-Throughput Real-Time PCR Profiling.

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.

Identification of reference genes for circulating microRNA analysis in colorectal cancer.

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.

An improved method for detecting circulating microRNAs with S-Poly(T) Plus real-time PCR.

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.

Development and reproductive potential of diamondback moth (Lepidoptera: Plutellidae) on selected wild crucifer species.

The diamondback moth, Plutella xylostella (L.), is an oligophagous insect that primarily feeds on members of the family Cruciferae. The development, survival, and reproductive potential of P. xylostella were studied on eight wild cruciferous species: Rorippa indica (L.) Hiern, Cardamine hirsuta L., Descurainia sophia (L.) Webb ex Prantl, Capsella bursa-pastoris (L.) Medic, Cardamine leucantha (Tausch) O. E. Schulz, Orychophragmus violaceus (L.) O. E. Schulz, Thlaspi arvense L., and Cardamine macrophylla Willd. Developmental durations of immatures from egg to adult emergence differed significantly among the plant species, with the longest period recorded on C. macrophylla (20.8 d) and the shortest on R. indica (15.8 d). The female pupae of P. xylostella reared on C. leucantha and T. arvense were lighter (4.2 and 4.3 mg/pupa) than those reared on other hosts (5.2-6.5 mg/pupa), and the male pupae from T. arvense were the lightest (3.1 mg/pupa) among all colonies. Survival from egg to adult emergence ranged from 95.7% on R. indica to 48.8% on T. arvense. The longevity (10.1 d) of P. xylostella female and the oviposition period (7.7 d) were the longest when larvae fed R. indica than those that fed on other wild hosts. Female adults of P. xylostella from O. violaceus, C. macrophylla, and Ca. bursa-pastoris had higher fecundity (305-351 eggs/female) than from other wild host plants, whereas that from R. indica had the lowest fecundity (134 eggs/female). C. hirsuta was the best wild host plant for P. xylostella because of the highest intrinsic rates of increase (rm = 0.2402), whereas T. arvense was the least favorable hosts with the lowest intrinsic rates of increase (rm = 0.1577). The results from this study will be useful for interpretation of the performance and population dynamics of P. xylostella on wild hosts and cultivated cruciferous vegetables.