CLT-seq as a universal homopolymer-sequencing concept reveals poly(A)-tail-tuned ncRNA regulation.

Dynamic tuning of the poly(A) tail is a crucial mechanism for controlling translation and stability of eukaryotic mRNA. Achieving a comprehensive understanding of how this regulation occurs requires unbiased abundance quantification of poly(A)-tail transcripts and simple poly(A)-length measurement using high-throughput sequencing platforms. Current methods have limitations due to complicated setups and elaborate library preparation plans. To address this, we introduce central limit theorem (CLT)-managed RNA-seq (CLT-seq), a simple and straightforward homopolymer-sequencing method. In CLT-seq, an anchor-free oligo(dT) primer rapidly binds to and unbinds from anywhere along the poly(A) tail string, leading to position-directed reverse transcription with equal probability. The CLT mechanism enables the synthesized poly(T) lengths, which correspond to the templated segment of the poly(A) tail, to distribute normally. Based on a well-fitted pseudogaussian-derived poly(A)-poly(T) conversion model, the actual poly(A)-tail profile is reconstructed from the acquired poly(T)-length profile through matrix operations. CLT-seq follows a simple procedure without requiring RNA-related pre-treatment, enrichment or selection, and the CLT-shortened poly(T) stretches are more compatible with existing sequencing platforms. This proof-of-concept approach facilitates direct homopolymer base-calling and features unbiased RNA-seq. Therefore, CLT-seq provides unbiased, robust and cost-efficient transcriptome-wide poly(A)-tail profiling. We demonstrate that CLT-seq on the most common Illumina platform delivers reliable poly(A)-tail profiling at a transcriptome-wide scale in human cellular contexts. We find that the poly(A)-tail-tuned ncRNA regulation undergoes a dynamic, complex process similar to mRNA regulation. Overall, CLT-seq offers a simplified, effective and economical approach to investigate poly(A)-tail regulation, with potential implications for understanding gene expression and identifying therapeutic targets.

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.

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.

PDGF/MEK/ERK axis represses Ca2+ clearance via decreasing the abundance of plasma membrane Ca2+ pump PMCA4 in pulmonary arterial smooth muscle cells.

Pulmonary arterial hypertension (PAH) is a rare and lethal disease characterized by vascular remodeling and vasoconstriction, which is associated with increased intracellular calcium ion concentration ([Ca2+]i). Platelet-derived growth factor-BB (PDGF-BB) is the most potent mitogen for pulmonary arterial smooth muscle cells (PASMCs) and is involved in vascular remodeling during PAH development. PDGF signaling has been proved to participate in maintaining Ca2+ homeostasis of PASMCs; however, the mechanism needs to be further elucidated. Here, we illuminate that the expression of plasma membrane calcium-transporting ATPase 4 (PMCA4) was downregulated in PASMCs after PDGF-BB stimulation, which could be abolished by restraining the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK). Functionally, suppression of PMCA4 attenuated the [Ca2+]i clearance in PASMCs after Ca2+ entry, promoting cell proliferation and elevating cell locomotion through mediating formation of focal adhesion. Additionally, the expression of PMCA4 was decreased in the pulmonary artery of monocrotaline (MCT)- or hypoxia-induced PAH rats. Moreover, knockdown of PMCA4 could increase the right ventricular systolic pressure (RVSP) and wall thickness (WT) of pulmonary artery in rats raised under normal conditions. Taken together, our findings demonstrate the importance of the PDGF/MEK/ERK/PMCA4 axis in intracellular Ca2+ homeostasis in PASMCs, indicating a functional role of PMCA4 in pulmonary arterial remodeling and PAH development.

A novel miR-200c/c-myc negative regulatory feedback loop is essential to the EMT process, CSC biology and drug sensitivity in nasopharyngeal cancer.

Overexpression of the c-Myc oncogene has been implicated in cancer stem cell - like (CSC) phenotypes and epithelial-to-mesenchymal transition (EMT) in cancer. However, the underlying molecular mechanism by which c-Myc regulates EMT and CSC potential in remains unclear. In the present study, we showed that the expression of c-Myc protein is inversely correlated with microRNA (miR)-200c expression in primary tumor samples from nasopharyngeal cancer (NPC) patients. We further demonstrated that Myc and miR-200c negatively regulate the expression each other in NPC cell lines. c-Myc transcriptionally repressed expression of miR-200c by directly binding to two E-box sites located within a 1 kb segment upstream of TSS of the miR-200c. In addition, miR-200c post-transcriptionally repressed expression of c-Myc by binding to its 3'-untranslated region, suggesting the existence of a negative feedback loop between Myc and miR-200c. Overexpression of c-Myc interfered with this feedback loop and activated the EMT program, induced CSC phenotypes, and enhanced drug sensitivity, whereas miR-200c could counteract these biological effects of c-Myc. Our results provide a novel mechanism governing c-Myc and miR-200c expression and indicate that either targeting c-Myc or restoring miR-200c expression would be a promising approach to overcome oncogenic role of c-Myc in NPC.

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.

The Long Noncoding RNA LnRPT Is Regulated by PDGF-BB and Modulates the Proliferation of Pulmonary Artery Smooth Muscle Cells.

Pulmonary artery hypertension (PAH) is a rare and fatal disorder that involves extensive remodeling of the pulmonary arteries mediated by hyperproliferation of pulmonary artery smooth muscle cells (PASMCs). Aberrant platelet-derived growth factor (PDGF) activity can lead to hyperproliferation of PASMCs; however, little is known about the role of long noncoding RNA (lncRNA) in this process. Using RNA sequencing, we identified 725 lncRNAs in rat PASMCs, 95 of which were expressed differentially in response to PDGF-BB treatment. Depletion of four lncRNAs affected the proliferation of rat PASMCs as measured by 5-ethynyl-2'-deoxyuridine incorporation assay. Among these, one lncRNA, named LnRPT (lncRNA regulated by PDGF and transforming growth factor ß), was found to be the most potent in promoting the proliferation of PASMCs when knocked down. In contrast, proliferation of PASMCs was repressed when LnRPT was overexpressed. Mechanistically, LnRPT inhibited the expression of two genes involved in the Notch signaling pathway (notch3 and jag1) as well as the cell-cycle-regulating gene ccna2. In addition, downregulation of LnRPT induced by PDGF-BB was abrogated when phosphatidylinositol 3'-kinase activity was inhibited with pictilisib. Downregulation of LnRPT was also observed in the pulmonary arteries of rats with monocrotaline-induced PAH. This study provides novel insights into the effects of PDGF-BB on lncRNA expression in PASMCs, and identifies one lncRNA, LnRPT, that plays a role in PAH development as a regulator of PASMC proliferation by mediating the Notch signaling pathway and cell cycle.

PDGFBB promotes proliferation and migration via regulating miR-1181/STAT3 axis in human pulmonary arterial smooth muscle cells.

Platelet-derived growth factor (PDGF) can induce hyperproliferation of pulmonary artery smooth muscle cells (PASMCs), which is a key causative factor to the occurrence and progression of pulmonary arterial hypertension (PAH). We previously identified that miR-1181 is significantly downregulated by PDGFBB in human PASMCs. In this work, we further explore the function of miR-1181 and underlying regulatory mechanisms in PDGF-induced PASMCs. First, the expression pattern of miR-1181 was characterized under PDGFBB treatment, and PDGF receptor/PKCß signaling was found to repress miR-1181 expression. Then, gain- and loss-of-function experiments were respectively conducted and revealed the prominent role of miR-1181 in inhibiting PASMC proliferation and migration. Flow cytometry analysis suggested that miR-1181 regulated the PASMC proliferation through influencing the cell cycle transition from G0/G1 to S phase. Moreover, we exhibited that miR-1181 targeting STAT3 formed a regulatory axis to modulate PASMC proliferation. Finally, serum miR-1181 expression was also observed to be reduced in adult and newborn patients with PAH. Overall, this study provides novel findings that the miR-1181/STAT3 axis mediated PDGFBB-induced dysfunction in human PASMCs, implying a potential use of miR-1181 as a therapeutic and diagnostic candidate for the vascular remodeling diseases.

A Critical Role for P2X7 Receptor-Induced VCAM-1 Shedding and Neutrophil Infiltration during Acute Lung Injury.

Pulmonary neutrophils are the initial inflammatory cells that are recruited during lung injury and are crucial for innate immunity. However, pathological recruitment of neutrophils results in lung injury. The objective of this study is to determine whether the novel neutrophil chemoattractant, soluble VCAM-1 (sVCAM-1), recruits pathological levels of neutrophils to injury sites and amplifies lung inflammation during acute lung injury. The mice with P2X7 receptor deficiency, or treated with a P2X7 receptor inhibitor or anti-VCAM-1 Abs, were subjected to a clinically relevant two-hit LPS and mechanical ventilation-induced acute lung injury. Neutrophil infiltration and lung inflammation were measured. Neutrophil chemotactic activities were determined by a chemotaxis assay. VCAM-1 shedding and signaling pathways were assessed in isolated lung epithelial cells. Ab neutralization of sVCAM-1 or deficiency or antagonism of P2X7R reduced neutrophil infiltration and proinflammatory cytokine levels. The ligands for sVCAM-1 were increased during acute lung injury. sVCAM-1 had neutrophil chemotactic activities and activated alveolar macrophages. VCAM-1 is released into the alveolar airspace from alveolar epithelial type I cells through P2X7 receptor-mediated activation of the metalloproteinase ADAM-17. In conclusion, sVCAM-1 is a novel chemoattractant for neutrophils and an activator for alveolar macrophages. Targeting sVCAM-1 provides a therapeutic intervention that could block pathological neutrophil recruitment, without interfering with the physiological recruitment of neutrophils, thus avoiding the impairment of host defenses.

miR-4632 mediates PDGF-BB-induced proliferation and antiapoptosis of human pulmonary artery smooth muscle cells via targeting cJUN.

MicroRNAs (miRNAs) can regulate the proliferative status of pulmonary artery smooth muscle cells (PASMCs), which is a core factor modulating pulmonary vascular remodeling diseases, such as atherosclerosis and pulmonary arterial hypertension (PAH). Our previous work has shown that miR-4632, a rarely reported miRNA, is significantly downregulated in platelet-derived growth factor (PDGF)-BB-stimulated human pulmonary artery smooth muscle cells (HPASMCs), yet its cell function and the underlying molecular mechanisms remain to be elucidated. Here, we find that miR-4632 is highly expressed in HPASMCs and its expression significantly decreased in response to different stimuli. Functional studies revealed that miR-4632 inhibited proliferation and promoted apoptosis of HPASMCs but had no effects on cell contraction and migration. Furthermore, the cJUN was identified as a direct target gene of miR-4632, while knockdown of cJUN was necessary for miR-4632-mediated HPASMC proliferation and apoptosis. In addition, the downregulation of miR-4632 by PDGF-BB was found to associate with histone deacetylation through the activation of PDGF receptor/phosphatidylinositol 3'-kinase/histone deacetylase 4 signaling. Finally, the expression of miR-4632 was reduced in the serum of patients with PAH. Overall, our results suggest that miR-4632 plays an important role in regulating HPASMC proliferation and apoptosis by suppression of cJUN, providing a novel therapeutic miRNA candidate for the treatment of pulmonary vascular remodeling diseases. It also implies that serum miR-4632 has the potential to serve as a circulating biomarker for PAH diagnosis.

miR-34b Modulates Skeletal Muscle Cell Proliferation and Differentiation.

Myogenesis involves myoblast proliferation and differentiation to myocytes, followed by fusion and hypertrophy to form myotubes during muscle development. Increasing evidence showed that microRNAs (miRNAs) play important roles in the regulation of myogenesis. We have previously revealed that miR-34b is steadily increased during this process. This miRNA regulates differentiation in various cell types, though its function in myogenesis remains to be elucidated. In this study, we show that miR-34b represses muscle cell proliferation and promotes myotube formation. Our quantitative iTRAQ-based proteomic analysis reveals 97 proteins are regulated by miR-34b in mouse myoblast C2C12. We identified that miR-34b targets 14-3-3 protein gamma, adenosylhomocysteinase and nucleolin by binding to their 3'UTR. Further analysis of these proteins expression patterns show that nucleolin is a cognate target of miR-34b during myogenic differentiation. Here, we proved that a moderate reduction of nucleolin in cells enhanced the myotube formation. However, nucleolin is required for myogenesis, as cells with low levels of nucleolin reduced cell proliferation rate and are unable to differentiate. Our data demonstrated that nucleolin regulates myogenesis in a protein-abundance-dependent manner. J. Cell. Biochem. 118: 4285-4295, 2017. © 2017 Wiley Periodicals, Inc.

MicroRNA-17-92 Regulates the Transcription Factor E2F3b during Myogenesis In Vitro and In Vivo.

Myogenic differentiation, which occurs during muscle development, is a highly ordered process that can be regulated by E2F transcription factors. Available data show that E2F3b, but not E2F3a, is upregulated and required for myogenic differentiation. However, the regulation of E2F3b expression in myogenic differentiation is not well understood. To investigate whether E2Fb expression is controlled by miRNAs, we used bioinformatics to combine the database of microRNAs downregulated during myogenesis and those predicted to target E2F3. This identified miR-17 and miR-20a as miRNAs potentially involved in E2F3 regulation. We found that miR-17-92 controls the expression of E2F3b in C2C12 cells during myogenic differentiation. Moreover, we confirmed that miR-20a regulates the expression of E2F3b proteins in vivo using a muscle regeneration model.

Multi-omics analysis reveals regulators of the response to PDGF-BB treatment in pulmonary artery smooth muscle cells.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a lethal disease with pronounced narrowing of pulmonary vessels due to abnormal cell proliferation. The platelet-derived growth factor BB (PDGF-BB) is well known as a potent mitogen for smooth muscle cell proliferation. To better understand how this growth factor regulates pulmonary arterial smooth muscle cells (PASMCs) proliferation, we sought to characterize the response to PDGF-BB stimulation at system-wide levels, including the transcriptome and proteome. RESULTS: In this study, we identified 1611 mRNAs (transcriptome), 207 proteins (proteome) differentially expressed in response to PDGF-BB stimulation in PASMCs based on RNA-sequencing and isobaric tags for relative and absolute quantification (iTRAQ) assay. Transcription factor (TF)-target network analysis revealed that PDGF-BB regulated gene expression potentially via TFs including HIF1A, JUN, EST1, ETS1, SMAD1, FOS, SP1, STAT1, LEF1 and CEBPB. Among them, SMAD1-involved BMPR2/SMADs axis plays a significant role in PAH development. Interestingly, we observed that the expression of BMPR2 was decreased in both mRNA and protein level in response to PDGF-BB. Further study revealed that BMPR2 is the direct target of miR-376b that is up-regulated upon PDGF-BB treatment. Finally, EdU incorporation assay showed that miR-376b promoted proliferation of PASMCs. CONCLUSION: This integrated analysis of PDGF-BB-regulated transcriptome and proteome was performed for the first time in normal PASMCs, which revealed a crosstalk between PDGF signaling and BMPR2/SMADs axis. Further study demonstrated that PDGF-BB-induced miR-376b upregulation mediated the downregulation of BMPR2, which led to expression change of its downstream targets and promoted proliferation of PASMCs.

Loss of microRNA-17∼92 in smooth muscle cells attenuates experimental pulmonary hypertension via induction of PDZ and LIM domain 5.

RATIONALE: Recent studies suggest that microRNAs (miRNAs) play important roles in regulation of pulmonary artery smooth muscle cell (PASMC) phenotype and are implicated in pulmonary arterial hypertension (PAH). However, the underlying molecular mechanisms remain elusive. OBJECTIVES: This study aims to understand the mechanisms regulating PASMC proliferation and differentiation by microRNA-17∼92 (miR-17∼92) and to elucidate its implication in PAH. METHODS: We generated smooth muscle cell (SMC)-specific miR-17∼92 and PDZ and LIM domain 5 (PDLIM5) knockout mice and overexpressed miR-17∼92 and PDLIM5 by injection of miR-17∼92 mimics or PDLIM5-V5-His plasmids and measured their responses to hypoxia. We used miR-17∼92 mimics, inhibitors, overexpression vectors, small interfering RNAs against PDLIM5, Smad, and transforming growth factor (TGF)-ß to determine the role of miR-17∼92 and its downstream targets in PASMC proliferation and differentiation. MEASUREMENTS AND MAIN RESULTS: We found that human PASMC (HPASMC) from patients with PAH expressed decreased levels of the miR-17∼92 cluster, TGF-ß, and SMC markers. Overexpression of miR-17∼92 increased and restored the expression of TGF-ß3, Smad3, and SMC markers in HPASMC of normal subjects and patients with idiopathic PAH, respectively. Knockdown of Smad3 but not Smad2 prevented miR-17∼92-induced expression of SMC markers. SMC-specific knockout of miR-17∼92 attenuated hypoxia-induced pulmonary hypertension (PH) in mice, whereas reconstitution of miR-17∼92 restored hypoxia-induced PH in these mice. We also found that PDLIM5 is a direct target of miR-17/20a, and hypertensive HPASMC and mouse PASMC expressed elevated PDLIM5 levels. Suppression of PDLIM5 increased expression of SMC markers and enhanced TGF-ß/Smad2/3 activity in vitro and enhanced hypoxia-induced PH in vivo, whereas overexpression of PDLIM5 attenuated hypoxia-induced PH. CONCLUSIONS: We provided the first evidence that miR-17∼92 inhibits PDLIM5 to induce the TGF-ß3/SMAD3 pathway, contributing to the pathogenesis of PAH.

miR-30e-5p and miR-15a Synergistically Regulate Fatty Acid Metabolism in Goat Mammary Epithelial Cells via LRP6 and YAP1.

MicroRNA (miRNA) regulates the expression of genes and influences a series of biological processes, including fatty acid metabolism. We screened the expression of miRNA in goat mammary glands during peak-lactation and non-lactating ("dry") periods, and performed an in vitro study with goat mammary epithelial cells (GMEC) prior to sequencing analysis. Results illustrated that miR-30e-5p and miR-15a were highly expressed. Utilizing a luciferase reporter assay and Western blot, low-density lipoprotein receptor-related protein 6 (LRP6) and Yes associated protein 1 (YAP1) genes were demonstrated to be a target of miR-30e-5p and miR-15a in GMEC. Moreover, we demonstrated that the overexpression of miR-30e-5p and miR-15a in GMEC promoted fat metabolism while their knockdown impaired fat metabolism. These findings extend the discovery of a key role of miR-30e-5p and miR-15a in mediating adipocyte differentiation by suggesting a role in promoting milk fat synthesis. In conclusion, our findings indicate that miR-30e-5p, together with miR-15a, represses expression of LRP6 and promotes fat metabolism in GMEC. The data expanded our knowledge on the function of miRNAs in milk fat metabolism and synthesis in ruminant mammary cells.

miR-9 enhances the transactivation of nuclear factor of activated T cells by targeting KPNB1 and DYRK1B.

The fast response to stimuli and subsequent activation of the nuclear factor of activated T cells (NFAT) signaling pathway play an essential role in human T cell functions. MicroRNAs (miRNAs) are increasingly implicated in regulation of numerous biological and pathological processes. In this study we demonstrate a novel function of miRNA-9 (miR-9) in regulation of the NFAT signaling pathway. Upon PMA-ionomycin stimulation, miR-9 was markedly increased, consistent with NFAT activation. Overexpression of miR-9 significantly enhanced NFAT activity and accelerated NFAT dephosphorylation and its nuclear translocation in response to PMA-ionomycin. Karyopherin-ß1 (KPNB1, a nucleocytoplasmic transporter) and dual-specificity tyrosine phosphorylation-regulated kinase 1B (DYRK1B) were identified as direct targets of miR-9. Functionally, miR-9 promoted IL-2 production in stimulated human lymphocyte Jurkat T cells. Collectively, our data reveal a novel role for miR-9 in regulation of the NFAT pathway by targeting KPNB1 and DYRK1B.

MicroRNA-124 suppresses the transactivation of nuclear factor of activated T cells by targeting multiple genes and inhibits the proliferation of pulmonary artery smooth muscle cells.

Abnormal proliferation and phenotypic modulation of pulmonary artery smooth muscle cells (PASMC) contributes to the pathogenesis of numerous cardiovascular disorders, including pulmonary arterial hypertension (PAH). The nuclear factor of activated T cells (NFAT) signaling pathway is linked to PASMC proliferation and PAH. MicroRNAs (miRNAs) are small non-coding RNAs that function in diverse biological processes. To systemically identify the specific miRNAs that regulate the NFAT pathway, a human primary miRNA library was applied for cell-based high throughput screening with the NFAT luciferase reporter system. Eight miRNAs were found to modulate NFAT activity efficiently. Of them, miR-124 robustly inhibited NFAT reporter activity and decreased both the dephosphorylation and the nuclear translocation of NFAT. miR-124 also inhibited NFAT-dependent transcription of IL-2 in Jurkat T cells. miR-124 exerted its effects by targeting multiple genes, including a known component of the NFAT pathway, NFATc1, and two new regulators of NFAT signaling, CAMTA1 (calmodulin-binding transcription activator 1) and PTBP1 (polypyrimidine tract-binding protein 1). Physiologically, miR-124 was down-regulated by hypoxia in human PASMC, consistent with the activation of NFAT during this process. Down-regulation of miR-124 was also observed in 3-week hypoxia-treated mouse lungs. Furthermore, the overexpression of miR-124 not only inhibited human PASMC proliferation but also maintained its differentiated phenotype by repressing the NFAT pathway. Taken together, our data provide the first evidence that miR-124 acts as an inhibitor of the NFAT pathway. Down-regulation of miR-124 in hypoxia-treated PASMC and its antiproliferative and prodifferentiation effects imply a potential value for miR-124 in the treatment of PAH.