A Novel Splicing Mutation c.335-1 G > A in the Cardiac Transcription Factor NKX2-5 Leads to Familial Atrial Septal Defect Through miR-19 and PYK2.

Mutations of NKX2-5 largely contribute to congenital heart diseases (CHDs), especially atrial septal defect (ASD). We identified a novel heterozygous splicing mutation c.335-1G > A in NKX2-5 gene in an ASD family via whole exome sequencing (WES) and linkage analysis. Utilizing the human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) as a disease model, we showed that haploinsufficiency of NKX2-5 contributed to aberrant orchestration of apoptosis and proliferation in ASD patient-derived hiPSC-CMs. RNA-seq profiling and dual-luciferase reporter assay revealed that NKX2-5 acts upstream of PYK2 via miR-19a and miR-19b (miR-19a/b) to regulate cardiomyocyte apoptosis. Meanwhile, miR-19a/b are also downstream mediators of NKX2-5 during cardiomyocyte proliferation. The novel splicing mutation c.335-1G > A in NKX2-5 and its potential pathogenic roles in ASD were demonstrated. Our work provides clues not only for deep understanding of NKX2-5 in cardia development, but also for better knowledge in the molecular mechanisms of CHDs.

Designed miR-19a/b sponge induces apoptosis in lung cancer cells through the PI3K-PTEN-Akt pathway regulation.

BACKGROUND: MicroRNAs (miRNAs) are one of the main factors in cancer development and can alter the activity of proto-oncogenic or tumor suppressor genes. The miR-17-92 cluster, which comprises miR-17, miR-18a, miR-19a/b, miR-20a, and miR-92a, has been identified as a biomarker in a variety of cancer types. Among them, miR-19a/b exerts an oncogenic effect by suppressing tumor suppressor genes, including PTEN and TP53INP1in numerous types of cancers, including NSCLC. An miRNA sponge is an mRNA with multiple repetitive sequences that prevents miRNAs from interacting with their targets, thereby inhibiting their action. METHODS AND RESULTS: In this study, we designed an miR-19a/b sponge plasmid and transfected it into A549 lung cancer cell lines and analyzed its effects on PTEN and TP53INP1 gene expression as the main miR-19a/b target and apoptosis rate in these cell lines. CONCLUSIONS: The findings revealed that miR-19a/b sponge significantly increased PTEN and TP53INP1 mRNA expression. The effect of the sponge on TP53INP1 was much greater than that on PTEN. This is because TP53INP1 is directly (sponge effect) and indirectly (AKT pathway is affected by the P53 gene) affected by this sponge. In addition, compared with the control group, the percentage of primary and secondary apoptosis increased significantly (P value < 0.0001).

CircFN1 upregulation initiated oxidative stress-induced apoptosis and inhibition of proliferation and migration in trophoblasts via circFN1-miR-19a/b-3p-ATF2 ceRNA network.

Recently, the etiopathogenesis of preeclampsia (PE) has been developed from the perspective of circular RNA, microRNA and their crosstalk in placental oxidative stress. Real-time quantitative PCR and western blotting detected expression of circular RNA-fibronectin 1 (circFN1; ID hsa_circ_0058152), microRNA (miR)-19a/b-3p and activating transcription factor 2 (ATF2). Receiver operating characteristic (ROC) curve analyzed the predictive value of circFN1. Oxidative stress, apoptosis, proliferation, and migration were measured using superoxide dismutase (SOD) and malonaldehyde (MDA) assay kits, Annexin V/Prodium iodide and western blotting methods, MTS and EdU assays, and scratch wound assay, respectively. Target relationships were retrieved by miRNA target prediction software and confirmed by dual-luciferase reporter assay, RNA immunoprecipitation and RNA pull-down. Expression of circFN1 was upregulated in the serum of PE pregnancies, and the area under the ROC curve of serum circFN1 was 0.7826 (95% confidence interval: 0.6495-0.9157; sensitivity 86.96%; specificity 56%). Functionally, its upregulation decreased SOD activity, cell viability, EdU incorporation, migration rate, and Bcl-2 expression in human trophoblast HTR-8/SV-neo cells, but meanwhile increased MDA level, apoptosis rate, and Bax and cleaved-caspase3 expression. Moreover, its downregulation played the opposite effects in HTR-8/SV-neo cells. Mechanistically, circFN1 functioned as "miRNA sponge" for miR-19a/b-3p and modulated the latter's target gene ATF2. There were feedback effects of miR-19a/b-3p restorations and ATF2 depletion on circFN1 actions in HTR-8/SV-neo cells. Oxidative injury-mediated placental trophoblast dysfunction in PE was through competing endogenous RNA (ceRNA) mechanism of CircFN1-miR-19a/b-3p-ATF2 axes.

Circulating miR-19a-3p and miR-19b-3p characterize the human aging process and their isomiRs associate with healthy status at extreme ages.

Blood circulating microRNAs (c-miRs) are potential biomarkers to trace aging and longevity trajectories to identify molecular targets for anti-aging therapies. Based on a cross-sectional study, a discovery phase was performed on 12 donors divided into four groups: young, old, healthy, and unhealthy centenarians. The identification of healthy and unhealthy phenotype was based on cognitive performance and capabilities to perform daily activities. Small RNA sequencing identified 79 differentially expressed c-miRs when comparing young, old, healthy centenarians, and unhealthy centenarians. Two miRs, that is, miR-19a-3p and miR-19b-3p, were found increased at old age but decreased at extreme age, as confirmed by RT-qPCR in 49 donors of validation phase. The significant decrease of those miR levels in healthy compared to unhealthy centenarians appears to be due to the presence of isomiRs, not detectable with RT-qPCR, but only with a high-resolution technique such as deep sequencing. Bioinformatically, three main common targets of miR-19a/b-3p were identified, that is, SMAD4, PTEN, and BCL2L11, converging into the FoxO signaling pathway, known to have a significant role in aging mechanisms. For the first time, this study shows the age-related increase of plasma miR-19a/b-3p in old subjects but a decrease in centenarians. This decrease is more pronounced in healthy centenarians and was confirmed by the modified pattern of isomiRs comparing healthy and unhealthy centenarians. Thus, our study paves the way for functional studies using c-miRs and isomiRs as additional parameter to track the onset of aging and age-related diseases using new potential biomarkers.

miR-19a/b-3p promotes inflammation during cerebral ischemia/reperfusion injury via SIRT1/FoxO3/SPHK1 pathway.

BACKGROUND: Stroke affects 3-4% of adults and kills numerous people each year. Recovering blood flow with minimal reperfusion-induced injury is crucial. However, the mechanisms underlying reperfusion-induced injury, particularly inflammation, are not well understood. Here, we investigated the function of miR-19a/b-3p/SIRT1/FoxO3/SPHK1 axis in ischemia/reperfusion (I/R). METHODS: MCAO (middle cerebral artery occlusion) reperfusion rat model was used as the in vivo model of I/R. Cultured neuronal cells subjected to OGD/R (oxygen glucose deprivation/reperfusion) were used as the in vitro model of I/R. MTT assay was used to assess cell viability and TUNEL staining was used to measure cell apoptosis. H&E staining was employed to examine cell morphology. qRT-PCR and western blot were performed to determine levels of miR-19a/b-3p, SIRT1, FoxO3, SPHK1, NF-κB p65, and cytokines like TNF-α, IL-6, and IL-1ß. EMSA and ChIP were performed to validate the interaction of FoxO3 with SPHK1 promoter. Dual luciferase assay and RIP were used to verify the binding of miR-19a/b-3p with SIRT1 mRNA. RESULTS: miR-19a/b-3p, FoxO3, SPHK1, NF-κB p65, and cytokines were elevated while SIRT1 was reduced in brain tissues following MCAO/reperfusion or in cells upon OGD/R. Knockdown of SPHK1 or FoxO3 suppressed I/R-induced inflammation and cell death. Furthermore, knockdown of FoxO3 reversed the effects of SIRT1 knockdown. Inhibition of the miR-19a/b-3p suppressed inflammation and this suppression was blocked by SIRT1 knockdown. FoxO3 bound SPHK1 promoter and activated its transcription. miR-19a/b-3p directly targeted SIRT1 mRNA. CONCLUSION: miR-19a/b-3p promotes inflammatory responses during I/R via targeting SIRT1/FoxO3/SPHK1 axis.

miR-19a/b promote EMT and proliferation in glioma cells via SEPT7-AKT-NF-κB pathway.

miR-19a/b belong to the miR-17-92 family. We have demonstrated previously that miR-19a/b are overexpressed in glioma and glioma cell lines. However, the role of miR-19a/b in glioma remains unclear. In the present study, we aim to identify the biological function and molecular mechanism of miR-19a/b in glioma cell proliferation and epithelial-mesenchymal transition (EMT). Knocking down miR-19a/b in LN308 glioblastoma (GBM) cells with higher expression of miR-19a/b inhibits cell proliferation and invasion, induces apoptosis, and suppresses EMT by downregulating the expression of Akt, phosphorylated p-Akt, nuclear factor κB (NF-κB), Snail, N-cadherin, and Vimentin and upregulating E-cadherin in vitro and in vivo. Enhanced proliferation and EMT are also observed when miR-19a/b are transfected into SNB19 GBM cells, with lowered expression of miR-19a/b. miR-19a is more effective than miR-19b in the regulation of biological behavior of glioma cells. miR-19a/b modulate molecular events for the promotion of EMT via the Akt-NF-κB pathway. SEPT7 has been confirmed as the target gene of miR-19a/b. The effect of miR-19a/b on proliferation and EMT of glioma cells and the Akt-NF-κB pathway could be reversed by transfection with SEPT7. Our study strongly suggests that miR-19a/b play a significant role in glioma progression and EMT through regulating target gene-SEPT7 and the SEPT7-Akt-NF-κB pathway.

PAI-1 is a novel component of the miR-17~92 signaling that regulates pulmonary artery smooth muscle cell phenotypes.

We have previously reported that miR-17~92 is critically involved in the pathogenesis of pulmonary hypertension (PH). We also identified two novel mR-17/20a direct targets, PDZ and LIM domain protein 5 (PDLIM5) and prolyl hydroxylase 2 (PHD2), and elucidated the signaling pathways by which PDLIM5 and PHD2 regulate functions of pulmonary artery smooth muscle cells (PASMCs). In addition, we have shown that plasminogen activator inhibitor-1 (PAI-1) is also downregulated in PASMCs that overexpress miR-17~92. However, it is unclear whether PAI-1 is a direct target of miR-17~92 and whether it plays a role in regulating the PASMC phenotype. In this study, we have identified PAI-1 as a novel target of miR-19a/b, two members of the miR-17~92 cluster. We found that the 3'-untranslated region (UTR) of PAI-1 contains a miR-19a/b binding site and that miR-19a/b can target this site to suppress PAI-1 protein expression. MiR-17/20a, two other members of miR-17~92, may also indirectly suppress PAI-1 expression through PDLIM5. PAI-1 is a negative regulator of miR-17~92-mediated PASMC proliferation. Silencing of PAI-1 induces Smad2/calponin signaling in PASMCs, suggesting that PAI-1 is a negative regulator of the PASMC contractile phenotype. We also found that PAI-1 is essential for the metabolic gene expression in PASMCs. Furthermore, although there is no significant change in PAI-1 levels in PASMCs isolated from idiopathic pulmonary arterial hypertension and associated pulmonary arterial hypertension patients, PAI-1 is downregulated in hypoxia/Sugen-induced hypertensive rat lungs. These results suggest that miR-17~92 regulates the PASMC contractile phenotype and proliferation coordinately and synergistically by direct and indirect targeting of PAI-1.

Oncogenic miR-19a and miR-19b co-regulate tumor suppressor MTUS1 to promote cell proliferation and migration in lung cancer.

MTUS1 (microtubule-associated tumor suppressor 1) has been identified that can function as a tumor suppressor gene in many malignant tumors. However, the function and mechanisms underlying the regulation of MTUS1 are unclear. In the present study, we reported that miR-19a and miR-19b (miR-19a/b) promote proliferation and migration of lung cancer cells by targeting MTUS1. First, MTUS1 was proved to function as a tumor suppressor in lung cancer and was linked to cell proliferation and migration promotion. Second, an inverse correlation between miR-19a/b expression and MTUS1 mRNA/protein expression was noted in human lung cancer tissues. Third, MTUS1 was appraised as a direct target of miR-19a/b by bioinformatics analysis. Fourth, direct MTUS1 regulation by miR-19a/b in lung cancer cells was experimentally affirmed by cell transfection assay and luciferase reporter assay. Finally, miR-19a/b were shown to cooperatively repress MTUS1 expression and synergistically regulate MTUS1 expression to promote lung cancer cell proliferation and migration. In conclusion, our findings have provided the first clues regarding the roles of miR-19a/b, which appear to function as oncomirs in lung cancer by downregulating MTUS1.

Oncogenic miR-19a and miR-19b co-regulate tumor suppressor MTUS1 to promote cell proliferation and migration in lung cancer

MTUS1 (microtubule-associated tumor suppressor 1) has been identified that can function as a tumor suppressor gene in many malignant tumors. However, the function and mechanisms underlying the regulation of MTUS1 are unclear. In the present study, we reported that miR-19a and miR-19b (miR-19a/b) promote proliferation and migration of lung cancer cells by targeting MTUS1. First, MTUS1 was proved to function as a tumor suppressor in lung cancer and was linked to cell proliferation and migration promotion. Second, an inverse correlation between miR-19a/b expression and MTUS1 mRNA/protein expression was noted in human lung cancer tissues. Third, MTUS1 was appraised as a direct target of miR-19a/b by bioinformatics analysis. Fourth, direct MTUS1 regulation by miR-19a/b in lung cancer cells was experimentally affirmed by cell transfection assay and luciferase reporter assay. Finally, miR-19a/b were shown to cooperatively repress MTUS1 expression and synergistically regulate MTUS1 expression to promote lung cancer cell proliferation and migration. In conclusion, our findings have provided the first clues regarding the roles of miR-19a/b, which appear to function as oncomirs in lung cancer by downregulating MTUS1.