Long Noncoding RNA HLA Complex Group 18 Improves the Cell Proliferation of Myocardial Fibroblasts by Regulating the Hsa-microRNA-133a/Epidermal Growth Factor Receptor Axis.

Hsa-microRNA (has-miR)-133a inactivates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and suppresses the cell proliferation of myocardial fibroblasts by downregulation of the epidermal growth factor receptor (EGFR) expression. Bioinformatics analysis exhibits extended noncoding RNA HLA complex group 18 (lncRNA-HCG18) binds to hsa-miR-133a. The purpose of the current experiment is to explore whether lncRNA-HCG18 adsorbed hsa-miR-133a through sponging, resulting in decreased inhibition of hsa-miR-133a on EGFR and ultimately promoting the proliferation of myocardial fibroblasts. To verify and study the correlation and mechanism between lncRNA-HCG18, hsa-miR-133a, and their target genes. Firstly, after overexpression/silencing of lncRNA-HCG18 in myocardial fibroblasts, the level of hsa-miR-133a expression was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and the EGFR, ERK1/2, and p-ERK1/2 expression levels were assessed by Western blotting to confirm that upregulation of EGFR and p-ERK1/2 protein levels by overexpression of lncRNA-HCG18, siRNA lncRNA-HCG18 (siHCG18) reduced the EGFR and p-ERK1/2 protein levels. Then, the luciferase reporter gene system was used to verify that lncRNA-HCG18 regulated EGFR expression by inhibiting the function of the hsa-miR-133a regulatory target gene. Then, a RAP assay was used to confirm that lncRNA-HCG18 interacted with hsa-miR-133a. Finally, the analysis of CCK-8 results indicated that the cell proliferation of myocardial fibroblasts was significantly reduced by siHCG18 while reversed by overexpression of lncRNA-HCG18. Thus, lncRNA-HCG18 inhibited cell viability of cardiac fibroblasts via the hsa-miR-133a/EGFR axis, which was regarded as a regulator of cell proliferation of cardiac fibroblasts in cardiovascular diseases.

BMPR2 promoter methylation and its expression in valvular heart disease complicated with pulmonary artery hypertension.

Valvular heart disease (VHD) is a common heart disease that affects blood flow. It usually requires heart surgery. Valvular heart disease complicated with pulmonary artery hypertension (VHD-PAH) may be lethal due to heart failure that results from increased heart burden. It is important for these patients to seek early treatment in order to minimize the heart damage. However, there is no reliable diagnosis method in VHD. In this study, we found DNA methylation was increased at the promoter of BMPR2 gene in the VHD patients compared with the healthy controls. This finding was confirmed by an independent cohort study of VHD patients and healthy controls. In addition, BMPR2 mRNA levels were reduced in the plasma of the VHD patients. There is strong correlation between BMPR2 promoter DNA methylation and the severity of VHD. Indeed, we found that both BMPR2 promoter DNA methylation and BMPR2 mRNA levels in the plasma are good biomarkers of VHD by themselves, with the respective AUC value of 0.879 and 0.725, respectively. When they were used in combination, the diagnostic value was even better, with the AUC value of 0.93. Consistent with the results in the VHD patients, we observed decreased BMPR2 and increased fibrosis in the lung of a PAH model mouse. BMPR2 was also decreased in the hearts of the PAH mice, whereas BMP4 was increased. Furthermore, BMPR2 was reduced in the heart valve tissue samples of human VHD patients after valve replacement with moderate/severe PAH compared with those with mild PAH. There was also increased apoptosis in the hearts of the PAH mice. BMPR2 promoter DNA methylation and its expression appear to be good biomarkers for VHD. Our results also suggest that DNA methylation may cause PAH through deregulation of BMP signaling and increased apoptosis.

Efficient detection of differentially methylated regions in the genome of patients with thoracic aortic dissection and association with MMP2 hypermethylation.

DNA methylation is known to regulate the expression of numerous genes but its role in the pathogenesis of thoracic aortic dissection (TAD) has remained largely elusive. In the present study, the DNA methylome of patients with TAD was analyzed using a methylation microarray and bisulfite pyrosequencing was used to determine whether the hypermethylation of matrix metalloproteinase 2 (MMP2) specifically is associated with TAD. Chip-based whole-DNA methylome analysis was performed on 4 male patients with TAD and 4 male healthy controls using an Illumina HumanMethylation EPIC 850K BeadChip. The resulting data were analyzed by clustering and principal component analysis, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed on the differentially methylated genes to interrogate their biological functions. Compared to the healthy controls, 3,362 loci were differentially methylated in the patients with TAD with a statistical significance of P<0.05, while 1,223 loci had a significance of P<0.01. Among these loci, 2,019 were hypermethylated and 1,343 were hypomethylated. From GO analysis within the biological process category, the MMP2, MMP14 and WNT2B genes were identified. enrichment was observed for loci involved in cellular component organization, enzyme-linked receptor protein signaling pathways (potentially having a key role in the development of cardiopulmonary function disorders) and vascular reconstruction. Bisulfite pyrosequencing of plasma samples indicated significantly increased methylation (P<0.01) of the CpG site at position 2 in the promoter of MMP2 in the TAD group relative to the healthy controls, and the mean methylation level of four CpG sites on the MMP2 gene in the TAD group was slightly higher than that in the control group, but not significantly. Hypermethylation of the MMP2 promoter may be a promising novel diagnostic and prognostic biomarker for TAD. Future studies on the epigenetics of biomarkers linked to vascular reconstruction and immune function may provide further insight into the pathogenesis and progression of TAD.

MicroRNA-9 Enhanced Cisplatin Sensitivity in Nonsmall Cell Lung Cancer Cells by Regulating Eukaryotic Translation Initiation Factor 5A2.

We determined the role of microRNA (miR)-9 in regulating cisplatin chemoresistance in nonsmall cell lung cancer (NSCLC) cells. miR-9 and eukaryotic translation initiation factor 5A2 (eIF5A2) levels were examined by reverse transcription-quantitative PCR. Cell Counting Kit-8 and the 5-ethynyl-2'-deoxyuridine (EdU) assay were used to determine the effects of miR-9 mimic or inhibitor on NSCLC cell proliferation and viability, respectively. Bioinformatics was used to analyze the relationship between miR-9 and eIF5A2. Flow cytometry was used to analyze the percentage of apoptotic cells. miR-9 mimic enhanced cisplatin sensitivity, while miR-9 inhibitor produced the opposite result. eIF5A2 was identified as a potential target of miR-9, where miR-9 regulated eIF5A2 expression at mRNA and protein level. miR-9 mimic decreased the expression of eIF5A2 mRNA and protein, while miR-9 inhibitor increased eIF5A2 expression. eIF5A2 knockdown resolved the effects of miR-9 mimic or inhibitor on cisplatin sensitivity. miR-9 may be a potential biomarker for enhancing cisplatin sensitivity by regulating eIF5A2 in NSCLC cells.

Differentially methylated regions in patients with rheumatic heart disease and secondary pulmonary arterial hypertension.

The aim of the present study was to identify differentially methylated regions (DMRs) in patients with rheumatic heart disease and secondary pulmonary arterial hypertension (RHD-PAH). A genome-wide DNA methylation assay was performed between 6 patients with RHD-PAH and 6 healthy controls using an Illumina Infinium HumanMethylation450 BeadChip kit. The Limma software package was subsequently used to identify significant DMRs. A total of 40 hypome-thylated and 64 hypermethylated CpG sites were identified between the RHD-PAH group and the control group. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes term and signaling pathway enrichment analyses revealed that the DMRs, mapped to the genes including protein kinase C α, protein kinase AMP-activated non-catalytic subunit γ2, sprouty related EVH1 domain containing 2 and LIF interleukin 6 family cytokine, were significantly enriched in the negative regulation of protein kinase/transferase activity and the positive regulation of protein amino acid phosphorylation/phosphate metabolic process. The identified DMRs may provide novel insights into the pathogenesis of RHD-PAH.

MicroRNA-9 regulates non-small cell lung cancer cell invasion and migration by targeting eukaryotic translation initiation factor 5A2.

MicroRNAs (miRNAs) play a critical role in cancer development and progression. Bioinformatics analyses has identified eukaryotic translation initiation factor 5A2 (eIF5A2) as a target of miR-9. In this study, we attempted to determine whether miR-9 regulates non-small cell lung cancer (NSCLC) cell invasion and migration by targeting eIF5A2 We examined eIF5A2 expression using reverse transcription-quantitative PCR (RT-qPCR) and subsequently transfected A549 and NCI-H1299 NSCLC cells with a miR-9 mimic or miR-9 inhibitor to determine the migration and invasive capability of the cells via wound healing assay and Transwell invasion assay, respectively. E-cadherin and vimentin expression was detected with western blotting. The miR-9 mimic significantly reduced NSCLC cell invasive and metastatic ability, and the miR-9 inhibitor enhanced NSCLC cell migration activity, increasing the number of migrated cells. There was no significant difference between the negative control siRNA and miR-9 mimic groups after knockdown of eIF5A2; western blotting showed that miR-9 regulated E-cadherin and vimentin expression. These data show that miR-9 regulates NSCLC cell invasion and migration through regulating eIF5A2 expression. Taken together, our findings suggest that the mechanism of miR-9-regulated NSCLC cell invasion and migration may be related to epithelial-mesenchymal transition.

Hypermethylation of brain natriuretic peptide gene is associated with the risk of rheumatic heart disease.

To investigate the contribution of brain natriuretic peptide (BNP) promoter DNA methylation to the risk of rheumatic heart disease (RHD) and the influence of warfarin anticoagulant therapy on BNP methylation levels for RHD patients after surgery. BNP methylation levels were determined by bisulfite pyrosequencing from plasma samples of RHD patients compared with healthy controls. Several factors influencing the RHD patients were included like age, smoking and cholesterol levels. A fragment of five CG sites (CpG1-5) in the promoter region of BNP gene was measured. BNP gene hypermethylation was found in CpG4 and CpG5 in RHD patients compared with non-RHD controls. A significant difference was also observed between RHD patients with long-term administration of warfarin and RHD patients who had recently undergone an operation. Moreover, single CpG4 and CpG5 analysis revealed a significant increase in methylation levels in men. BNP gene body hypermethylation is associated with the risk of RHD, and also influenced by the warfarin anticoagulant therapy of RHD patients after surgery, which could represent novel and promising targets for therapeutic development.

Detection of Differentially Expressed MicroRNAs in Rheumatic Heart Disease: miR-1183 and miR-1299 as Potential Diagnostic Biomarkers.

This study compared microRNA (miRNA) expression profiles between rheumatic heart disease (RHD) patients and healthy controls to investigate their differential expression and help elucidate their mechanisms of action. Microarray analysis was used to measure miRNA expression, and a total of 133 miRNAs were shown to be significantly upregulated in RHD patients compared with controls, including miR-1183 and miR-1299. A total of 137 miRNAs, including miR-4423-3p and miR-218-1-3p, were significantly downregulated in RHD patients. Quantitative real-time-PCR confirmed microarray findings for miR-1183 and miR-1299 in both tissue and plasma. Bioinformatic predictions were also made of differentially expressed miRNAs as biomarkers in RHD by databases and GO/pathway analysis. Furthermore, we investigated miR-1183 and miR-1299 expression in RHD patients with secondary pulmonary hypertension (PAH). Our findings identified an important role for miR-1299 as a direct regulator of RHD, while the observed difference in expression of miR-1183 between RHD-PAH patients with high or low pulmonary artery pressure suggests that miR-1183 overexpression may reflect pulmonary artery remodeling. miR-1183 and miR-1299 appear to play distinct roles in RHD pathogenesis accompanied by secondary PAH and could be used as potential biological markers for disease development.

Cisplatin sensitivity is enhanced in non-small cell lung cancer cells by regulating epithelial-mesenchymal transition through inhibition of eukaryotic translation initiation factor 5A2.

BACKGROUND: Epithelial-mesenchymal transition (EMT) has been believed to be related with chemotherapy resistance in non-small cell lung cancer (NSCLC). Recent studies have suggested eIF5A-2 may function as a proliferation-related oncogene in tumorigenic processes. METHODS: We used cell viability assays, western blotting, immunofluorescence, transwell-matrigel invasion assay, wound-healing assay combined with GC7 (a novel eIF5A-2 inhibitor) treatment or siRNA interference to investigate the role of eIF5A-2 playing in NSCLC chemotherapy. RESULTS: We found low concentrations of GC7 have little effect on NSCLC viability, but could enhance cisplatin cytotoxicity in NSCLC cells. GC7 also could reverse mesenchymal phenotype in NCI-H1299 and prevented A549 cells undergoing EMT after TGF-ß1 inducement. eIF5A-2 knockdown resulted in EMT inhibition. CONCLUSION: Our data indicated GC7 enhances cisplatin cytotoxicity and prevents the EMT in NSCLC cells by inhibiting eIF5A-2.

Down-regulation of eIF5A-2 prevents epithelial-mesenchymal transition in non-small-cell lung cancer cells.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is believed to be the critical process in malignant tumor invasion and metastases, and has a great influence on improving the survival rate in non-small-cell lung cancer (NSCLC) patients. Recent studies suggested that eukaryotic initiation factor 5A-2 (eIF5A-2) might serve as an adverse prognostic marker of survival. We detected eIF5A-2 in NSCLC A549 cells, and found that the invasive capability correlates with the eIF5A-2 expression. METHODS: Transforming growth factor (TGF)-ß1 was used to induce EMT in A549 cells. Western blotting, immunofluorescence, wound healing assay, and transwell-matrigel invasion chambers were used to identify phenotype changes. Western blotting was also used to observe changes of the expression of eIF5A-2. We down-regulated the eIF5A-2 expression using an eIF5A-2 siRNA and identified the phenotype changes by western blotting and immunofluorescence. We tested the change of migration and invasion capabilities of A549 cells by the wound healing assay and transwell-matrigel invasion chambers. RESULTS: After stimulating with TGF-ß1, almost all A549 cells changed to the mesenchymal phenotype and acquired more migration and invasion capabilities. These cells also had higher eIF5A-2 protein expression. Down-regulation of eIF5A-2 expression with eIF5A-2 siRNA transfection could change the cells from mesenchymal to epithelial phenotype and decrease tumor cell migration and invasive capabilities significantly. CONCLUSIONS: The expression of eIF5A-2 was up-regulated following EMT phenotype changes in A549 cells, which correlated with enhanced tumor invasion and metastatic capabilities. Furthermore, in the A549 cell line, the process of EMT phenotype change could be reversed by eIF5A-2 siRNA, with a consequent weakening of both invasive and metastatic capabilities.