A 10-Gene Signature to Predict the Prognosis of Early-Stage Triple-Negative Breast Cancer.

Purpose: Triple-negative breast cancer (TNBC) is a particularly challenging subtype of breast cancer, with a poorer prognosis compared to other subtypes. Unfortunately, unlike luminal type cancers, there is no validated biomarker to predict the prognosis of patients with early-stage TNBC. Accurate biomarkers are needed to establish effective therapeutic strategies. Materials and Methods: In this study, we analyzed gene expression profiles of tumor samples from 184 TNBC patients (training cohort, n=76; validation cohort, n=108) using RNA sequencing. Results: By combining weighted gene expression, we identified a 10-gene signature (DGKH, GADD45B, KLF7, LYST, NR6A1, PYCARD, ROBO1, SLC22A20P, SLC24A3, and SLC45A4) that stratified patients by risk score with high sensitivity (92.31%), specificity (92.06%), and accuracy (92.11%) for invasive disease-free survival. The 10-gene signature was validated in a separate institution cohort and supported by meta-analysis for biological relevance to well-known driving pathways in TNBC. Furthermore, the 10-gene signature was the only independent factor for invasive disease-free survival in multivariate analysis when compared to other potential biomarkers of TNBC molecular subtypes and T-cell receptor ß diversity. 10-gene signature also further categorized patients classified as molecular subtypes according to risk scores. Conclusion: Our novel findings may help address the prognostic challenges in TNBC and the 10-gene signature could serve as a novel biomarker for risk-based patient care.

hsa-miR-CHA2, a novel microRNA, exhibits anticancer effects by suppressing cyclin E1 in human non-small cell lung cancer cells.

Despite considerable therapeutic advancements, the global survival rate for lung cancer patients remains poor, posing challenges in developing an effective treatment strategy. In many cases, microRNAs (miRNAs) exhibit abnormal expression levels in cancers, including lung cancer. Dysregulated miRNAs often play a crucial role in the development and progression of cancer. Therefore, understanding the mechanisms underlying aberrant miRNA expression during carcinogenesis may provide crucial clues to develop novel therapeutics. In this study, we identified and cloned a novel miRNA, hsa-miR-CHA2, which is abnormally downregulated in non-small cell lung cancer (NSCLC)-derived cell lines and tissues of patients with NSCLC. Furthermore, we found that hsa-miR-CHA2 regulates the post-transcriptional levels of Cyclin E1 (CCNE1) by binding to the 3'-UTR of CCNE1 mRNA. CCNE1, a cell cycle regulator involved in the G1/S transition, is often amplified in various cancers. Notably, hsa-miR-CHA2 overexpression led to the alteration of the Rb-E2F pathway, a significant signaling pathway in the cell cycle, by targeting CCNE1 in A549 and SK-LU-1 cells. Subsequently, we confirmed that hsa-miR-CHA2 induced G1-phase arrest and exhibited an anti-proliferative effect by targeting CCNE1. Moreover, in subcutaneous xenograft mouse models, intra-tumoral injection of polyplexed hsa-miR-CHA2 mimic suppressed tumor growth and development. In conclusion, hsa-miR-CHA2 exhibited an anticancer effect by targeting CCNE1 both in vitro and in vivo. These findings suggest the potential role of hsa-miR-CHA2 as an important regulator of cell proliferation in molecular-targeted therapy for NSCLC.

A Unique Immune-Related Gene Signature Represents Advanced Liver Fibrosis and Reveals Potential Therapeutic Targets.

Innate and adaptive immune responses are critically associated with the progression of fibrosis in chronic liver diseases. In this study, we aim to identify a unique immune-related gene signature representing advanced liver fibrosis and to reveal potential therapeutic targets. Seventy-seven snap-frozen liver tissues with various chronic liver diseases at different fibrosis stages (1: n = 12, 2: n = 12, 3: n = 25, 4: n = 28) were subjected to expression analyses. Gene expression analysis was performed using the nCounter PanCancer Immune Profiling Panel (NanoString Technologies, Seattle, WA, USA). Biological meta-analysis was performed using the CBS Probe PINGSTM (CbsBioscience, Daejeon, Korea). Using non-tumor tissues from surgically resected specimens, we identified the immune-related, five-gene signature (CHIT1_FCER1G_OSM_VEGFA_ZAP70) that reliably differentiated patients with low- (F1 and F2) and high-grade fibrosis (F3 and F4; accuracy = 94.8%, specificity = 91.7%, sensitivity = 96.23%). The signature was independent of all pathological and clinical features and was independently associated with high-grade fibrosis using multivariate analysis. Among these genes, the expression of inflammation-associated FCER1G, OSM, VEGFA, and ZAP70 was lower in high-grade fibrosis than in low-grade fibrosis, whereas CHIT1 expression, which is associated with fibrogenic activity of macrophages, was higher in high-grade fibrosis. Meta-analysis revealed that STAT3, a potential druggable target, highly interacts with the five-gene signature. Overall, we identified an immune gene signature that reliably predicts advanced fibrosis in chronic liver disease. This signature revealed potential immune therapeutic targets to ameliorate liver fibrosis.

The novel microRNA hsa-miR-CHA1 regulates cell proliferation and apoptosis in human lung cancer by targeting XIAP.

OBJECTIVES: MicroRNAs have critical roles in cancer development by regulating the expression of oncogenes or tumor suppressor genes. We identified and characterized a novel miRNA, miR-CHA1, in human lung cancer cells. The aim of this study was to investigate its novel function in human lung cancer by targeting XIAP. MATERIAL AND METHODS: Novel miRNA cloning, Real-time qRT-PCR, western blotting, dual luciferase assay, miRNA transfection, proliferation and apoptosis assay were carried on human lung cancer cell line A549. Fifteen paired NSCLC tissues and noncancerous lung tissues were collected. In vivo xenograft assay was performed. RESULTS: Expression of miR-CHA1 was downregulated in human lung cancer cell lines and tissues compared with normal cells and tissues. We identified a putative target gene, XIAP, whose expression was regulated by miR-CHA1 overexpression. XIAP is an inhibitor of apoptosis that represses the activation of caspase 3 and 9. XIAP mRNA and protein levels were directly suppressed by miR-CHA1. XIAP has an important role in carcinogenesis, and previous studies suggest that it may regulate cell survival and proliferation by its anti-apoptotic ability. CONCLUSION: Taken together, miR-CHA1 inhibited cell proliferation and induced apoptosis in vitro and in vivo by targeting XIAP. These data can be applied to identify novel therapeutic targets for lung cancer therapy.

Alteration of microRNA 340-5p and Arginase-1 Expression in Peripheral Blood Cells during Acute Ischemic Stroke.

Acute stroke alters the systemic immune response as can be observed in peripheral blood; however, the molecular mechanism by which microRNA (miRNA) regulates target gene expression in response to acute stroke is unknown. We performed a miRNA microarray on the peripheral blood of 10 patients with acute ischemic stroke and 11 control subjects. Selected miRNAs were quantified using a TaqMan assay. After searching for putative targets from the selected miRNAs using bioinformatic analysis, functional studies including binding capacity and protein expression of the targets of the selected miRNAs were performed. The results reveal a total of 30 miRNAs that were differentially expressed (16 miRNAs were upregulated and 14 miRNAs were downregulated) during the acute phase of stroke. Using prediction analysis, we found that miR-340-5p was predicted to bind to the 3'-untranslated region of the arginase-1 (ARG1) gene; a luciferase reporter assay confirmed the binding of miR-340-5p to ARG1. miR-340-5p was downregulated whereas ARG1 mRNA was upregulated in peripheral blood in patients experiencing acute stroke. Overexpression of miR-340-5p in human neutrophil and mouse macrophage cell lines induced downregulation of the ARG1 protein. Transfection with miR-340-5p increased nitric oxide production after LPS treatment in a mouse macrophage cell line. Our results suggest that several miRNAs are dynamically altered in the peripheral blood during the acute phase of ischemic stroke, including miR-340-5p. Acute stroke induces the downregulation of miR-340-5p, which subsequently upregulates ARG1 protein expression.

The novel hsa-miR-12528 regulates tumourigenesis and metastasis through hypo-phosphorylation of AKT cascade by targeting IGF-1R in human lung cancer.

Lung cancer cases are increasing yearly; however, few novel therapeutic strategies for treating this disease have been developed. Here the dysregulation between microRNAs and oncogenes or tumour-suppressor genes forms a close connection-loop to the development or progression in human lung carcinogenesis. That is, the relationship between microRNAs and carcinogenic mechanism may find the critical clue to improve the treatment efficacy. Accordingly, we identified and characterised a novel microRNA, hsa-miR-12528, in A549 cells. The miR-12528 expression was aberrantly downregulated in cancer cell lines and in the patient tissues derived from human non-small cell lung cancer. In addition, we found that miR-12528 post-transcriptionally controls the translation of the insulin-like growth factor 1 receptor (IGF-1R) gene by directly targeting the 3'-untranslated region of IGF-1R mRNA. Notably, the IGF-1R gene is elevated in the majority of cancers and may be an attractive therapeutic target for anticancer therapy because elevated IGF-1R mediates the signalling amplification of a major oncogenic pathway in neoplasia. In A549 cells, miR-12528 overexpression epigenetically altered the downstream phosphorylation of the primary IGF-1R networks, negatively regulated proliferation, apoptosis and migratory activity, and consequently inhibited tumourigenesis and metastasis in vivo. Therefore, our discovery of hsa-miR-12528 may be able to be applied to the development of molecular-target therapeutic strategies and diagnosis-specific biomarkers for human lung cancer.

Discovery and characterization of miRNA during cellular senescence in bone marrow-derived human mesenchymal stem cells.

Cellular senescence is an irreversible cell cycle arrest in which specific mRNAs and miRNAs are involved in senescence progression. miRNAs interact with specific mRNAs to regulate various cellular mechanisms, including metabolism, proliferation, apoptosis, senescence and differentiation. In this study, we identify and characterize miRNAs during cellular senescence in mesenchymal stem cells (MSCs). Using previously reported miRNAs, expression profiling of 23 miRNAs was performed using real-time PCR analysis. Among these miRNAs, 19 miRNAs showed upregulated expression patterns in senescent MSCs compared with young MSCs, and 5 miRNAs were downregulated. These miRNAs have not been previously identified as being related to cellular senescence but seem to be related. miR-103-2*, miR-140-5p and miR-330-5p are highly upregulated, while miR-29b and miR-199b-5p are significantly downregulated in senescent MSCs. We identify unique functions of 5 miRNAs and predict putative target genes of 5 miRNAs using our previous report. Among them, miR-199b-5p directly suppressed LAMC1 expression, as shown in a luciferase assay. miR-199b-5p significantly regulates translational activity but does not control post-transcriptional activity. Likewise, miR-199b-5p modulates LAMC networks, which demonstrates the resulting phenomenon during cellular senescence, namely, that miR-199b-5p indirectly regulates cellular senescence in MSCs.

Blood genomic profiling in extracranial- and intracranial atherosclerosis in ischemic stroke patients.

OBJECTIVE: Extracranial- and intracranial atherosclerosis (ECAS and ICAS) have been suggested to have different pathogeneses. Blood genomic profiling may identify their unique molecular signatures. METHODS: Whole gene microarray of peripheral blood was performed in 24 patients with acute ischemic stroke (ECAS, n=12; ICAS, n=12) and 12 healthy controls. Differential gene expression and gene set enrichment analysis (GSEA) were conducted. Plasma resistin levels were compared across independent samples of stroke patients with ECAS (n=39), ICAS (n=20), and small vessel disease (SVD, n=57). RESULTS: Microarray revealed that 144 and 24 transcripts were altered in ECAS and ICAS, respectively, compared to controls. All the transcripts that were differentially expressed in ICAS were also differentially expressed in ECAS. A total of 120 transcripts were differentially expressed only in ECAS. Gene sets related to immune response and protein metabolism were altered in both ECAS and ICAS, but the magnitude of gene alteration was higher in ECAS than in ICAS. Several genes of interest including RETN, IRF5, CD163, and CHST13 were more highly expressed in ECAS than in ICAS. Circulating resistin levels were elevated in independent samples of ECAS, but not in those of ICAS, compared to those of SVDs. CONCLUSIONS: ECAS showed prominent genomic alteration related to immune response compared to ICAS. Although there was no ECAS-specific gene to be identified on microarray, the level of resistin expression was high on peripheral blood in ECAS, suggesting that resistin is associated with the pathogenesis of ECAS.

Changes in PTTG1 by human TERT gene expression modulate the self-renewal of placenta-derived mesenchymal stem cells.

In addition to their differentiation potential, self-renewal capability is an important characteristic of stem cells. The limited self-renewal activity of mesenchymal stem cells is the greatest obstacle to the application of stem cell therapy in regenerative medicine. The human TERT gene enhances the self-renewal of MSCs, but the mechanism of self-renewal and the interactions among TERT-gene-related molecules remain unknown. The objectives of this study were to generate immortalized MSCs derived from MSCs isolated from placenta (naive) by human TERT gene transfection with the AMAXA gene delivery system, to compare their characteristics, and to investigate whether increased TERT expression affected the pituitary tumor transforming gene (PTTG1; also known as securin), which is involved in chromosome segregation during mitosis. TERT-immortalized cells (TERT+) with a prolonged life span displayed high PTTG1 expression. TERT+ cells also retained the stemness capacity and multipotency of naive cells and displayed high PTTG1 expression. However, down-regulation of PTTG1 by treatment with short interfering RNA induced cell senescence and decreased telomerase activity. Moreover, TERT bound to PTTG1 formed complexes with chaperones such as Ku70 and heat shock protein 90. Thus, placental MSCs immortalized by TERT gene transfection display differentiation potential and exhibit enhanced self-renewal through a balanced interaction of PTTG1 and chaperones. The interaction between TERT and PTTG1 by association of Ku70 might be important for the enhancement of the limited self-renewal activity of MSCs and for understanding the regulatory mechanisms of self-renewal.

Novel anti-nociceptive effects of cardamonin via blocking expression of cyclooxygenase-2 and transglutaminase-2.

Recently, we reported that Alpinia katsumadai (AK) has anti-nociceptive activity in vivo and that cardamonin (CDN) from AK suppresses the activity and expression of transglutaminase-2 (Tgase-2). However, it remains unknown whether CDN contributes to the anti-nociceptive activities of AK in vivo. We examined the anti-inflammatory effects of CDN in MG63 osteoblast-like cells and Raw264.7 macrophage-like cells treated with interleukin-1ß treatment. CDN suppressed the expression of Tgase-2, cyclooxygenase-2 (COX-2), and p65 (nuclear factor-κB) in a concentration-dependent manner, and restored the expression of IκB in MG63 and Raw264.7 cells. However, CDN did not inhibit the activity of COX-2. Gene silencing of Tgase-2 reduced the COX-2 expression in MG63 cells. Phenylbenzoquinone (PBQ)-induced writhing, carrageenan-induced hyperalgesia, and rota-rod test were used to evaluate the anti-nociceptive activity in vivo. CDN (3-30 mg/kg, orally administered) significantly inhibited PBQ-induced writhing. CDN also produced a significant, dose-dependent increase in the withdrawal response latencies in carrageenan-induced hyperalgesia. The effects of CDN on PBQ-induced writhing were not caused by impaired motor functions. These results suggest that CDN might be helpful in controlling the pain from inflammatory diseases.

Predictive value of circulating interleukin-6 and heart-type fatty acid binding protein for three months clinical outcome in acute cerebral infarction: multiple blood markers profiling study.

INTRODUCTION: There is no single blood marker for predicting the prognosis in ischemic stroke. A combination of multiple blood markers may enhance the ability to predict long-term outcome following ischemic stroke. METHODS: Blood concentrations of neuronal markers (neuron-specific enolase, visinin-like protein 1, heart type fatty acid binding protein (hFABP) and neuroglobin), astroglial markers (S100B and glial fibrillary acidic protein), inflammatory markers (IL-6, TNF-α, and C-reactive protein), blood-brain barrier marker (matrix metalloproteinase 9), and haemostatic markers (D-dimer and PAI-1) were measured within 24 hours after stroke onset. The discrimination and reclassification for favorable and poor outcome were compared after adding individual or a combination of blood markers to the clinical model of stroke outcome. RESULTS: In multivariate analysis, natural log-transformed (log) IL-6 (odds ratio (OR): 1.75, 95% CI: 1.25 to 2.25, P=0.001) and loghFABP (OR: 3.23, 95% CI: 1.44 to 7.27, P=0.005) were independently associated with poor outcome. The addition of a single blood marker to the clinical model did not improve the discriminating ability of the clinical model of stroke outcome. However, the addition of the combination of logIL-6 and loghFABP to the clinical model showed improved discrimination (area under receiver operating characteristic (AUROC) curve: 0.939 versus 0.910, P=0.03) and reclassification performance (net reclassification improvement index: 0.18, P=0.005). CONCLUSIONS: A combination of circulating IL-6 and hFABP level has an additive clinical value for the prediction of stroke outcome.

Limited clinical value of multiple blood markers in the diagnosis of ischemic stroke.

OBJECTIVES: No ideal blood marker exists for the diagnosis of ischemic stroke. Combined use of multiple blood markers would enhance the ability of clinical diagnosis of ischemic stroke. DESIGN AND METHODS: Blood concentrations of neuronal markers (NSE, VSNL-1, hFABP, and Ngb), astroglial markers (S100B and GFAP), inflammatory markers (IL-6 and TNF-α), blood-brain barrier marker (MMP-9), and hemostatic markers (PAI-1) were measured within 6-24 h of stroke onset. The area under the receiver operator characteristic (AUROC) curve of patients with ischemic stroke and stroke-mimic was compared after adding individual or a combination of blood markers to the clinical diagnostic assessment (age, atrial fibrillation, and Face-Arm-Speech Test [FAST]). RESULTS: Despite acute elevations of blood IL-6, S100B, MMP-9, hFABP, and PAI-1 in univariate analysis, only IL-6, S100B, and MMP-9 were independently associated with ischemic stroke in multivariate analysis. The addition of biomarkers (IL-6, S100B, and MMP-9) did not improve the diagnostic performance of baseline clinical models with added biomarkers versus baseline clinical models alone (AUROC, 0.865 vs. 0.837, p=0.069). CONCLUSIONS: IL-6, S100B, and MMP-9 markers are elevated in the peripheral blood during the acute phase of ischemic stroke. However, the clinical usefulness of these biomarkers is limited due to low discriminating ability when compared to clinical parameters alone in diagnosis of ischemic stroke.

Expression profiles of subtracted mRNAs during cellular senescence in human mesenchymal stem cells derived from bone marrow.

Cellular senescence is an irreversible cell cycle arrest that limits the replicative lifespan of cells. Senescence suppresses development of tumors by regulating aging factors, such as cyclin dependent kinase inhibitor (CKI) and telomerase. Suppression subtractive hybridization (SSH) was used to identify genes that were differentially expressed between young human mesenchymal stem cells (Y-hMSCs) and senescent human mesenchymal stem cells (S-hMSCs). We selected positive clones that were functionally characterized by referring to public databases using NCBI BLAST tool. This search revealed that 19 genes were downregulated, and 43 genes were upregulated in S-hMSCs relative to Y-hMSCs. Among subtracted clones in Y-hMSCs, most of genes markedly were related to metabolic functions. These genes, PDIA3, WDR1, FSTL1, COPG1, LMAN1, and PDIA6, significantly downregulated. Conversely, genes for subtracted clones in S-hMSCs were mostly associated with cell adhesion. In particular, the expression levels of 9 genes, HSP90B1, EID1, ATP2B4, DDAH1, PRNP, RAB1A, PGS5, TM4SF1 and SSR3, gradually increased during senescence. These genes have not previously been identified as being related to cellular senescence, but they seemed to be potentially affected during cellular senescence.

miR-7641 modulates the expression of CXCL1 during endothelial differentiation derived from human embryonic stem cells.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that negatively regulate gene expression through binding to 3' untranslated region. We identified and characterized the novel miRNA, miR-7641, in human mesenchymal stem cells. The expression of miR-7641 was downregulated during differentiation from human embryonic stem cells to endothelial cells. The CXCL1, a member of the CXC chemokine family, is known as promoting neovascularization by binding G-protein coupled receptors and is related to endothelial cells biogenesis such as angiogenesis, and it was predicted as target gene of miR-7641 by computerized analysis and the luciferase reporter assay. The miR-7641 significantly suppressed CXCL1 of transcriptional and post-translational levels. These data suggest that miR-7641 might be related with differentiation of human endothelial cells.

Effect of Immortalization-Upregulated Protein-2 (IMUP-2) on Cell Death of Trophoblast.

Trophoblasts, in the placenta, play a role for placental development as well as implantation in the early pregnancy. The characteristics and functions of trophoblast are identified by their localization and potency for proliferation, differentiation, and invasion. Thus, inadequate trophoblast cell death induces trophoblast dysfunction resulting in abnormal placental development and several gynecological diseases. Recently, it was reported that increased immortalization-upregulated protein-2 (IMUP-2) by hypoxia influences trophoblast apoptosis. However, IMUP-2 function on autophagy, which is type II programmed cell death remains unclear. In this study, we analyzed IMUP-2 expression in trophoblast cells (HTR8-SVneo) and compared IMUP-2 effects on cell death including apoptosis and autophagy in trophoblast regardless of IMUP-2 expression. Increased IMUP-2 in trophoblast by IMUP-2 gene transfection induces cell death, especially, apoptosis increases more than autophagy (p<0.05). However, the decreased IMUP-2 in trophoblasts after siRNA treatment decreased apoptosis with the decreased activities of caspase 3 and 7. The expressions of LC3 and MDC as an autophagosome makers and phosphorylated mTOR, which is a negative regulator for autophagy, increased. In addition, the S phase of cell cycle increased in trophoblasts when IMUP-2 expression decreased. Taken together, the alteration of IMUP-2 can control the balance between apoptosis and autophagy of trophoblasts resulting in functional involvement in placental development and in gynecological diseases by regulating the function of trophoblasts.

The novel miR-7515 decreases the proliferation and migration of human lung cancer cells by targeting c-Met.

MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression in human diseases, including lung cancer. miRNAs have oncogenic and nononcogenic functions in lung cancer. In this study, we report the identification of a novel miRNA, miR-7515, from lung cancer cells. The novel miR-7515 was characterized using various predictive programs and experimental methods. miR-7515 was able to forming a stem-loop structure and its sequence was conserved in mammals. The expression level of miR-7515 in lung cancer cells and tissues was profiled using TaqMan miRNA assays. miR-7515 was downregulated in lung cancer compared with normal human lung cells and tissues. The target of miR-7515 was determined using a dual luciferase reporter assay. Expression of the target gene was determined by quantitative RT-PCR and Western blot analysis after transfection with miR-7515. miR-7515 directly suppressed human mesenchymal-epithelial transition factor (c-Met) by binding to the 3' untranslated region (UTR). Overexpression of miR-7515 significantly decreased cell-cycle-related proteins downstream of c-Met through c-Met inhibition. Cell proliferation and migration were examined using the XTT proliferation assay and the Transwell migration assay. miR-7515 led to decreased cell proliferation, migration and invasion in a lung cancer cell line. These results suggest that miR-7515 plays an important role in the proliferation and migration of lung cancer cells through c-Met regulation.

Hypoxia-induced downregulation of XIAP in trophoblasts mediates apoptosis via interaction with IMUP-2: implications for placental development during pre-eclampsia.

The regulation of trophoblast apoptosis is essential for normal placentation, and increased placental trophoblast cell apoptosis is the cause of pathologies such as intrauterine growth retardation (IUGR) and pre-eclampsia. X-linked inhibitor of apoptosis (XIAP) is expressed in trophoblasts, but little is known about the role of XIAP in placental development. In the present study, the function of XIAP in the placenta and in HTR-8/SVneo trophoblasts under hypoxic conditions was examined. In addition, the correlation between XIAP and immortalization-upregulated protein-2 (IMUP-2) was demonstrated in HTR-8/SVneo trophoblasts under hypoxia, based on a previous study showing that increased IMUP-2 induces trophoblast apoptosis and pre-eclampsia. XIAP was downregulated in pre-eclamptic placentas (P < 0.05). In HTR-8/SVneo trophoblasts, XIAP expression was decreased and the expression of apoptosis-related genes was increased in response to hypoxia. Ectopic expression of hypoxia inducible factor (HIF)-1α in HRT-8 SV/neo cells induced the nuclear translocation of XIAP and alterations of XIAP protein stability. Furthermore, hypoxia induced nuclear translocated XIAP co-localized with upregulated IMUP-2 in trophoblast nuclei, and the interaction between XIAP and IMUP-2 induced apoptosis in HRT-8 SV/neo cells. The present results suggest that hypoxia-induced down-regulation of XIAP mediates apoptosis in trophoblasts through interaction with increased IMUP-2, and that this mechanism underlies the pathogenesis of pre-eclampsia.

Alteration of immunologic responses on peripheral blood in the acute phase of ischemic stroke: blood genomic profiling study.

OBJECTIVE: Peripheral blood cells and inflammatory mediators have a detrimental effect on brain during cerebral ischemia. We investigated the immunologic changes on peripheral blood in the acute phase of ischemic stroke using RNA microarray. METHODS: mRNA microarray and real time-polymerase chain reaction (RT-PCR) for genes of interest in microarray data were analyzed in 12 stroke patients and 12 controls. Plasma matrix metalloproteinase-9 (MMP-9) concentrations were measured in 120 stroke patients and 82 controls. RESULTS: In microarray analysis, a total of 11 genes of interest showed different expression in patients with ischemic stroke. The three most highly expressed genes were C19orf59 (chromosome 19 open reading frame 59), MMP9 and IL18RAP (interleukin-18 receptor accessory protein), whereas gene with the lowest expression was GNLY (granulysin). The expression patterns of three selected genes (MMP9, IL18RAP and GNLY) were validated by RT-PCR. The plasma concentration of MMP-9 was significantly elevated in the stroke patients, and showed a weakly positive correlation with infarct volume. Gene set enrichment analysis (GSEA) showed that gene sets related to immunity and defense, signal transduction, transport and cell adhesion were significant in acute ischemic stroke. CONCLUSIONS: In the peripheral blood, numerous genes of inflammatory mediators, including MMP9, IL18RAP and GNLY, are altered in the acute phase of ischemic stroke. This stroke-specific gene expression profiling provides valuable information about the role of peripheral inflammation to the pathophysiological mechanism of ischemic stroke.

Discovery and characterization of novel microRNAs during endothelial differentiation of human embryonic stem cells.

MicroRNAs (miRNAs) are small RNAs that participate in the regulation of genes associated with the differentiation and proliferation. In this study, 5 novel miRNAs were identified from human mesenchymal stem cells and characterized using various analyses. To investigate the potential functions associated with the regulation of cell differentiation, the differences in miRNA expression were examined in undifferentiated and differentiated human embryonic stem (ES) cells using reverse transcription (RT)-PCR analysis. Specifically, 3 miRNAs exhibited decreased expression levels in human umbilical vein endothelial cells (HUVECs) and endothelial cells derived from human ES cells. Putative target genes related to differentiation or maturation of endothelial cells were predicted by seed sequences of 2 novel miRNAs and analyzed for their expression via miRNA-mediated regulation using a luciferase assay. In HUVECs, CDH5 gene expression was directly repressed by hsa-miR-6086. Similarly, hsa-miR-6087 significantly downregulated endoglin expression. Therefore, the roles of these 2 miRNAs may be to directly suppress their target genes, popularly known as endothelial cell markers. Taken together, our results demonstrate that several novel miRNAs perform critical roles in human endothelial cell development.

Molecular imaging of a cancer-targeting theragnostics probe using a nucleolin aptamer- and microRNA-221 molecular beacon-conjugated nanoparticle.

MicroRNAs (miRNA, miR) have been reported as cancer biomarkers that regulate tumor suppressor genes. Hence, simultaneous detecting and inhibiting of miRNA function will be useful as a cancer theragnostics probe to minimize side effects and invasiveness. In this study, we developed a cancer-targeting therangostics probe in a single system using an AS1411 aptamer - and miRNA-221 molecular beacon (miR-221 MB)-conjugated magnetic fluorescence (MF) nanoparticle (MFAS miR-221 MB) to simultaneously target to cancer tissue, image intracellularly expressed miRNA-221 and treat miRNA-221-involved carcinogenesis. AS1411 aptamer-conjugated MF (MFAS) nanoparticles displayed a great selectivity and delivery into various cancer cell lines. The miR-221 MB detached from the MFAS miR-221 MB in the cytoplasm of C6 cells clearly imaged miRNA-221 biogenesis and simultaneously resulted in antitumor therapeutic effects by inhibiting miRNA function, indicating a successful astrocytoma-targeting theragnostics. MFAS miRNA MB can be easily applied to other cancers by simply changing a targeted miRNA highly expressed in cancers.