Jin Kyeoung Kim

Reactive oxygen species-responsive miR-210 regulates proliferation and migration of adipose-derived stem cells via PTPN2.

Hypoxia enhances the proliferation and migration of adipose-derived stem cells (ASCs) via the generation of reactive oxygen species (ROS). Therefore, this study primarily investigated whether or not ROS generation could regulate microRNA-210 (miR-210) expression, and increase proliferation/migration of ASCs. In addition, we tried to identify the signaling pathways involved in miR-210 upregulation and the direct target genes of miR-210 that mediate these functions. Various sources of ROS generation such as hypoxia, antimycin, rotenone, and platelet-derived growth factor (PDGF)-BB upregulated miR-210 expression, and increased the proliferation/migration of ASCs. There is a positive feed-forward loop between ROS generation and miR-210, and miR-210 itself increases ROS generation by downregulation of iron–sulfur cluster scaffold homolog 2 (ISCU2). Although hypoxia-inducible factor-1α was not involved in miR-210 expression, pharmacological or small interfering RNA (siRNA)-driven inhibition of Akt and ERK1/2 molecules reduced miR-210 expression. Transfection of siRNAs of NF-κB and Elk1 also reduced miR-210 expression, indicating that these signaling pathways mediate miR-210 upregulation. Protein tyrosine phosphatase, non-receptor type 2 (PTPN2) was selected for miR-210 target gene, and it was downregulated by ROS generators or miR-210 mimic treatment. PTPN2 was first proven to be a direct miR-210 target in luciferase activity assay, and pharmacological inhibition or overexpression of PTPN2 regulated the proliferation and migration of ASC. In conclusion, ROS generation from diverse sources induces miR-210 expression in ASCs via PDGFR-β, Akt and ERK pathways. Transcription of miR-210 expression is regulated by NF-κB and Elk1, and miR-210 increases the proliferation and migration of ASCs via ISCU2 and PTPN2 downregulation.

Human chorionic-plate-derived mesenchymal stem cells and Wharton’s jelly-derived mesenchymal stem cells: a comparative analysis of their potential as placenta-derived stem cells

Placenta-derived stem cells (PDSCs) have gained interest as an alternative source of stem cells for regenerative medicine because of their potential for self-renewal and differentiation and their immunomodulatory properties. Although many studies have characterized various PDSCs biologically, the properties of the self-renewal and differentiation potential among PDSCs have not yet been directly compared. We consider the characterization of chorionic-plate-derived mesenchymal stem cells (CP-MSCs) and Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) among various PDSCs and the assessment of their differentiation potential to be important for future studies into the applicability and effectiveness of PDSCs in cell therapy. In the present study, the capacities for self-renewal and multipotent differentiation of CP-MSCs and WJ-MSC isolated from normal term placentas were compared. CP-MSCs and WJ-MSCs expressed mRNAs for the pluripotent stem cell markers Oct-4, Nanog, and Sox-2. Additionally, HLA-G for immunomodulatory effects was found to be expressed at both the mRNA and protein levels in both cell types. The CP-MSCs and WJ-MSCs also had the capacities to differentiate into cells of mesodermal (adipogenic and osteogenic) and endodermal (hepatogenic) lineages. Expression of adipogenesis-related genes was higher in CP-MSCs than in WJ-MSCs, whereas WJ-MSCs accumulated more mineralized matrix than CP-MSCs. The WJ-MSCs expressed more of CYP3A4 mRNA, a marker for mature hepatocytes, than CP-MSCs. Thus, we propose that CP-MSCs and WJ-MSCs are useful sources of cells for appropriate clinical applications in the treatment of various degenerative diseases.

Long-term proliferation and characterization of human spermatogonial stem cells obtained from obstructive and non-obstructive azoospermia under exogenous feeder-free culture conditions.

Objectives:  The aim of the present study was to improve efficiency of isolation and to optimize proliferative potential of human spermatogonial stem cells (SSCs) obtained from obstructive azoospermic (OA) and non‐obstructive azoospermic (NOA) patients, and further, to characterize these cells for potential use in infertility treatment or study of reproductive biology.

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.

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. Mol Cancer Res; 11(1); 43–53. ©2012 AACR.

Expression profiles of SV40‐immortalization‐associated genes upregulated in various human cancers

Immortalization is an early and essential step of human carcinogenesis which is associated with alterations in gene expression and regulation. Suppression subtractive hybridization (SSH) was successfully performed to identify immortalization‐associated genes upregulated in SV40‐immortalized lung fibroblasts. We identified 116 known genes which were related to diverse functions, with 32.8% relevant for cell cycle or proliferation indicating the potential involvement of these genes in immortalization. We chose eight known genes located on the overrepresented chromosomes of non‐small‐cell lung cancers (NSCLCs). ASPM, RFC4, C3orf26, BXDC2, C15orf44, AURKA, C20orf77, and RBMX were upregulated in immortalized cells, cancer cells, and non‐small‐cell lung cancer (NSCLC) tissues. We additionally cloned two novel genes (CHA‐V‐97 and CHA‐V‐165) which showed similar upregulated expression patterns in cells and tissues examined. Identification and further characterization of these genes may provide insights of novel players for immortalization and human carcinogenesis. J. Cell. Biochem. 106: 703–713, 2009.

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.

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.

In vitro screening system for hepatotoxicity: comparison of bone-marrow-derived mesenchymal stem cells and Placenta-derived stem cells.

Stem cells have unique properties such as self‐renewal, plasticity to generate various cell types, and availability of cells of human origin. The characteristics are attentive in the toxicity screening against chemical toxicants. Placenta‐derived stem cells (PDSCs) have been spotlighted as a new cell source in stem cell research recently because they are characterized by their capacity to differentiate into multilineages. However, the use of PDSCs as an in vitro screening model for potential drug candidates has not yet been studied. Here, we analyzed the potentials for bone‐marrow‐derived mesenchymal stem (BM‐MSCs), which is a representative adult stem cells and PDSCs as an in vitro hepatotoxicity screening system, using well‐known hepatotoxicants. BM‐MSCs and PDSCs were analyzed to the potential for hepatogenic differentiation and were cultured with different concentrations of hepatotoxicants for time courses. The viability and ATP‐binding cassette (ABC) transporters were measured by the MTT assay and RT‐PCR, respectively. The sensitivities of PDSCs to hepatotoxicants are more sensitive than those of BM‐MSCs. The viability (IC50) to in PDSCs was less than that of BM‐MSCs after 48 and 72 h (P < 0.05) of CCl4 exposure. The toxicities of CCl4 were decreased by fourfold in hepatogenic differentiation inducing PDSCs compared to the undifferentiated cells. The alteration of ABCGs was observed in PDSCs during differentiation. These findings suggest that the naïve PDSCs expressing ABCGs can be used as a source for in vitro screening system as well as the expression patterns of ABCG1 and ABCG2 might be involved in the sensitivity of PDSCs to hepatotoxicants. J. Cell. Biochem. 112: 49–58, 2011.

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. J. Cell. Biochem. 114: 89–98, 2012.