International Journal of Molecular Medicine

Hata, Akiko ; Kang, Hara

doi: 10.3892/ijmm.2015.2060pmid: 25571950

MicroRNAs (miRNAs or miRs) have emerged as key regulators of gene expression in essential cellular processes, such as cell growth, differentiation and development. Recent findings have established that the levels of miRNAs are modulated by cell signaling mechanisms, including the bone morphogenetic protein (BMP) signaling pathway. The BMP signaling pathway controls diverse cellular activities by modulating the levels of miRNAs, indicating the complexity of gene regulation by the BMP signaling pathway. The tight regulation of the levels of miRNAs is critical for maintaining normal physiological conditions, and dysregulated miRNA levels contribute to the development of diseases. In the present review, we discuss different insights (provided over the past decade) into the regulation of miRNAs governed by the BMP signaling pathway and the implications of this regulation on the understanding of the cellular differentiation of vascular smooth muscle cells (VSMCs), osteoblasts and neuronal cells.

Qi, Quan ; Ding, Chenhui ; Hong, Pingping ; Yang, Gang ; Xie, Yanxin ; Wang, Jing ; Huang, Sunxing ; He, Ke ; Zhou, Canquan

doi: 10.3892/ijmm.2014.2044pmid: 25524499

The present study aimed to investigate the X chromosome inactivation (XCI) status in long-term cultured human parthenogenetic embryonic stem cells. One human embryonic stem (hES) cell line and 2 human parthenogenetic embryonic stem (hPES) cell lines were subjected to long-term culture in vitro (>50 passages). Karyotyping, array-based comparative genomic hybridization (aCGH), X-inactive specific transcript (XIST) RNA, immunofluorescence staining and real-time PCR were used to assess the chromosome karyotypes of these cells and the XCI status. X chromosome microdeletion was observed in the hPES-2 cells following culture for 50 passages. As early as 20 passages, XIST RNA expression was detected in the hPES-2 cells and was followed by low X-linked gene expression. The XIST RNA expression level was higher in the differentiated hPES-2 cells. The hPES-2' cells that were subclones of hPES-2 retained the XCI status, and had low XIST and X-linked gene expression. XIST RNA expression remained at a low level in the differentiated hPES-2' cells. The human biparental embryonic stem (hBES)-1 and hPES-1 cells did not exhibit XCI, and the differentiated hPES-1 cells had high expression levels of XIST RNA. In conclusion, the chromosome karyotypes of some hPES cell lines revealed instabilities. Similar to the hES cells, the hPES cells exhibited 3 XCI statuses. The unstable XCI status of the hPES-2 line may have been related to chromosome instability. These unstable chromosomes renedered these cells susceptible to environmental conditions and freezing processes, which may be the result of environmental adaptations.

Yang, Zhen ; Hao, Jie ; Hu, Zhen‑Ming

doi: 10.3892/ijmm.2014.2051pmid: 25543998

The aim of the present study was to examine the microRNA (miRNA or miR) expression profiles during the chondrogenic differentiation of human adipose‑derived stem cells (hADSCs) and identify the potential mechanisms through which miRNAs may affect the process of chondrogenesis. hADSCs were isolated and cultured. The expression levels of chondrogenic markers was detected by FACS analysis and immunohistochemistry. The miRNA expression profiles were then obtained through a miRNA array and confirmed through northern blot analysis. Putative targets of the miRNAs were predicted and validated through a luciferase reporter assay. The comparison of hADSCs following the induction of chondrogenic differentiation with undifferentiated hADSCs revealed 20 miRNAs that were differentially expressed by at least 2‑fold, and these miRNAs included 12 upregulated miRNAs and 8 downregulated miRNAs. Northern blot analysis further confirmed the miRNA expression levels. Of these miRNAs, the expression of miR‑490‑5p was gradually downregulated following the induction of chondrogenic differentiation. The overexpression of miR‑490‑5p increased the expression of the chondrogenic markers, collagen, type II, alpha 1 (Col2A1), collagen, type X, alpha 1 (Col10A1) and aggrecan. Furthermore, it was confirmed that miR‑490‑5p directly targets bone morphogenetic protein receptor type 2 (BMPR2). In conclusion, in this study, we identified a set of miRNAs that may play key roles in the regulation of the chondrogenic differentiation of hADSCs. Our results may provide a basis for the further investigations into the molecular mechanisms of action of miRNAs in hADSC chondrogenesis.

Huang, Qi ; Wu, Yuan ; Qin, Chao ; He, Wenwu ; Wei, Xing

doi: 10.3892/ijmm.2014.2047pmid: 25543670

The phospholipase A (PLA)2 family is the most complex gene family of phospholipases and plays a crucial role in a number of physiological activities. However, the phylogenetic background of the PLA2 gene family and the amino acid residues of the PLA2G7 gene following positive selection gene remain undetermined. In this study, we downloaded 49 genomic data sets of PLA from different species, including the human, house mouse, Norway rat, pig, dog, chicken, cattle, African clawed frog, Sumatran orangutan and the zebrafish species. Phylogenetic relationships were determined using the neighbor-joining (NJ), minimum evolution (ME) and maximum parsimony (MP) methods, as well as the Bayesian information criterion. The results were then presented as phylogenetic trees. Positive selection sites were detected using site, branch and branch‑site models. These methods led us to the following assumptions: i) closer lineages were observed between PLA2G16 and PLA2G6, PLA2G7 and PLA2G4, PLA2G3 and PLA2G12, as well as among PLA2G10, PLA2G5 and PLA2G15; ii) PLA2G5 appeared to be the origin of the PLA2 family, and PLA2G7 was one of the most evolutionarily distant PLA2 proteins; iii) 16 positive-selection sites were detected and were marked in the PLA2G7 protein sequence as 327D, 257Q, 276G, 34s, 66G, 67C, 319S, 28N, 50S, 54T, 58R, 75T, 88Q, 92R, 179H and 191K.

Wen, Ming ; Yan, Yong ; Yan, Ning ; Chen, Xiao Shan; Liu, Shi Yong; Feng, Zhan Hui

doi: 10.3892/ijmm.2015.2061pmid: 25571999

Mutations in RNA‑binding Fox 1 (RBFOX1) are known to be associated with neurodevelopmental disorders including epilepsy, mental retardation and autism spectrum disorder. The deletion of the Rbfox1 gene in mice has been shown to result in heightened susceptibility to seizures. However, other studies have revealed mutations or the downregulation of RBFOX1 in specimens obtained from patients with epilepsy or malformations of cortical development (MCD). Generally, the expression of RBFOX1 varies according to tissue type. In this study, we demonstrated the upregulation of RBFOX1 protein in the cortex of patients with MCD and intractable epilepsy. Electrophysiological recordings of cultured rat cortical neurons with increased Rbfox1 expression also revealed a significantly increased amplitude of action potential (AP) and Na+ current density. Some of these neurons (26.32%) even displayed spontaneous, recurrent, epileptiform discharges (SREDs). Additionally, certain Rbfox1 target transcripts associated with epilepsy, including glutamate receptor, ionotropic, N-methyl D-aspartate 1 (Grin1, also known as N-methyl-D-aspartate receptor subunit NR1 (NMDAR1)), synaptosomal-associated protein, 25 kDa (SNAP‑25 or Snap25) and sodium channel, voltage gated, type VIII, alpha subunit (Scn8a, also known as Nav1.6) were identified to be upregulated in these cultured cortical neurons with an upregulated Rbfox1 expression. These data suggest that the upregulation of RBFOX1 contributes to neuronal hyperexcitation and seizures. The upregulation of NMDAR1 (Grin1), SNAP‑25 (Snap25) and Scn8a may thus be involved in Rbfox1‑related neuronal hyperexcitation.

Liu, Jia ; Guo, Wenjie ; Li, Jing ; Li, Xiang ; Geng, Ji ; Chen, Qiuyun ; Gao, Jing

doi: 10.3892/ijmm.2015.2073pmid: 25604962

In this study, the antitumor activity of the novel manganese (II) compound, Adpa-Mn {((Adpa)Mn(Cl)(H2O)) (Adpa=bis(2-pyridylmethyl)amino-2-propionic acid)}, and its possible mechanisms of action were investigated. In vitro, the growth inhibitory effects of Adpa-Mn (with IC50 values lower than 15 µM) on tumor cell lines were examined by MTT assay. We found that this compound was more selective against cancer cells than the popular chemotherapeutic reagent, cisplatin. We then found that Adpa-Mn achieved its selectivity against cancer cells through the transferrin (Tf)-transferrin receptor (TfR) system, which is highly expressed in tumor cells. Furthermore, Adpa-Mn induced both apoptosis and autophagy, as indicated by chromatin condensation, the activation of poly(ADP-ribose) polymerase (PARP), Annexin V/propidium iodide staining, an enhanced fluorescence intensity of monodansylcadaverine (MDC), as well as the elevated expression of the autophagy-related protein, microtubule-associated protein 1 light chain 3 (LC3). In addition, Adpa-Mn induced the generation of intracellular reactive oxygen species (ROS) and its anticancer effects were significantly reduced following pre-treatment with the antioxidant, N-acetyl cysteine, indicating that ROS triggered cell death. In vivo, the induction of apoptosis and autophagy in tumor tissue was confirmed following treatment with Adpa-Mn, which contributed to its significant antitumor activity against hepatocellular carcinoma (Hep-A cell) xenografts at 10 mg/kg. Taken together, these data suggest the possible use of Adpa-Mn as a novel anticancer drug.

Zhao, Xiuhua ; Zhao, Qian ; Luo, Zhen ; Yu, Yan ; Xiao, Na ; Sun, Xuan ; Cheng, Lamei

doi: 10.3892/ijmm.2015.2067pmid: 25585915

The spontaneous immortalization of cells in vitro is a rare event requiring genomic instability, such as alterations in chromosomes and mutations in genes. In the present study, we report a spontaneously immortalized liver sinusoidal endothelial cell (LSEC) line generated from mouse liver. These immortalized LSECs showed typical LSEC characteristics with the structure of transcellular fenestrations, the expression of von Willebrand factor (VWF) and the ability to uptake DiI-acetylated-low density lipoprotein (DiI-Ac-LDL). However, these immortalized LSECs lost the ability to form capillary-like structures, and showed clonal and multilayer growth without contact inhibition. Moreover, their proliferation rate increased with the increase in the number of passages. In addition, these cells obained the expression of CD31 and desmin, and showed an upregulation of p53 protein expression; however, their karyotype was normal, and they could not form colonies in soft agar or tumors in SCID mice. In conclusion, in the present study, we successfully established a spontaneously immortalized LSEC line.

Qiu, Lei ; Yin, Guojian ; Cheng, Li ; Fan, Yuting ; Xiao, Wenqin ; Yu, Ge ; Xing, Miao ; Jia, Rongrong ; Sun, Ruiqing ; Ma, Xiuying ; Hu, Guoyong ; Wang, Xingpeng ; Tang, Maochun ; Zhao, Yan

Xue, Tao ; Wei, Li ; Zha, Ding-Jun ; Qiao, Li ; Lu, Lian-Jun ; Chen, Fu-Quan ; Qiu, Jian-Hua

doi: 10.3892/ijmm.2015.2075pmid: 25605314

Stem cell therapy has attracted widespread attention for a number of diseases. Recently, neural stem cells (NSCs) from the cochlear nuclei have been identified, indicating a potential direction for the treatment of sensorineural hearing loss. Acoustic stimuli play an important role in the development of the auditory system. In this study, we aimed to determine whether acoustic stimuli induce NSC development and differentiation through the upregulation of clusterin (CLU) in NSCs isolated from the cochlear nuclei. To further clarify the underlying mechanisms involved in the development and differentiation of NSCs exposed to acoustic stimuli, we successfully constructed animal models in which was CLU silenced by an intraperitoneal injection of shRNA targeting CLI. As expected, the NSCs from rats treated with LV-CLU shRNA exhibited a lower proliferation ratio when exposed to an augmented acoustic environment (AAE). Furthermore, the inhibition of cell apoptosis induced by exposure to AAE was abrogated after silencing the expression of the CLU gene. During the differentiation of acoustic stimuli-exposed stem cells into neurons, the number of astrocytes was significantly reduced, as evidenced by the expression of the cell markers, microtubule associated protein‑2 (MAP-2) and glial fibrillary acidic protein (GFAP), which was markedly inhibited when the CLU gene was silenced. Our results indicate that acoustic stimuli may induce the development and differentiation of NSCs from the cochlear nucleus mainly through the CLU pathway. Our study suggests that CLU may be a novel target for the treatment of sensorineural hearing loss.

Reikvam, Håkon ; Ryningen, Anita ; Sæterdal, Lars Rune ; Nepstad, Ina Rune ; Foss, Brynjar Rune ; Bruserud, Øystein Rune

doi: 10.3892/ijmm.2014.2045pmid: 25529637

Bone marrow stromal cells support both normal and malignant hematopoiesis. Τhis support is mediated through the local cytokine network and by direct cell‑cell interactions mediated via adhesion molecules and the formation of gap junctions by connexins. Previous studies on connexins in human acute myeloid leukemia (AML) have mainly focused on the investigation of leukemia cell lines. In the present study, we therefore investigated the expression of various connexins at the protein (i.e., cell surface expression) and mRNA level in primary human AML cells. The cell surface expression of the connexins, Cx26, Cx32, Cx37, Cx43 and Cx45, varied considerably between patients, and detectable levels were observed only for subsets of patients. On the whole, Cx43 and Cx45 showed the highest cell surface expression. Connexin expression was dependent on AML cell differentiation, but showed no association with cytogenetic abnormalities or mutations of the fms-related tyrosine kinase 3 (FLT3) or nucleophosmin (NPM)‑1 genes. By contrast, only Cx45 showed a significant variation between patients at the mRNA level. A high Cx45 expression was associated with the altered regulation of the mitogen‑activated protein kinase (MAPK) pathway and the release of pro-inflammatory cytokines (interleukin (IL)‑17, tumor necrosis factor (TNF), interferon‑γ), whereas a low Cx45 expression was associated with the altered regulation of protein functions (i.e., ligase activity, protein folding and catabolism). There was no significant correlation observed between the connexin mRNA and protein levels. Thus, differences in connexin expression can be used to subclassify AML patients. Differences in connexin cell surface expression profiles are not reflected at the mRNA level and have to be directly examined, whereas variations in Cx45 mRNA expression are associated with differences in cell signaling and the regulation of protein functions.