• Journal of Life Science
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• v.30 no.9
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• pp.772-782
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• 2020

Skeletal muscle differentiation or myogenesis is important to maintain muscle mass and metabolic homeostasis. Muscle-specific microRNAs (miRNAs) are known to play a critical role in skeletal myogenic differentiation. In this study, we examined the expression profiling of miRNAs during myogenic differentiation in rat L6 myoblasts using rat miRNA microarrays. We identified the upregulated expression of miR-128 as well as several well-known myogenic miRNAs, including miR-1, miR-133b, and miR-206. We additionally confirmed the increased expression of miR-128 observed on microarray through quantitative real-time PCR (qRT-PCR), which showed similarly upregulated expression of both primary miR-128 and mature miR-128, consistent with the microarray findings. Furthermore, transfection of miR-128 into rat L6 myoblasts induced gene expression of myogenic markers such as muscle creatine kinase (MCK), myogenin, and myosin heavy chain (MHC). Protein expression of MHC was increased as well. Inhibition of miR-128 by inhibitory peptide nucleic acids (PNAs) blocked the expression of those myogenic markers. In addition, the transfection of miR-128 into rat L6 myoblasts enhanced the phosphorylation of Erk and Akt proteins stimulated by insulin, while simultaneously reversing the inhibited phosphorylation of Erk and Akt due to insulin resistance. These findings suggest that miR-128 may play important roles in myogenic differentiation and insulin signaling.

• Biomolecules & Therapeutics
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• v.25 no.6
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• pp.609-617
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• 2017

Pancreatic cancer is one of the most lethal and aggressive cancers in the world. However, no effective treatment is currently available for pancreatic cancer. The objective of this study was to determine the anti-pancreatic cancer effect of ${\alpha}$-mangostin (${\alpha}M$) and ${\gamma}$-mangostin (${\gamma}M$) extracted from the pericarp of Garcinia mangostana L.. Both ${\alpha}$M and ${\gamma}M$ reduced the viability of pancreatic cancer cells MIA PaCa-2 and PANC-1 in a dose-dependent manner. These compounds induced apoptosis by increasing c-PARP and c-Caspase 3 levels. They also induced autophagy by increasing levels of microtubule-associated protein 1A/1B light chain 3B (LC3II) in both cell lines while decreasing sequestosome 1 (p62) in MIA PaCa-2. Both ${\alpha}$M and ${\gamma}M$ induced autophagy through increasing phosphorylation levels of AMP-activated protein kinase (p-AMPK) and p38-mitogen activated protein kinase (p-p38) while decreasing phosphorylation level of mammalian target of rapamycin complex 1 (p-mTOR). Of various microRNAs (miRNA), miR-18a was found to be a putative regulatory miRNA for autophagy induced by ${\alpha}$M or ${\gamma}M$. In combination with gemcitabine, a compound frequently used in pancreatic cancer treatment, ${\alpha}$M and ${\gamma}M$ showed synergistic anti-cancer effects in MIA PaCa-2. Collectively, these results suggest that ${\alpha}$M and ${\gamma}M$ can induce apoptosis and autophagy in pancreatic cancer cells and that their anti-cancer effect is likely to be associated with miR-18a. In conclusion, ${\alpha}$M and ${\gamma}M$ might be used as a potential new therapy for pancreatic cancer.

• Molecular & Cellular Toxicology
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• v.5 no.1
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• pp.67-74
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• 2009

Exposures to environmental chemicals that mimic endogenous hormones are proposed for a number of adverse health effects, including infertility, abnormal prenatal and childhood development and above all cancers. In addition, recently miRNA (micro RNA) has been recognized to play an important role in various diseases and in cellular and molecular responses to toxicants. In this study, endocrine disrupting environmental toxicant, nonylphenol (NP) was treated to MCF-7 (Human breast cancer cell) and HepG2 (Human hepatocellular liver carcinoma) cell line at 3 hrs and 48 hrs time point and miRNA analysis using $mirVana^{TM}$ miRNA bioarray was performed and compared with total mRNA microarray data for the same cell line and treatment. Robust data quality was achieved through the use of dye-swap. Analysis of microarray data identifies a total of 20 and 11 miRNA expressions at 3 hrs and 48 hrs exposure to NP in MCF-7 cell line and a total of 14 and 47 miRNA expression at 3 hrs and 48 hrs exposure respectively to NP in HepG2 cell line. Expression profiling of the selected miRNA (let-7c, miR-16, miR-195, miR-200b, miR200c, miR-205, and miR-589) reveals changes in the expression of target genes related to metabolism, immune response, apoptosis, and cell differentiation. The present study can be informative and helpful to understand the role of miRNA in molecular mechanism of chemical toxicity and their influence on hormone dependent disease. Also this study may prove to be a valuable tool for screening potential estrogen mimicking pollutants in the environment.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.08a
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• pp.277.1-277.1
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• 2013

Molecular diagnostics consists of three processes, which are a sample pretreatment, a nucleic acid amplification, and an amplicon detection. Among three components, sample pretreatment is an important process in that it can increase the limit of detection by purifying nucleic acid in biological sample from contaminants that may interfere with the downstream genetic analysis such as nucleic acid amplification and detection. To achieve point-of-care virus detection system, the sample pretreatment process needs to be simple, rapid, and automatic. However, the commercial RNA extraction kits such as Rneasy (Qiagen) or MagnaPure (Roche) kit are highly labor-intensive and time-consuming due to numerous manual steps, and so it is not adequate for the on-site sample preparation. Herein, we have developed a rotary microfluidic system to extract and purify the RNA without necessity of external mechanical syringe pumps to allow flow control using microfluidic technology. We designed three reservoirs for sample, washing buffer, and elution buffer which were connected with different dimensional microfluidic channels. By controlling RPM, we could dispense a RNA sample solution, a washing buffer, and an elution buffer successively, so that the RNA was captured in the sol-gel solid phase, purified, and eluted in the downstream. Such a novel rotary sample preparation system eliminates some complicated hardwares and human intervention providing the opportunity to construct a fully integrated genetic analysis microsystem.

• International Journal of Oral Biology
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• v.36 no.3
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• pp.109-116
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• 2011

Periodontitis results from the activation of host immune and inflammatory defense responses to subgingival plaque bacteria, most of which are gram-negative rods with lipopoly-saccharides (LPSs) in their cell walls. LPSs have been known to induce proinflammatory responses and recently it was reported also that they induce the expression of microRNAs (miRNAs) in host cells. In our current study therefore, we aimed to examine and compare the miRNA expression patterns induced by the LPSs of major periodontopathogens in the human gingival epithelial cell line, Ca9-22. The cells were treated with 1 ${\mu}g$/ml of E. coli (Ec) LPS or 5 ${\mu}g$/ml of an LPS preparations from four periodontopathogens Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Aggregatibacter actinomycetemcomitans (Aa), and Fusobacterium nucleatum (Fn) for 24 h. After small RNA extraction from the treated cells, miRNA microarray analysis was carried out and characteristic expression profiles were observed. Fn LPS most actively induced miRNAs related to inflammation, followed by Aa LPS, Pi LPS, and Ec LPS. In contrast, Pg LPS only weakly activated miRNAs related to inflammation. Among the miRNAs induced by each LPS, miR-875-3p, miR-449b, and miR-520d-3p were found to be commonly up-regulated by all five LPS preparations, although at different levels. When we further compared the miRNA expression patterns induced by each LPS, Ec LPS and Pi LPS were the most similar although Fn LPS and Aa LPS also induced a similar miRNA expression pattern. In contrast, the miRNA profile induced by Pg LPS was quite distinctive compared with the other bacteria. In conclusion, miR-875-3p, miR-449b, and miR-520d-3p miRNAs are potential targets for the diagnosis and treatment of periodontal inflammation induced by subgingival plaque biofilms. Furthermore, the observations in our current study provide new insights into the inflammatory miRNA response to periodontitis.

• Molecules and Cells
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• v.40 no.4
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• pp.291-298
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• 2017

MicroRNAs (miRNAs) are short non-coding RNAs that regulate genes posttranscriptionally. Past studies have reported that miR-210 is up-regulated in many cancers including cervical cancer, and plays a pleiotropic role in carcinogenesis. However, its role in regulating response towards anti-cancer agents has not been fully elucidated. We have previously reported that the natural compound 1'S-1'-acetoxychavicol acetate (ACA) is able to induce cytotoxicity in various cancer cells including cervical cancer cells. Hence, this study aims to investigate the mechanistic role of miR-210 in regulating response towards ACA in cervical cancer cells. In the present study, we found that ACA down-regulated miR-210 expression in cervical cancer cells, and suppression of miR-210 expression enhanced sensitivity towards ACA by inhibiting cell proliferation and promoting apoptosis. Western blot analysis showed increased expression of mothers against decapentaplegic homolog 4 (SMAD4), which was predicted as a target of miR-210 by target prediction programs, following treatment with ACA. Luciferase reporter assay confirmed that miR-210 binds to sequences in 3'UTR of SMAD4. Furthermore, decreased in SMAD4 protein expression was observed when miR-210 was overexpressed. Conversely, SMAD4 protein expression increased when miR-210 expression was suppressed. Lastly, we demonstrated that overexpression of SMAD4 augmented the anti-proliferative and apoptosis-inducing effects of ACA. Taken together, our results demonstrated that down-regulation of miR-210 conferred sensitivity towards ACA in cervical cancer cells by targeting SMAD4. These findings suggest that combination of miRNAs and natural compounds could provide new strategies in treating cervical cancer.

• BMB Reports
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• v.48 no.7
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• pp.371-372
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• 2015

MicroRNAs (miRNAs) can regulate the expression of genes that are involved in multiple cellular pathways. However, their targets and mechanism of action associated with the autophagy pathway are not fully investigated yet. EWSR1 (EWS RNA-Binding Protein 1/Ewing Sarcoma Break Point Region 1) gene encodes a RNA/DNA binding protein that is ubiquitously expressed and plays roles in numerous cellular processes. Recently, our group has shown that EWSR1 deficiency leads to developmental failure and accelerated senescence via processing of miRNAs, but its role in the regulation of autophagy remains elusive. In this context, we further investigated and found that EWSR1 deficiency triggers the activation of the DROSHA-mediated microprocessor complex and increases the levels of miR125a and miR351, which directly target Uvrag. Interestingly, the miR125a- and miR351-targeted reduction of Uvrag led to the inhibition of autophagy in both ewsr1 knockout (KO) MEFs and ewsr1 KO mice. In summary, our study demonstrates that EWSR1 is associated with the posttranscriptional regulation of Uvrag via miRNA processing. The regulation of autophagy pathway in miRNAs-Uvrag-dependent manner provides a novel mechanism of EWSR1 deficiency-related cellular dysfunction. [BMB Reports 2015; 48(7): 371-372]

• Journal of Ginseng Research
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• v.41 no.4
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• pp.477-486
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• 2017

Background: Our previous studies have demonstrated that ginsenoside-Rg1 can promote angiogenesis in vitro and in vivo through activation of the glucocorticoid receptor (GR). Furthermore, microRNA (miRNA) expression profiling has shown that Rg1 can modulate the expression of a subset of miRNAs to induce angiogenesis. Moreover, Rb1 was shown to be antiangiogenic through activation of a different pathway. These studies highlight the important functions of miRNAs on ginseng-regulated physiological processes. The aim of this study was to determine the angiogenic properties of Korean Red Ginseng extract (KGE). Methods and Results: Combining in vitro and in vivo data, KGE at $500{\mu}g/mL$ was found to induce angiogenesis. According to the miRNA sequencing, 484 differentially expressed miRNAs were found to be affected by KGE. Among them, angiogenic-related miRNAs; miR-15b, -23a, -214, and -377 were suppressed by KGE. Meanwhile, their corresponding angiogenic proteins were stimulated, including vascular endothelial growth factor, vascular endothelial growth factor receptor-2, endothelial nitric oxide synthase, and MET transmembrane tyrosine kinase. The miRNAs-regulated signaling pathways of KGE were then found by Cignal 45-Pathway Reporter Array, proving that KGE could activate GR. Conclusion: KGE was found capable of inducing angiogenesis both in vivo and in vitro models through activating GR. This study provides a valuable insight into the angiogenic mechanisms depicted by KGE in relation to specific miRNAs.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.10
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• pp.5915-5920
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• 2013

Aim: Although aberrant miRNA expression has been documented, altered miR-101 expression in cervical cancer and its carcinogenic effects and mechanisms remain unexplored. The aim of our study was to investigate the role of miR-101 alteration in cervical carcinogenesis. Methods: Expression of miR-101 was examined by quantitative real-time reverse transcriptase PCR (qRT-PCR) in Hela cells. After modulating miR-101 expression using miR-101 mimics, cell growth, apoptosis and proliferation, and migration were tested separately by MTT or flow cytometry and cell wound healing assay and protein expression was detected by qRT-PCR. The expression of COX-2 in Hela cell was also examined by immunohistochemical staining and the correlation with miR-101 expression was analysed. Results: The miR-101 demonstrated significantly low expression in Hela cell. When we transfected miR-101 mimics into Hela cells, the modulation of miR-101 expression remarkably influenced cell proliferation, cycling and apoptosis: 1) The expression of microRNA-101 tended to increase after transfection; 2) Overexpression of miR-101 was able to promote cell apoptosis, the apoptosis rate being markedly higher (97.6%) than that seen pre-transfection (12.2%) (P<0.05); 3) The miR-101 negatively regulates cell migration and invasion, scratch results being lower ($42.7um{\pm}2um$) than that observed pre-transfection ($181.4um{\pm}2um$); 4) miRNA-101 inhibits the proliferation of Hela cells as well as the level of COX-2 protein, which was negatively correlated with miR-101 expression. Conclusions: Overexpression of miR-101 has obvious inhibitory effects on cell proliferation, migration and invasion. Thus reduced miR-101 expression could participate in the development of cervical cancer at least partly through loss of inhibition of target gene COX-2, which probably occurs in a relative late phase of carcinogenesis. Our data suggest an important role of miR-101 in the molecular etiology of cancer and indicate potential application of miR-101 in cancer therapy.

• The Korean Journal of Physiology and Pharmacology
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• v.23 no.3
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• pp.171-179
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• 2019

Pituitary tumors are usually benign but can occasionally exhibit hormonal and proliferative behaviors. Dysregulation of the G1/S restriction point largely contributes to the over-proliferation of pituitary tumor cells. F-box protein S-phase kinase-interacting protein-2 (SKP2) reportedly targets and inhibits the expression of $p27^{Kip1}$, a well-known negative regulator of G1 cell cycle progression. In this study, SKP2 expression was found to be upregulated while $p27^{Kip1}$ expression was determined to be downregulated in rat and human pituitary tumor cells. Furthermore, SKP2 knockdown induced upregulation of $p27^{Kip1}$ and cell growth inhibition in rat and human pituitary tumor cells, while SKP2overexpression elicited opposite effects on $p27^{Kip1}$ expression and cell growth. The expression of microRNA-186 (miR-186) was reported to be reduced in pituitary tumors. Online tools predicted SKP2 to be a direct downstream target of miR-186, which was further confirmed by luciferase reporter gene assays. Moreover, miR-186 could modulate the cell proliferation and $p27^{Kip1}$-mediated cell cycle alternation of rat and human pituitary tumor cells through SKP2. As further confirmation of these findings, miR-186 and $p27^{Kip1}$ expression were downregulated, while SKP2 expression was upregulated in human pituitary tumor tissue samples; thus, SKP2 expression negatively correlated with miR-186 and $p27^{Kip1}$ expression. In contrast, miR-186 expression positively associated with $p27^{Kip1}$ expression. Taken together, we discovered a novel mechanism by which miR-186/SKP2 axis modulates pituitary tumor cell proliferation through $p27^{Kip1}$-mediated cell cycle alternation.