• BMB Reports
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• v.51 no.11
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• pp.602-607
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• 2018

Aberrant expression of microRNAs (miRNAs) plays important roles in carcinogenesis and tumor progression. However, the expression and biological role of miR-301b in triple-negative breast cancer (TNBC) remains unclear. Here we aimed to evaluate the roles and mechanisms of miR-301b in TNBC cells. miR-301b expression was assessed in TNBC specimens and cell lines by quantitative Real-Time PCR (qRT-PCR). TNBC cells were transfected with miR-301b mimics, inhibitors or Cylindromatosis (CYLD) small interfering RNA (siRNA) using Lipofectamine 2000. The functional roles of miR-301b were determined by cell proliferation, colony formation, and apoptosis assays. Western blots and qRT-PCR were used to measure the expression of mRNAs and proteins in the cells. We found that miR-301b was upregulated in TNBC specimens and cell lines. Overexpression of miR-301b promoted cell proliferation in TNBC cells, while inhibited the apoptosis induced by 5-FU. CYLD was downregulated by miR-301b at both mRNA and protein levels in TNBC cells. Dual-luciferase report assay confirmed that miR-301b downregulated CYLD by direct interaction with the 3'-untranslated region(3'-UTR) of CYLD mRNA. $NF-{\kappa}B$ activation was mechanistically associated with miR-301b-mediated downregulation of CYLD. However, inhibition of miR-301b reversed all the effects of miR-301b. In conclusion, miR-301b plays an oncogenic role in TNBC possibly by downregulating CYLD and subsequently activating $NF-{\kappa}B$ p65, and this may provide a novel therapeutic approach for TNBC.

• Journal of Life Science
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• v.18 no.10
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• pp.1395-1399
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• 2008

We have recently reported the PRC1 promoter as a promoter candidate to control expression of transcriptionally targeted genes for breast cancer gene therapy. We tested whether the PRC1 promoter could be also applied for the lung cancer gene therapy. In the transient transfection assay with naked plasmids containing the luciferase fused to the PRC1 promoter, the promoter showed little activity in the normal lung cell line, MRC5. However, in the lung cancer A549 cells, PRC1 showed approximately 30-fold activation which was similar to the survivin promoter, the gene whose promoter has been already reportedas a candidate for the gene therapy of lung cancer. In viral systems, the PRC1 promoter showed approximately 75% and 66% of transcriptional activity compared to the CMV promoter in the adeno-associated virus (AAV) and the adenovirus (AV) systems, respectively. However, the PRC1 promoter in either AAV or AV showed approximately 20% activity compared to the CMV promoter in the normal lung cells. In addition, human lung tumor xenograft mice showed that the PRC1 promoter activity was as strong as the CMV activity in vivo. Taken together, these results suggested that PRC1 might be a potential promoter candidate for transcriptionally targeted lung cancer gene therapy.

• BMB Reports
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• v.44 no.2
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• pp.123-128
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• 2011

Leucine-rich repeat containing protein 10 (LRRC10) is characterized as a cardiac-specific gene, suggesting a role in heart development and disease. A severe cardiac morphogenic defect in zebrafish morphants was recently reported but a contradictory result was found in mice, suggesting a more complicated molecular mechanism exists during mouse embryonic development. To elucidate how LRRC10 is regulated, we analyzed the 5'enhancer region approximately 3 kilo bases (kb) upstream of the Lrrc10 start site using luciferase reporter gene assays. Our characterization of the Lrrc10 promoter indicates it possesses complicated cis-and trans-acting elements. We show that GATA4 and MEF2C could both increase transcriptional activity of Lrrc10 promoter individually but that they do not act synergistically, suggesting that there exists a more complex regulation pattern. Surprisingly, knockout of Gata4 and Mef2c binding sites in the 5’enhancer region (-2,894/-2,889) didn't change the transcriptional activity of the Lrrc10 promoter and the likely GATA4 binding site identified was located in a region only 100 base pair (bp) upstream of the promoter. Our data provides insight into the molecular regulation of Lrrc10 expression, which probably also contributes to its tissue-specific expression.

• BMB Reports
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• v.39 no.3
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• pp.304-310
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• 2006

Transcription factor NF-E2-related factor 2 (Nrf2) regulates the induction of Phase II detoxifying enzymes and antioxidant enzymes in response to many cancer chemopreventive compounds. In this study, we investigated the role of receptor associated coactivator (RAC3) or steroid receptor coactivator-3 (SRC3) and other nuclear co-regulators including CBP/p300 (CREB-binding protein), CARM1 (Coactivator-associated arginine methyltransferase), PRMT1 (Protein arginine methyl-transferase 1), and p/CAF (p300/CBP-associated factor) in the transcriptional activation of a chimeric Gal4-Nrf2-Luciferase system containing the transactivation domain (TAD) of Nrf2 in HepG2 cells. The results indicated that RAC3 up-regulated the transactivation activity of Gal4-Nrf2-(1-370) in a dose-dependent manner. The enhancement of transactivation domain activity of Gal4-Nrf2-(1-370) by RAC3 was dampened in the presence of dominant negative mutants of RAC3. Next we studied the effects of other nuclear co-regulators including CBP/p300, CARM1, PRMT1 and p/CAF, and the results showed that they had different level of positive effects on this transactivation domain activity of Gal4-Nrf2-(1-370). But importantly, synergistic effects of these co-regulators in the presence of RAC3/SRC3 on the transactivation activity of Gal4-Nrf2-(1-370) were observed. In summary, our present study showed for the first time that the 160 RAC3/SRC3 is involved in the functional transactivation of TAD of Nrf2 and that the other nuclear co-regulators such as CBP/p300, CARM1, PRMT1 and p/CAF can also transcriptionally activate this TAD of Nrf2 and that they could further enhance the transactivation activity mediated by RAC3/SRC3.

• The Journal of Korean Medicine
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• v.29 no.2
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• pp.151-164
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• 2008

Objective: To investigate the effects of Yeongdamsagantang (YDGT) on apoptosis of neuronal cells that can result in dementia. Method: The water extract of the YDGT was tested in vitro for its beneficial effects on neuronal survival and neuroprotective functions, particularly in connection with $A{\beta}$ oligomer-related dementias. $A{\beta}$ oligomers derived from proteolytic processing of the ${\beta}-amyloid$ precursor protein (APP), including the $amyloid-{\beta}$ peptide $(A{\beta})$, play a critical role in the pathogenesis of Alzheimer's disease. A neuroblastoma cell line stably expressing an $A{\beta}$ oligomerassociated neuronal degeneration was used to investigate if YDGT inhibits formation of $A{\beta}$ oligomer. To measure the ATP generating level in mitochondrial membrane, luciferin/luciferase luminescence kit (Promega) and luminator was used, and to survey the protein's apparition, confocal microscopy was used. Result: $A{\beta}oligomer$ had a profound attenuation in the increase in CT105 expressing neuro2A cells from YDGT. Experimental evidence indicates that YDGT protected against neuronal damage from cells, but its cellular and molecular mechanisms remain unknown. We demonstrated that YDGT inhibited formation of $amyloid-{\beta}$ $(A{\beta})$ oligomers, which were the behavior, and possibly causative, features of AD. The decreased $A{\beta}$ oligomer in the presence of YDGT was observed in the conditioned medium of this $A{\beta}oligomer-secreting$ cell line under in vitro. In the cells, YDGT significantly attenuated mitochondrion-initiated apoptosis. Conclusion: (i) a direct $A{\beta}$ oligomer toxicity and the apoptosis initiated by the mitochondria; and (ii) multiple cellular and molecular neuroprotective mechanisms, including attenuation of apoptosis and direct inhibition of $A{\beta}$ oligomer aggregation, underlie the neuroprotective effects of YDGT.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.3
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• pp.398-407
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• 2020

Objective: The Yip1 domain family (YIPF) proteins were proposed to function in endoplasmic reticulum (ER) to Golgi transport and maintenance of the morphology of the Golgi, which were homologues of yeast Yip1p and Yif1p. YIPF3, the member 3 of YIPF family was a homolog of Yif1p. The aim of present study was to investigate the expression and regulation mechanism of porcine YIPF3. Methods: Quantitative realtime polymerase chain reaction (qPCR) was used to analyze porcine YIPF3 mRNA expression pattern in different tissues and pig kidney epithelial (PK15) cells stimulated by polyinosine-polycytidylic acid (poly [I:C]). Site-directed mutations combined with dual luciferase reporter assays and electrophoretic mobility shift assay (EMSA) were employed to reveal transcription regulation mechanism of porcine YIPF3. Results: Results showed that the mRNA of porcine YIPF3 (pYIPF3) was widely expressed with the highest levels in lymph and lung followed by spleen and liver, while weak in heart and skeletal muscle. Subcellular localization results indicated that it expressed in Golgi apparatus and plasma membranes. Upon stimulation with poly (I:C), the level of this gene was dramatically up-regulated in a time- and concentration-dependent manner. pYIPF3 core promoter region harbored three cis-acting elements which were bound by ETS proto-oncogene 2 (ETS2), zinc finger and BTB domain containing 4 (ZBTB4), and zinc finger and BTB domain containing 14 (ZBTB14), respectively. In which, ETS2 and ZBTB4 both promoted pYIPF3 transcription activity while ZBTB14 inhibited it, and these three transcription factors all played important regulation roles in tumorigenesis and apoptosis. Conclusion: The pYIPF3 mRNA expression was regulated by ETS2, ZBTB4, and ZBTB14, and its higher expression in immune organs might contribute to enhancing ER to Golgi transport of proteins, thus adapting to the immune response.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.23
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• pp.10439-10444
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• 2015

Many chemotherapeutic agents have been successfully used to treat hepatocellular carcinoma (HCC); however, the development of chemoresistance in liver cancer cells usually results in a relapse and worsening of prognosis. It has been demonstrated that DNA methylation and histone modification play crucial roles in chemotherapy resistance. Currently, extensive research has shown that there is another potential mechanism of gene expression control, which is mediated through the function of short noncoding RNAs, especially for microRNAs (miRNAs), but little is known about their roles in cancer cell drug resistance. In present study, by taking advantage of miRNA effects on the resistance of human hepatocellular carcinoma cells line to cisplatin, it has been demonstrated that miR-340 were significantly downregulated whereas Nrf2 was upregulated in HepG2/CDDP (cisplatin) cells, compared with parental HepG2 cells. Bioinformatics analysis and luciferase assays of Nrf2-3'-untranslated region-based reporter constructor indicated that Nrf2 was the direct target gene of miR-340, miR-340 mimics suppressing Nrf2-dependent antioxidant pathway and enhancing the sensitivity of HepG2/CDDP cells to cisplatin. Interestingly, transfection with miR-340 mimics combined with miR-340 inhibitors reactivated the Nrf2 related pathway and restored the resistance of HepG2/CDDP cells to CDDP. Collectively, the results first suggested that lower expression of miR-340 is involved in the development of CDDP resistance in hepatocellular carcinoma cell line, at least partly due to regulating Nrf2-dependent antioxidant pathway.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.16
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• pp.6949-6954
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• 2014

Background: Recent studies have indicated that microRNA-15a (miR-15a) is dysregulated in breast cancer (BC). We aimed to evaluate the expression of miR-15a in BC tissues and corresponding para-carcinoma tissues. We also focused on effects of miR-15a on cellular behavior of MDA-MB-231 and expression of its target gene synuclein-${\gamma}$ (SNCG). Materials and Methods: The expression levels of miR-15a were analysed in BC formalin fixed paraffin embedded (FFPE) tissues by microarray and quantitative real-time PCR. CCK-8 assays, cell cycle and apoptosis assays were used to explore the potential functions of miR-15a in MDA-MB-231 human BC cells. A luciferase reporter assay confirmed direct targets. Results: Downregulation of miR-15a was detected in most primary BCs. Ectopic expression of miR-15a promoted proliferation and suppressed apoptosis in vivo. Further studies indicated that miR-15a may directly interact with the 3'-untranslated region (3'-UTR) of SNCG mRNA, downregulating its mRNA and protein expression levels. SNCG expression was negatively correlated with miR-15a expression. Conclusions: MiR-15a has a critical role in mediating cell cycle arrest and promoting cell apoptosis of BC, probably by directly targeting SNCG. Thus, it may be involved in development and progression of BC.

• Journal of Pharmacopuncture
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• v.15 no.3
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• pp.13-18
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• 2012

Objectives: The purpose of this study is to examine whether Hominis Placental pharmacopuncture solution (HPPS) combined with zinc-oxide nanoparticles (ZnO NP) activates RAW 264.7 cells. Methods: We soaked ZnO nanoparticles in the Hominis Placenta pharmacopuncture solution, thereby making a combined form (ZnO NP HPPS). The effect of ZnO NP HPPS on the intracellular reactive oxygen species (ROS) production was measured by 2', 7'-dichlorofluorescin diacetate (DCFH-DA) assay. The effect of ZnO NP HPPS on NF-${\kappa}B$ was measured by using a luciferase assay. The effect of ZnO NP HPPS on the cytokine expression was assessed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The cellular uptake of ZnO NP HPPS was measured by using a flow cytometric analysis, and cellular structural alterations were analyzed by using transmission electron microscopy (TEM). Results: Neither the HPPS nor the ZnO NPs induced intracellular ROS production in RAW 264.7 cells. Neither of the materials activated NF-${\kappa}B$ or it's dependent genes, such as TNF-${\alpha}$, IL-1, and MCP-1. However, ZnO NP HPPS, the combined form of ZnO NPs and HPPS, did induce the intracellular ROS production, as well as prominently activating NF-${\kappa}B$ and it's dependent genes. Also, compared to ZnO NPs, it effectively increa-sed the uptake by RAW 264.7 cells. In addition, cellular structural alterations were observed in groups treated with ZnO NP HPPS. Conclusions: Neither ZnO NP nor HPPS activated RAW 264.7 cells, which is likely due to a low cellular uptake. The ZnO NP HPPS, however, significantly activated NF-${\kappa}B$ and up-regulated its dependent genes such as TNF-${\alpha}$, IL-1, and MCP-1. ZnO NP HPPS was also more easily taken into the RAW 264.7 cells than either ZnO NP or HPPS.

• Journal of Nutrition and Health
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• v.42 no.5
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• pp.434-441
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• 2009

Ferroportin-1 (FPN) is a transporter protein that is known to mediate iron export from macrophages. The purpose of this study was to investigate the effect of copper on the regulation of FPN gene expression in J774 mouse macrophage cells. J774 cells were treated with various concentrations of $CuSO_4$ and RT-PCR analyses were performed to measure the steady-state levels of mRNAs for FPN and divalent metal transporter 1 (DMT1, an iron importer). Copper treatment significantly increased FPN mRNAs in a dose-dependent manner, but didn't change the levels of DMT1 mRNA. Experiments with transcriptional inhibitor actinomycin D (0.5 ${\mu}g$/mL) revealed that copper treatment did not affect the half-life of FPN mRNAs in J774 cells. On the other hand, results from luciferase reporter assays showed that copper directly stimulated the promoter activity of FPN. In summary, our data showed copper induced FPN mRNA of macrophages via a transcriptional rather than post-transcriptional mechanisms.