• Journal of Life Science
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• v.24 no.3
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• pp.323-328
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• 2014

MicroRNAs comprise a family of small noncoding RNAs that modulate physiological processes, including adipogenesis. MicroRNA-17 (miR-17) promotes adipocyte differentiation and enhances lipid accumulation. The transcriptional regulation of miR-17 during adipogenesis remains unknown. In this study, we investigated whether miR-17 is a target of peroxisome proliferator-activated receptor ${\gamma}$ ($PPAR{\gamma}$), which is a key regulator of adipogenesis. The levels of miR-17 and the expression of $PPAR{\gamma}$ increased after the induction of adipocyte differentiation. Three putative peroxisome proliferator response elements (PPREs) were identified in the miR-17 promoter region. Using chromatin immunoprecipitation and luciferase reporter assays, we observed the interaction of $PPAR{\gamma}$ with the miR-17 promoter. Mutagenesis experiments showed that the -677/-655 region of the miR-17 promoter could function as a PPRE site. These results suggest that $PPAR{\gamma}$ is essential for transcriptional activation of the miR-17 gene, thereby contributing to understanding the molecular mechanism of adipogenesis in adipocytes.

• Archives of Pharmacal Research
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• v.29 no.8
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• pp.633-644
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• 2006

In last couple of decades the use of natural compounds like flavonoids as chemopreventive agents has gained much attention. Our current study focuses on identifying chemopreventive flavonoids and their mechanism of action on human prostate cancer cells. Human prostate cancer cells (PC3), stably transfected with activator protein 1 (AP-1) luciferase reporter gene were treated with four main classes of flavonoids namely flavonols, flavones, flavonones, and isoflavones. The maximum AP-1 luciferase induction of about 3 fold over control was observed with $20\;{\mu}M$ concentrations of quercetin, chrysin and genistein and $50\;{\mu}M$ concentration of kaempferol. At higher concentrations, most of the flavonoids demonstrated inhibition of AP-1 activity. The MTS assay for cell viability at 24 h showed that even at a very high concentration $(500\;{\mu}M)$, cell death was minimal for most of the flavonoids. To determine the role of MAPK pathway in the induction of AP-1 by flavonoids, Western blot of phospho MAPK proteins was performed. Four out of the eight flavonoids namely kaempferol, apigenin, genistein and naringenin were used for the Western Blot analysis. Induction of phospho-JNK and phospho-ERK activity was observed after two hour incubation of PC3-AP1 cells with flavonoids. However no induction of phospho-p38 activity was observed. Furthermore, pretreating the cells with specific inhibitors of JNK reduced the AP-1 luciferase activity that was induced by genistein while pretreatment with MEK inhibitor reduced the AP-1 luciferase activity induced by kaempferol. The pharmacological inhibitors did not affect the AP-1 luciferase activity induced by apigenin and naringenin. These results suggest the possible involvement of JNK pathway in genistein induced AP-1 activity while the ERK pathway seems to play an important role in kaempferol induced AP-1 activity.

• Animal Bioscience
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• v.36 no.4
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• pp.555-569
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• 2023

Objective: The objective of this study was to investigate the effects of N⁶-Methyladenosine modification-circRNA-zinc finger protein 638 (m⁶A-circRNA-ZNF638) on the induced activation of secondary hair follicle (SHF) stem cells with its potential mechanisms in cashmere goats. Methods: The m⁶A modification of ZNF638 was analyzed using methylation immunoprecipitation with real-time quantitative polymerase chain reaction technique in SHF stem cells. The effects of circRNA-ZNF638 on the induced activation of SHF stem cells in m⁶A dependence were evaluated through the overexpression of circRNA-ZNF638/its m⁶A-deficient mutants in circRNA-ZNF638 knockdown SHF stem cells. The competitive binding of miR-361-5p to circRNA-ZNF638/Wnt5a 3'- untranslated region was analyzed through Dual-luciferase reporter assay. Results: The m⁶A-circRNA-ZNF638 had significantly higher transcription at anagen SHF bulge of cashmere goats compared with that at telogen, as well as it positively regulated the induced activation of SHF-stem cells in cashmere goats. Mechanismly, m⁶A-circRNA-ZNF638 sponged miR-361-5p to heighten the transcriptional expression of Wnt5a gene in SHF-stem cells. We further demonstrated that the internal m⁶A modification within circRNA-ZNF638 is required for mediating the miR-361-5p/Wnt5a pathway to regulate the induced activation of SHF stem cells through an introducing of m⁶A-deficient mutant of circRNA-ZNF638. Conclusion: The circRNA-ZNF638 contributes the proper induced activation of SHF-stem cells in cashmere goats in m⁶A-dependent manner through miR-361-5p/Wnt5a axis.

• Journal of Plant Biotechnology
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• v.35 no.3
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• pp.169-176
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• 2008

Salicylic acid(SA) is a phytohormone that is related to plant defense mechanism. The SA accumulation is triggered by abiotic and biotic stresses. SA acts as a signal molecular compound mediating systemic acquired resistance and hypersensitive response in plant. Although the role of SA has been studied extensively, an understanding of the SA regulatory mechanism is still lacking in plants. In order to comprehend SA regulatory mechanism, we have been transformed with a SID2 promoter:GUS::LUC fusion construct into siz1-2 mutant and wild plant(Col-0). SIZ1 encodes SUMO E3 ligase and negatively regulates SA accumulation in plants. SID2(SALICYLIC ACID INDUCTION DEFICIENT2) is a crucial enzyme of SA biosynthesis. The Arabidopsis SID2 gene encodes isochorismate synthase(ICS) that controls SA level by conversion of chorismate to isochorismate. We compared the regulation of SID2 in wild-type and siz1-2 transgenic plants that express SID2 promoter:GUS::LUC constructs respectively. The expressions of $\beta$-GLUCURONIDASE and LUCIFERASE were higher in siz 1-2 transgenic plant without any stress treatment. SID2 promoter:GUS::LUC/siz1-2 transgenic plant will be used as a starting material for isolation of siz1-2 suppressor mutants and genes involved in SA-mediated stress signaling pathway.

• Journal of Life Science
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• v.30 no.3
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• pp.313-319
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• 2020

Recently, cattle epidemic diseases are caused by a pathogen such as a virus or bacterium. Such diseases can spread through various pathways, such as feed intake, respiration, and contact between livestock. Diagnosis based on the ELISA (Enzyme-linked immunosorbent assay) and PCR (Polymerase chain reaction) methods has limitations because these traditional diagnostic methods are time consuming assays that require multiple steps and dedicated equipment. In this review, we propose the use of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas system based on DNA and RNA levels for early point-of-care diagnosis in cattle. In the CRISPR/Cas system, Cas effectors are classified into two classes and six subtypes. The Cas effectors included in class 2 are typically Cas9 in type II, Cas12 in type V (Cas12a and Cas12b) and Cas13 in type VI (Cas13a and Cas13b). The CRISPR/Cas system uses reporter molecules that are attached to the ssDNA strands. When the Cas enzyme cuts the ssDNA, these reporters either fluoresce or change color, indicating the presence of a specific disease marker. There are several steps in the development of a CRISPR/Cas system. The first is to select the Cas enzyme depending on DNA or RNA from pathogens (viruses or bacteria). Based on that, the next step is to integrate the optimal amplification, transducing method, and signal reporter. The CRISPR/Cas system is a powerful diagnostic tool using a gene-editing method, which is faster, better, and cheaper than traditional methods. This system could be used for early diagnosis of epidemic cattle diseases and help to control their spread.

• Journal of Life Science
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• v.16 no.4
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• pp.632-639
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• 2006

Here we examined the expression patterns and regulation of seven photosynthesis (PS) genes (puf, puc, puhA, bchC, bchE, bchF, and bchI) in the anoxygenic photosynthetic bacterium, Rhodobacter sphaeroides, based on lacZ reporter gene assay. Expression of the tested PS genes, except puhA and bchI, were strongly induced in R. sphaeroides grown under anaerobic conditions relative to that under aerobic conditions. The puhA and bchI genes appear to form the operons together with bchFNBHLM-RSP0290 and crtA, respectively. Expression of the puf, puc, and bchCXYZ operons in R. sphaeroides grown photosynthetically was proportional to the incident light intensity, whereas that of bchFNBHLM(RSP0290-puhA) was inversely related to light intensity. Expression of bchEJG was lowest under medium-light photosynthetic conditions $(10\;W/m^2)$ and highest under high light conditions $(100\;W/m^2)$. The regulation of PS genes by the three major regulatory systems involved in oxygen- and light-sensing in R. sphaeroides is as following: puf and bchC are regulated by both the PpsR repressor and the PrrBA two-component system. The puc operon is under control of PpsR, FnrL, and PrrBA system. Expression of bchE is controlled by FnrL and PrrBA two-component system, whereas bchF is regulated exclusively by PpsR. It was demonstrated that the PpsR repressor is responsible for high-light repression of bchF and that FnrL might be involved in perceiving the cellular redox state in addition to sensing $O_2$ itself.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.18
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• pp.7849-7855
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• 2014

Purpose: To investigate the effect of deacetylase inhibitory trichostatin A (TSA) on anti HepG2 liver carcinoma cells and explore the underlying mechanisms. Materials and Methods: HepG2 cells exposed to different concentrations of TSA for 24, 48, or 72h were examined for cell growth inhibition using CCK8, changes in cell cycle distribution with flow cytometry, cell apoptosis with annexin V-FTIC/PI double staining, and cell morphology changes under an inverted microscope. Expression of ${\beta}$-catenin, HDAC1, HDAC3, H3K9, CyclinD1 and Bax proteins was tested by Western blotting. Gene expression for ${\beta}$-catenin, HDAC1and HDAC3 was tested by q-PCR. ${\beta}$-catenin and H3K9 proteins were also tested by immunofluorescence. Activity of Renilla luciferase (pTCF/LEF-luc) was assessed using the Luciferase Reporter Assay system reagent. The activity of total HDACs was detected with a HDACs colorimetric kit. Results: Exposure to TSA caused significant dose-and time-dependent inhibition of HepG2 cell proliferation (p<0.05) and resulted in increased cell percentages in G0/G1 and G2/M phases and decrease in the S phase. The apoptotic index in the control group was $6.22{\pm}0.25%$, which increased to $7.17{\pm}0.20%$ and $18.1{\pm}0.42%$ in the treatment group. Exposure to 250 and 500nmol/L TSA also caused cell morphology changes with numerous floating cells. Expression of ${\beta}$-catenin, H3K9and Bax proteins was significantly increased, expression levels of CyclinD1, HDAC1, HDAC3 were decreased. Expression of ${\beta}$-catenin at the genetic level was significantly increased, with no significant difference in HDAC1and HDAC3 genes. In the cytoplasm, expression of ${\beta}$-catenin fluorescence protein was not obvious changed and in the nucleus, small amounts of green fluorescence were observed. H3K9 fluorescence protein were increased. Expression levels of the transcription factor TCF werealso increased in HepG2 cells following induction by TSA, whikle the activity of total HDACs was decreased. Conclusions: TSA inhibits HDAC activity, promotes histone acetylation, and activates Wnt/${\beta}$-catenin signaling to inhibit proliferation of HepG2 cell, arrest cell cycling and induce apoptosis.

• Journal of Life Science
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• v.26 no.9
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• pp.991-998
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• 2016

NF-κB acts as a critical transcription factor for the survival of cells via the induction of antiapoptotic genes. Constitutive activation of NF-κB in many types of solid tumors suggests that the inhibition of NF-κB might prevent or inhibit tumorigenesis. Although a number of studies demonstrated that Hsp70 regulated NF-κB activity, the exact mechanism is not clear. This study investigated the functional relationship of Hsp70 and IKKγ in the regulation of NF-κB activation using expression plasmids of components of the IKK complex. Wild-type and deletion mutants of IKKγ were expressed together with Hsp70, and the combined regulatory effect of Hsp70 and IKKγ on NF-κB activation was assayed. Hsp70 suppressed the activation of NF-κB in a reporter plasmid assay. Hsp70 also suppressed the phosphorylation and degradation of IκBα. The suppressive effect of Hsp70 on NF-κB activation was synergistically elevated by IKKγ. The N-terminal IKKβ binding site, C-terminal leucine zipper, and zinc finger domains of IKKγ were not necessary for the suppressive effect. Furthermore, Hsp70 and IKKγ synergistically suppressed the induction of COX-2 expression by lipopolysaccharides in RAW264.7 cells. These results suggest that overexpression of Hsp70 and IKKγ may be a strategic method for inhibition of NF-κB and related diseases.