• Asian-Australasian Journal of Animal Sciences
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• 제32권8호
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• pp.1095-1103
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• 2019

Objective: Among stress responses, the unfolded protein response (UPR) is a well-known mechanism related to endoplasmic reticulum (ER) stress. ER stress is induced by a variety of external and environmental factors such as starvation, ischemia, hypoxia, oxidative stress, and heat stress. Inositol requiring enzyme $1{\alpha}$ ($IRE1{\alpha}$)-X-box protein 1 (XBP1) is the most conserved pathway involved in the UPR and is the main component that mediates $IRE1{\alpha}$ signalling to downstream ER-associated degradation (ERAD)- or UPR-related genes. XBP1 is a transcription factor synthesised via a novel mechanism called 'frame switch splicing', and this process has not yet been studied in the horse XBP1 gene. Therefore, the aim of this study was to confirm the frame switch splicing of horse XBP1 and characterise its dynamics using Thoroughbred muscle cells exposed to heat stress. Methods: Primary horse muscle cells were used to investigate heat stress-induced frame switch splicing of horse XBP1. Frame switch splicing was confirmed by sequencing analysis. XBP1 amino acid sequences and promoter sequences of various species were aligned to confirm the sequence homology and to find conserved cis-acting elements, respectively. The expression of the potential XBP1 downstream genes were analysed by quantitative real-time polymerase chain reaction. Results: We confirmed that splicing of horse XBP1 mRNA was affected by the duration of thermal stress. Twenty-six nucleotides in the mRNA of XBP1 were deleted after heat stress. The protein sequence and the cis-regulatory elements on the promoter of horse XBP1 are highly conserved among the mammals. Induction of putative downstream genes of horse XBP1 was dependent on the duration of heat stress. We confirmed that both the mechanisms of XBP1 frame switch splicing and various binding elements found in downstream gene promoters are highly evolutionarily conserved. Conclusion: The frame switch splicing of horse XBP1 and its dynamics were highly conserved among species. These results facilitate studies of ER-stress in horse.

• Journal of Microbiology and Biotechnology
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• 제30권12호
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• pp.1876-1884
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• 2020

Ethanol often accumulates during the process of wine fermentation, and mitophagy has critical role in ethanol output. However, the relationship between mitophagy and ethanol stress is still unclear. In this study, the expression of ATG11 and ATG32 genes exposed to ethanol stress was accessed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The result indicated that ethanol stress induced expression of the ATG11 and ATG32 genes. The colony sizes and the alcohol yield of atg11 and atg32 were also smaller and lower than those of wild type strain under ethanol whereas the mortality of mutants is higher. Furthermore, compared with wild type, the membrane integrity and the mitochondrial membrane potential of atg11 and atg32 exhibited greater damage following ethanol stress. In addition, a greater proportion of mutant cells were arrested at the G1/G0 cell cycle. There was more aggregation of peroxide hydrogen (H2O2) and superoxide anion (O2•-) in mutants. These changes in H2O2 and O2•- in yeasts were altered by reductants or inhibitors of scavenging enzyme by means of regulating the expression of ATG11 and ATG32 genes. Inhibitors of the mitochondrial electron transport chain (mtETC) also increased production of H2O2 and O2•- by enhancing expression of the ATG11 and ATG32 genes. Further results showed that activator or inhibitor of autophagy also activated or inhibited mitophagy by altering production of H2O2 and O2•. Therefore, ethanol stress induces mitophagy which improves yeast the tolerance to ethanol and the level of mitophagy during ethanol stress is regulated by ROS derived from mtETC.

• Asian-Australasian Journal of Animal Sciences
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• 제31권10호
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• pp.1550-1557
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• 2018

Objective: Circular RNAs (circRNAs) are a newfound class of non-coding RNA in animals and plants. Recent studies have revealed that circRNAs play important roles in cell proliferation, differentiation, autophagy and apoptosis during development. However, there are few reports about muscle development-related circRNAs in livestock. Methods: RNA sequencing analysis was employed to identify and annotate circRNAs from longissimus dorsi of sheep. Reverse transcription followed by real-time quantitative (q) polymerase chain reaction (PCR) analysis verified the presence of these circRNAs. Targetscan7.0 and miRanda were used to analyse the interaction of circRNA-microRNA (miRNA). To investigate the function of circRNAs, an experiment was conducted to perform enrichment analysis hosting genes of circRNAs using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. Results: About 75.5 million sequences were obtained from RNA libraries of sheep skeletal muscle. These sequences were mapped to 729 genes in the sheep reference genome. We identified 886 circRNAs, including numerous circular intronic RNAs and exonic circRNAs. Reverse transcription PCR (RT-PCR) and DNA sequencing analysis confirmed the presence of several circRNAs. Real-Time RT-PCR analysis exhibited resistance of sheep circRNAs to RNase R digestion. We found that many circRNAs interacted with muscle-specific miRNAs involved in growth and development of muscle, especially circ776. The GO and KEGG enrichment analysis showed that hosting genes of circRNAs was involved in muscle cell development and signaling pathway. Conclusion: The study provides comprehensive expression profiles of circRNAs in sheep skeletal muscle. Our study offers a large number of circRNAs to facilitate a better understanding of their roles in muscle growth. Meanwhile, we suggested that circ776 could be analyzed in future study.

• 원예과학기술지
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• 제32권6호
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• pp.845-852
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• 2014

다양한 비생물적 스트레스 중 토양 염 집적은 식물의 광합성 효율, 생장 및 수확량의 감소를 초래한다. 최근 염 저항성 향상을 위한 많은 유전자들이 보고되고 있다. 본 연구의 목적은 형질전환 배추를 이용하여 아직 기능이 밝혀져 있지 않지만 완전장이 보고된 Brassica rapa Salt Stress Resistance(BrSSR) 유전자의 기능을 검정하는 것이다. BrSSR의 생리적 역할을 분석하기 위해, BrSSR의 과발현 vector인 pSL94 vector를 이용하여 내혼계 배추('CT001')를 형질전환하였다. Quantitative real-time RT-PCR 분석에서 형질전환체의 BrSSR 발현량은 대조군 대비 2.59배까지 증가하였다. 한편, 염 처리 후 표현형 분석에서 BrSSR이 과발현된 형질전환체들이 정상적인 생장을 보여줌으로써 염 스트레스에 내성을 가지는 것을 확인할 수 있었다. Microarray 분석을 통해 구축된 염 스트레스 저항성 관련 유전자들의 발현 네트워크 상에서 BrSSR은 기존에 염 저항성 관련 유전자로 보고되어 있는 ERD15(AT2G41430), protein containing PAM2(AT4G14270), GABA-T(AT3G22200)와 매우 밀접하게 연결되어 있는 것으로 분석되었다. 위 결과들을 바탕으로 BrSSR은 염 스트레스 발생 시 식물의 생장 및 저항성에 관련된 중요한 역할을 하는 것으로 판단된다.

• Animal Bioscience
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• 제35권1호
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• pp.115-125
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• 2022

Objective: Intramuscular fat (IMF) is a critical economic indicator of pork quality. Studies on IMF among different pig breeds have been performed via high-throughput sequencing, but comparisons within the same pig breed remain unreported. Methods: This study was performed to explore the gene profile and identify candidate long noncoding RNA (lncRNAs) and mRNAs associated with IMF deposition among Laiwu pigs with different IMF contents. Based on the longissimus dorsi muscle IMF content, eight pigs from the same breed and management were selected and divided into two groups: a high IMF (>12%, H) and low IMF group (<5%, L). Whole-transcriptome sequencing was performed to explore the differentially expressed (DE) genes between these two groups. Results: The IMF content varied greatly among Laiwu pig individuals (2.17% to 13.93%). Seventeen DE lncRNAs (11 upregulated and 6 downregulated) and 180 mRNAs (112 upregulated and 68 downregulated) were found. Gene Ontology analysis indicated that the following biological processes played an important role in IMF deposition: fatty acid and lipid biosynthetic processes; the extracellular signal-regulated kinase cascade; and white fat cell differentiation. In addition, the peroxisome proliferator-activated receptor, phosphatidylinositol-3-kinase-protein kinase B, and mammalian target of rapamycin pathways were enriched in the pathway analysis. Intersection analysis of the target genes of DE lncRNAs and mRNAs revealed seven candidate genes associated with IMF accumulation. Five DE lncRNAs and 20 DE mRNAs based on the pig quantitative trait locus database were identified and shown to be related to fat deposition. The expression of five DE lncRNAs and mRNAs was verified by quantitative real time polymerase chain reaction (qRT-PCR). The results of qRT-PCR and RNA-sequencing were consistent. Conclusion: These results demonstrated that the different IMF contents among pig individuals may be due to the DE lncRNAs and mRNAs associated with lipid droplets and fat deposition.

• Animal Bioscience
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• 제35권10호
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• pp.1489-1498
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• 2022

Objective: The objective of this study was to identify polymorphism in olfactomedin like 3 (OLFML3) gene, and association analysis with meat quality, carcass characteristics, retail meat cut, and fatty acid composition in sheep, and expression quantification of OLFML3 gene in phenotypically divergent sheep. Methods: A total of 328 rams at the age of 10 to 12 months with an average body weight of 26.13 kg were used. A novel polymorphism was identified using high-throughput sequencing in sheep and genotyping of OLFML3 polymorphism was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Among 328 rams, 100 rams representing various sheep genotypes were used for association study and proc general linear model was used to analyse association between genotypes and phenotypic traits. Quantitative real-time polymerase chain reaction (qRT-PCR) was used for the expression analysis of OLFML3 mRNA in phenotypically divergent sheep population. Results: The findings revealed a novel polymorphism in the OLFML3 gene (g.90317673 C>T). The OLFML3 gene revealed three genotypes: CC, CT, and TT. The single nucleotide polymorphism (SNP) was found to be significantly (p<0.05) associated with meat quality traits such as tenderness and cooking loss; carcass characteristics such as carcass length; retail meat cut such as pelvic fat in leg, intramuscular fat in loin and tenderloin, muscle in flank and shank; fatty acids composition such as tridecanoic acid (C13:0), palmitoleic acid (C16:1), heptadecanoic acid (C17:0), ginkgolic acid (C17:1), linolenic acid (C18:3n3), arachidic acid (C20:0), eicosenoic acid (C20:1), arachidonic acid (C20:4n6), heneicosylic acid (C21:0), and nervonic acid (C24:1). The TT genotype was associated with higher level of meat quality, carcass characteristics, retail meat cut, and some fatty acids composition. However, the mRNA expression analysis was not different among genotypes. Conclusion: The OLFML3 gene could be a potential putative candidate for selecting higher quality sheep meat, carcass characteristics, retail meat cuts, and fatty acid composition in sheep.

• Asian Pacific Journal of Cancer Prevention
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• 제15권14호
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• pp.5577-5582
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• 2014

Objective: To observe the effects of metastasis-associated tumor gene family 2 (MTA2) depletion on human breast cancer cell proliferation and metastasis. Methods: A short-hairpin RNA targeting MTA2 was chemically synthesized and transfected into a lentivirus to construct Lv-shMTA2 for infection into the MDA-MB231 human breast cancer cell line. At 48 hours after infection cells were harvested and mRNA and protein levels of MTA2 were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively. Cell viability and metastasis were assessed by CCK-8, wound-healing assay and Transwell assay, respectively. In addition, a xenograft model of human breast cancer was constructed to investigate cancerous cell growth and capacity for metastasis. Results: After infection with Lv-shMTA2, mRNA and protein levels of MTA2 was significantly reduced (p<0.05) and MDA-MB231 cell proliferation and metastasis were inhibited (p<0.05). In addition, mean tumor size was smaller than that in control group nude mice (p<0.05) and numbers of metastatic deposits in lung were lower than in control group mice (p<0.05). Depletion of MTA2 affected MMP-2 and apoptosis-related protein expression. Conclusions: For the first time to our knowledge we showed that MTA2 depletion could significantly inhibit human breast cancer cell growth and metastasis, implying that MTA2 might be involved in the progression of breast cancer. The role of MTA2 in breast cancer growth and metastasis might be linked with regulation of matrix metalloproteinase and apoptosis.

• Animal Bioscience
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• 제36권10호
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• pp.1488-1498
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• 2023

Objective: α_S1-Casein is more closely associated with milk allergic reaction than other milk protein components. microRNA (miRNA) is a class of small non-coding RNAs that modulate multiple biological progresses by the target gene. However, the post-transcriptional regulation of α_S1-casein expression by miRNA in ruminants remains unclear. This study aims to explore the regulatory roles of miR-380-3p on α_S1-casein synthesis in goat mammary epithelial cells (GMEC). Methods: α_S1-Casein gene and miR-380-3p expression was measured in dairy goat mammary gland by quantitative real-time polymerase chain reaction (qRT-PCR). miR-380-3p overexpression and knockdown were performed by miR-380-3p mimic or inhibitor in GMEC. The effect of miR-380-3p on α_S1-casein synthesis was detected by qRT-PCR, western blot, luciferase and chromatin immunoprecipitation assays in GMEC. Results: Compared with middle-lactation period, α_S1-casein gene expression is increased, while miR-380-3p expression is decreased during peak-lactation of dairy goats. miR-380-3p reduces α_S1-casein abundance by targeting the 3'-untranslated region (3'UTR) of α_S1-casein mRNA in GMEC. miR-380-3p enhances β-casein expression and signal transducer and activator of transcription 5a (STAT5a) activity. Moreover, miR-380-3p promotes β-casein abundance through target gene α_S1-casein, and activates β-casein transcription by enhancing the binding of STAT5 to β-casein gene promoter region. Conclusion: miR-380-3p decreases α_S1-casein expression and increases β-casein expression by targeting α_S1-casein in GMEC, which supplies a novel strategy for reducing milk allergic potential and building up milk quality in ruminants.

• 한국해양생명과학회지
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• 제5권2호
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• pp.35-42
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• 2020

17β-estradiol (E₂)는 난소로부터 방출되는 호르몬으로 가정 및 축산 오폐수에 포함되어 환경으로 지속적으로 유출된다. E₂는 높은 에스트로겐 활성을 가지고 있어 갑각류의 발달과 생식에 영향을 미치는 내분비계교란물질로 알려져 있다. 갑각류의 발달은 탈피호르몬(ecdysteroid)의 신호 전달 과정에 의해 이루어지지만 E₂가 소형 갑각류의 탈피호르몬 경로 유전자를 어떻게 조절하는지에 대한 연구는 매우 미흡하다. 본 연구에서는 기수산 물벼룩 Diaphanosoma celebensis에서 E₂에 대한 급성 독성 시험을 통해 24-h LC_x 값을 도출하였고, E₂ 노출에 따른 탈피호르몬 경로에 관여하는 7개의 유전자(CYP314a1, EcRA, EcRB, USP, ERR, Vtg, VtgR)의 시간별 발현 변화를 quantitative real time polymerase chain reaction (qRT-PCR)을 이용하여 분석하였다. D. celebensis의 24-h LC₅₀ 값은 9.581 mg/l (95% C.I.: 7.697~11.927 mg/l), 24 h-LC₁₀ 값은 4.842 mg/l(95% C.I.: 3.683~6.366 mg/l)로 나타났다. CYP314a1, EcRA, USP, VtgR 유전자의 발현이 12시간 또는 24시간에 유의하게 증가하는 양상을 보였다. 이러한 결과는 E₂가 D. celebensis의 탈피호르몬 경로에 관련하는 유전자의 발현을 조절함으로써 탈피와 생식에 영향을 미칠 수 있을 것임을 시사한다. 본 연구는 소형 갑각류에서 내분비계교란물질이 탈피 경로에 미치는 영향에 대한 분자 기전을 이해하는데 도움이 될 것이다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권5호
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• pp.1851-1855
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• 2015

Background: There are increasing data about microRNAs (miRNA) in the literature, providing abundant evidence that they play important roles in pathogenesis and development of colorectal cancer. In this study, we aimed to investigate the miRNA expression profiles in surgically resected specimens of patients with recurrent and non-recurrent colorectal cancer. Materials and Methods: The study population included 40 patients with stage II colorectal cancer (20 patients with recurrent tumors, and 20 sex and age matched patients without recurrence), who underwent curative colectomy between 2004 and 2011 without adjuvant therapy. Expression of 16 miRNAs (miRNA-9, 21, 30d, 31, 106a, 127, 133a, 133b, 135b, 143, 145, 155, 182, 200a, 200c, 362) was verified by quantitative real-time polymerase chain reaction (qRT-PCR) in all resected colon cancer tissue samples and in corresponding normal colonic tissues. Data analyses were carried out using SPSS 15 software. Values were statistically significantly changed in 40 cancer tissues when compared to the corresponding 40 normal colonic tissues (p<0.001). MiR-30d, miR-133a, miR-143, miR-145 and miR-362 expression was statistically significantly downregulated in 40 resected colorectal cancer tissue samples (p<0.001). When we compared subgroups, miRNA expression profiles of 20 recurrent cancer tissues were similar to all 40 cancer tissues. However in 20 non-recurrent cancer tissues, miR-133a expression was not significantly downregulated, moreover miR-133b expression was significantly upregulated (p<0.05). Conclusions: Our study revealed dysregulation of expression of ten miRNAs in Turkish colon cancer patients. These miRNAs may be used as potential biomarkers for early detection, screening and surveillance of colorectal cancer, with functional effects on tumor cell behavior.