• BMB Reports
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• 제50권4호
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• pp.220-225
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• 2017

Antisense transcripts were initially identified as transcriptional noise, but have since been reported to play an important role in the quality control of miRNA functions. In this report, we tested the hypothesis that heterogeneous nuclear ribonucleoprotein K (hnRNPK) regulates miRNA function via competitive endogenous RNAs, such as pseudogenes, long non-coding RNAs, and antisense transcripts. Based on analyses of RNA sequencing data, the knockdown of hnRNPK decreased the antisense PTOV1-AS1 transcript which harbors five binding sites for miR-1207-5p. We identified heme oxygenase-1 (HO-1) mRNA as a novel target of miR-1207-5p by western blotting and Ago2 immunoprecipitation. The knockdown of hnRNPK or PTOV1-AS1 suppressed HO-1 expression by increasing the enrichment of HO-1 mRNA in miR-1207-5p-mediated miRISC. Downregulation of HO-1 by a miR-1207-5p mimic or knockdown of hnRNPK and PTOV1-AS1 inhibited the proliferation and clonogenic ability of HeLa cells. Taken together, our results demonstrate that hnRNPK-regulated PTOV1-AS1 modulates HO-1 expression via miR-1207-5p.

• Asian-Australasian Journal of Animal Sciences
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• 제30권2호
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• pp.236-245
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• 2017

Objective: The study was to investigate the effects of alanyl-glutamine (Ala-Gln) and glutamine (Gln) supplementation on the intestinal mucosa barrier in piglets. Methods: A total of 180 barrows with initial weight $10.01{\pm}0.03kg$ were randomly allocated to three treatments, and each treatment consisted of three pens and twenty pigs per pen. The piglets of three groups were fed with control diet [0.62% alanine (Ala)], Ala-Gln diet (0.5% Ala-Gln), Gln diet (0.34% Gln and 0.21% Ala), respectively. Results: The results showed that in comparison with control diet, dietary Ala-Gln supplementation increased the height of villi in duodenum and jejunum (p<0.05), Gln supplementation increased the villi height of jejunum (p<0.05), Ala-Gln supplementation up-regulated the mRNA expressions of epidermal growth factor receptor and insulin-like growth factor 1 receptor in jejunal mucosa (p<0.05), raised the mRNA expressions of Claudin-1, Occludin, zonula occludens protein-1 (ZO-1) and the protein levels of Occludin, ZO-1 in jejunal mucosa (p<0.05), Ala-Gln supplementation enlarged the number of goblet cells in duodenal and ileal epithelium (p<0.05), Gln increased the number of goblet cells in duodenal epithelium (p<0.05) and Ala-Gln supplementation improved the concentrations of secretory immunoglobulin A and immunoglobulin G in the jejunal mucosa (p<0.05). Conclusion: These results demonstrated that dietary Ala-Gln supplementation could maintain the integrity of small intestine and promote the functions of intestinal mucosa barriers in piglets.

• Journal of Microbiology and Biotechnology
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• 제32권2호
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• pp.149-159
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• 2022

Cancers of the lung and liver are the top 10 leading causes of cancer death worldwide. Thus, it is essential to identify the genes specifically expressed in these two cancer types to develop new therapeutics. Although many messenger RNA (mRNA) sequencing data related to these cancer cells are available due to the advancement of next-generation sequencing (NGS) technologies, optimized data processing methods need to be developed to identify the novel cancer-specific genes. Here, we conducted an analytical comparison between Bowtie2, a Burrows-Wheeler transform-based alignment tool, and Kallisto, which adopts pseudo alignment based on a transcriptome de Bruijn graph using mRNA sequencing data on normal cells and lung/liver cancer tissues. Before using cancer data, simulated mRNA sequencing reads were generated, and the high Transcripts Per Million (TPM) values were compared. mRNA sequencing reads data on lung/liver cancer cells were also extracted and quantified. While Kallisto could directly give the output in TPM values, Bowtie2 provided the counts. Thus, TPM values were calculated by processing the Sequence Alignment Map (SAM) file in R using package Rsubread and subsequently in python. The analysis of the simulated sequencing data revealed that Kallisto could detect more transcripts and had a higher overlap over Bowtie2. The evaluation of these two data processing methods using the known lung cancer biomarkers concludes that in standard settings without any dedicated quality control, Kallisto is more effective at producing faster and more accurate results than Bowtie2. Such conclusions were also drawn and confirmed with the known biomarkers specific to liver cancer.

• Reproductive and Developmental Biology
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• 제37권4호
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• pp.175-183
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• 2013

Cathepsin B is abundantly expressed peptidase of the papain family in the lysosomes, and closely related to the cell degradation system such as apoptosis, necrosis and autophagy. Abnormal degradation of organelles often occurs due to release of cathepsin B into the cytoplasm. Many studies have been reported that relationship between cathepsin B and intracellular mechanisms in various cell types, but porcine embryos has not yet been reported. Therefore, this study evaluated the effect of cathepsin B inhibitor (E-64) on preimplantation developmental competence and quality of porcine embryos focusing on apoptosis and oxidative stress. The expression of cathepsin B mRNA in porcine embryos was gradually decreased in inverse proportion to E-64 concentration by using real-time RT-PCR. When putative zygotes were cultured with E-64 for 24 h, the rates of early cleavage and blastocyst development were decreased by increasing E-64 concentration. However, the rate of blastocyst development in $5{\mu}M$ treated group was similar to the control. On the other hand, both the index of apoptotic and reactive oxygen species (ROS) of blastocysts were significantly decreased in the $5{\mu}M$ E-64 treated group compared with control. We also examined the mRNA expression levels of apoptosis related genes in the blastocysts derived from $5{\mu}M$ E-64 treated and non-treated groups. Expression of the pro-apoptotic Bax gene was shown to be decreased in the E-64 treated blastocyst group, whereas expression of the anti-apoptotic Bcl-xL gene was increased. Taken together, these results suggest that proper inhibition of cathepsin B at early development stage embryos improves the quality of blastocysts, which may be related to not only the apoptosis reduction but also the oxidative stress reduction in porcine embryos.

• Asian-Australasian Journal of Animal Sciences
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• 제23권4호
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• pp.456-464
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• 2010

RNA interference (RNAi) is an acknowledged useful and effective tool to study gene function in various cells. Here, we suppressed the Connexin 43 (Cx 43) gene expression during in vitro development of ovine pre-implantation embryos using the RNAi method. The 353 bp Cx 43 double-stranded RNA was microinjected into in vitro fertilized ovine zygotes, and the levels of target mRNA and protein were investigated. Control groups included uninjected zygotes or those injected with RNase-free water. The dsRNA injection resulted in the specific reduction of Cx 43 transcripts as analyzed by quantitative real-time RT-PCR and decreased protein levels as shown by Western blot analysis at the blastocyst stage. Microinjection of Cx 43 dsRNA led to 20.3%, 21.7% and 34.5% blastocyst rates and 19.2%, 37.5% and 41.3% hatched blastocyst rates in Cx 43 dsRNA-injected, water-injected and uninjected groups, respectively. Then the RNAi could not significantly affect cell number and cell death rates of blastocysts. Therefore, suppression of Cx 43 dsRNA and proteins did not apparently affect the development potential of ovine pre-implantation embryos but may play a role in embryo quality. RNAi technology is a promising approach to study gene function in early ovine embryogenesis.

• Animal Bioscience
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• 제34권4호
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• pp.546-557
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• 2021

Objective: If fertilization does not occur within a specific period, the quality of unfertilized oocytes in the oviduct (in vivo aging) or in culture (in vitro aging) will deteriorate over time. Icariin (ICA), found in all species of Epimedium herbs, has strong antioxidant activity, and is thought to exert anti-aging effects in vitro. We asked whether ICA protects oocytes against age-related changes in vitro. Methods: We analyzed the reactive oxygen species (ROS) levels and expression of antioxidant, maternal, and estrogen receptor genes, and along with spindle morphology, and the developmental competence and quality of embryos in the presence and absence of ICA. Results: Treatment with 5 μM ICA (ICA-5) led to a significant reduction in ROS activity, but increased mRNA expression of glutathione and antioxidant genes (superoxide dismutase 1 [SOD1], SOD2, peroxiredoxin 5, and nuclear factor erythroid 2-like 2), during aging in vitro. In addition, ICA-5 prevented defects in spindle formation and chromosomal alignment, and increased mRNA expression of cytoplasmic maturation factor genes (bone morphogenetic protein 15, cyclin B1, MOS proto-oncogene, serine/threonine kinase, and growth differentiation factor-9). It also prevented apoptosis, increased mRNA expression of antiapoptotic genes (BCL2-like 1 and baculoviral IAP repeat-containing 5), and reduced mRNA expression of pro-apoptotic genes (BCL2 antagonist/killer 1 and activation of caspase-3). Although the maturation and cleavage rates were similar in all groups, the total cell number per blastocyst and the percentage of apoptotic cells at the blastocyst stage were higher and lower, respectively, in the control and ICA-5 groups than in the aging group. Conclusion: ICA protects oocytes against damage during aging in vitro; therefore, it can be used to improve assisted reproductive technologies.

• Asian-Australasian Journal of Animal Sciences
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• 제33권12호
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• pp.2008-2020
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• 2020

Objective: This study was conducted to investigate the effects of dietary corn resistant starch (RS) on the intestinal morphology and barrier functions of broilers. Methods: A total of 320 one-day-old broilers were randomly allocated to 5 dietary treatments: one normal corn-soybean (NC) diet, one corn-soybean-based diet supplementation with 20% corn starch (CS), and 3 corn-soybean-based diets supplementation with 4%, 8%, and 12% corn resistant starch (RS) (identified as 4% RS, 8% RS, and 12% RS, respectively). Each group had eight replicates with eight broilers per replicate. After 21 days feeding, one bird with a body weight (BW) close to the average BW of their replicate was selected and slaughtered. The samples of duodenum, jejunum, ileum, caecum digesta, and blood were collected. Results: Birds fed 4% RS, 8% RS and 12% RS diets showed lower feed intake, BW gain, jejunal villus height (VH), duodenal crypt depth (CD), jejunal VH/CD ratio, duodenal goblet cell density as well as mucin1 mRNA expressions compared to the NC group, but showed higher concentrations of cecal acetic acid and butyric acid, percentage of jejunal proliferating cell nuclear antigen-positive cells and delta like canonical Notch ligand 4 (Dll4), and hes family bHLH transcription factor 1 mRNA expressions. However, there were no differences on the plasma diamine oxidase activity and D-lactic acid concentration among all groups. Conclusion: These findings suggested that RS could suppress intestinal morphology and barrier functions by activating Notch pathway and inhibiting the development of goblet cells, resulting in decreased mucins and tight junction mRNA expression.

• 한국식품영양과학회지
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• 제42권7호
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• pp.1133-1138
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• 2013

미나리 유통과정중 생성되는 부산물의 활용방안과 청국장의 품질 및 기호성 개선 방안을 모색하기 위해 청국장의 제조시 전처리 방법을 달리한 미나리 분말 첨가에 따른 발효 및 품질 특성을 비교 검토하였다. 발효균주로 사용한 Bacillus subtilis RS-9은 볏짚으로부터 분리된 30 균주 중 대두 분해력, 단백 분해력, 전분 분해력, 항균활성이 가장 우수한 균주를 16S rRNA 염기서열 분석을 통해 동정한 후 사용하였다. 전처리 방법을 달리한 미나리 분말을 0.5%, 1%로 혼합하여 제조한 청국장의 발효 및 품질특성을 조사한 결과, 미나리 분말 혼합 청국장의 pH는 발효 전 기간 동안 대조구보다 낮은 경향을 나타내었으며, 첨가 미나리의 전처리 방법에 따라서는 뚜렷한 차이를 관찰할 수 없었다. 청국장의 발효 후 총균수는 8.70~8.88 log CFU/mL이었으며, 발효기간이 경과함에 따라 모든 처리구에서 증가하였다. 미나리 혼합 청국장의 아미노태 질소와 점질물 함량은 발효 전 기간 동안 대조구보다 낮은 경향을 나타내었다. 총 폴리페놀 함량은 발효후 모든 군이 증가하였으며, 스팀처리 미나리 첨가구가 $590.24{\mu}g/mL$로 가장 높은 함량을 나타내었다. ABTS radical 소거능은 대조구에 비해 뚜렷이 높았으며, 발효 후 모든 처리구에서 활성이 증가하였고 스팀처리 미나리 첨가구가 82.16%로 가장 우수하였다. 관능검사 결과는 생미나리 분말 0.5% 첨가군이 맛과 종합적 기호도에서 가장 우수하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제28권10호
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• pp.1380-1387
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• 2015

This study aims to determine the polymorphism and mRNA expression pattern of the heart-type fatty acid-binding protein (H-FABP) gene and their association with intramuscular fat (IMF) content in the breast and leg muscles of Baicheng oil chicken (BOC). A total of 720 chickens, including 240 black Baicheng oil chicken (BBOC), 240 silky Baicheng oil chicken (SBOC), and 240 white Baicheng oil chicken (WBOC) were raised. Three genotypes of H-FABP gene second extron following AA, AB, and BB were detected by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) strategy. The G939A site created AA genotype and G956A site created BB genotype. The content of IMF in AA genotype in breast muscle of BBOC was significantly higher than that of AB (p = 0.0176) and the genotype in leg muscle of WBOC was significantly higher than that of AB (p = 0.0145). The G939A site could be taken as genetic marker for higher IMF content selecting for breast muscle of BBOC and leg muscle of WBOC. The relative mRNA expression of H-FABP was measured by real-time PCR at 30, 60, 90, and 120 d. The IMF content significantly increased with age in both muscles. The mRNA expression level of H-FABP significantly decreased with age in both muscles of the three types of chickens. Moreover, a significant negative correlation between H-FABP abundance and IMF content in the leg muscles of WBOC (p = 0.035) was observed. The mRNA expression of H-FABP negatively correlated with the IMF content in both breast and leg muscles of BOC sat slaughter time.

• 한국수정란이식학회지
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• 제29권1호
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• pp.73-81
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• 2014

돼지 수정란의 체외 생산 효율성 향상을 위해서는 배발생율과 더불어 고품질의 배를 조기에 선별해야 한다. 체외 배 발생율에 대한 보고는 많지만, 고품질의 배를 선별할 수 있는 기술에 대한 연구는 거의 없었다. 본 연구에서는 돼지 난포란 유래 수정란의 체외배양에 있어서 배반포로의 배 발달과 생존에 미치는 Vitamin $K_1$(vit $K_1$) 첨가 농도, 시기 및 시간의 효과를 검토하였다. $1.0{\mu}M$, $3.0{\mu}M$ 및 $6.0{\mu}M$ vit $K_1$을 배양 1일째 24시간 첨가한 결과, 배반포 발달율이 시험군이 $14.5{\pm}4.3$, 0.0 및 0.0%로써 대조군의 $35.5{\pm}3.2%$에 비하여 유의하게 낮았고(p<0.05), 배반포의 생존율도 대조군이 $31.8{\pm}2.6%$로써 시험군의 $22.2{\pm}2.9$, 0.0 및 0.0%에 비하여 유의하게 높았다(p<0.05). 상기 첨가 농도에서 첨가 시간을 달리한 결과, $1.0{\mu}M$ 농도에서 6시간 처리군의 배반포 발달율과 생존율이 각각 $26.5{\pm}2.9%$ 및 $47.2{\pm}2.8%$로써 가장 높았고 특히, 12시간 처리군보다 유의하게 높았다(p<0.05). $3.0{\mu}M$ 농도에서는 대조군의 배발달율이 $36.4{\pm}3.1%$로 가장 높았으나, 생존율은 3.0시간 첨가군이 $41.7{\pm}3.2%$로 대조군에 비하여 유의하게 높았다(p<0.05). $6.0{\mu}M$ 농도에서도 배발달율은 대조군($32.0{\pm}2.8%$), 생존율은 0.5시간 첨가군($42.9{\pm}1.8%$)이 가장 높았다. 각각의 vit $K_1$ 첨가 농도와 시간을 기준으로 서로 다른 배양 시기에 첨가한 결과, $1.0{\mu}M$ 6시간 첨가군에서는 배반포 발달율은 배양 4일째 첨가군, 생존율은 배양 2일째 첨가군이 가장 높았다. 한편, $3.0{\mu}M$ 3.0시간 및 $6.0{\mu}M$ 0.5시간 첨가군에서는 배양 4일째 첨가군의 배반포 발달율($59.5{\pm}4.1%$ 및 $50.0{\pm}3.6%$)과 생존율($72.7{\pm}5.4%$ 및 $79.2{\pm}4.0%$)이 대조군과 다른 시험군에 비하여 유의하게 높았다(p<0.05). 한편, vit $K_1$ 첨가에 따른 배반포의 세포 수를 조사한 결과, 첨가군($1.0{\mu}M$ 6시간 배양 2일째, $3.0{\mu}M$ 3.0시간 배양 4일째 및 $6.0{\mu}M$ 0.5시간 배양 6일째)이 $53.4{\pm}5.8$, $49.4{\pm}3.8$ 및 $51.5{\pm}4.5$개로써 대조군의 $40.2{\pm}2.3$개에 비하여 유의하게 많았다(p<0.05). 그러나 사멸세포 수는 시험군이 $3.2{\pm}0.9{\sim}3.7{\pm}2.1$개로써 대조군의 $4.2{\pm}1.2$개보다 적었으나, 유의차는 없었다. 세포 사멸 유도 유전자인 Bax mRNA 발현은 처리군과 대조군은 비슷하였으나, 세포 사멸 억제 유전자인 Bcl-xL mRNA 발현은 처리군이 대조군보다 높았고 특히, $6.0{\mu}M$ 0.5시간 배양 4일째 첨가군이 가장 높았다. 이상의 결과로부터 돼지 미성숙 난포란 유래 수정란의 체외 배양에 vit $K_1$의 첨가는 배반포의 생존율과 세포수 증가에 효과적이었다. 그 이유에 대해서는 아직 많은 부분이 밝혀져야 되겠지만, 고품질의 배반포 조기 선발에는 활용이 가능할 것으로 생각된다.