Adenoviral Vector Mediates High Expression Levels of Human Lactoferrin in the Milk of Rabbits

( Zeng Sheng Han ),( Qing Wang Li ),( Zhi Ying Zhang ),( Yong Sheng Yu ),( Bo Xiao ),( Shu Yun Wu ),( Zhong Liang Jiang ),( Hong Wei Zhao ),( Rui Zhao ),( Jian Li ) 한국미생물 · 생명공학회 2008 Journal of microbiology and biotechnology Vol.18 No.1

The limitations in current technology for generating transgenic animals, such as the time and the expense, hampered its extensive use in recombinant protein production for therapeutic purpose. In this report, we present a simple and less expensive alternative by directly infusing a recombinant adenovirus vector carrying human lactoferrin cDNA into rabbit mammary glands. The milk serum was collected from the infected mammary gland 48 h post-infection and subjected to a 10% SDS-PAGE and Western blotting. An 80-kDa protein was visualized after viral vector infection. With this method, we obtained a high level of expressed human lactoferrin of up to 2.3 mg/ml in the milk. Taken together, the method is useful for the transient high-level expression recombinant proteins, and the approach established here is probably one of the most economical and efficient ways for large-scale production of recombinant proteins of biopharmaceutical interest.

A Hypoxia-Induced SCFFBXL1 E3 Ligase Ubiquitinates and Degrades the MEN1 Tumor Suppressor to Promote Colorectal Cancer Tumorigenesis

Jun Zeng,Xiao-qing Xiao,Zhi-yong Zhou 대한암학회 2022 Cancer Research and Treatment Vol.54 No.2

Purpose Emerging evidence has shown that SKP1-cullin-1-F-box-protein (SCF) E3 ligases contribute to the pathogenesis of different cancers by mediating the ubiquitination and degradation of tumor suppressors. However, the functions of SCF E3 ligases in the pathogenesis of colorectal cancer (CRC) remain obscure. Materials and Methods The cancerous and adjacent noncancerous tissues from CRC patients were collected, and protein levels were analyzed. Lentiviral short hairpin RNA (shRNA) and plasmid transfection were used to knock down and overexpress gene expression in CRC cell lines. Immunoprecipitation (IP), mass spectrometry, and co-IP analyses were used to determine protein interactions and the assembly of the SCF complex. Cell proliferation, migration, and tumor xenograft assays were performed to examine the effects of SCF members on CRC cell growth in vitro and in vivo. Results Hypoxia activated the docking of hypoxia-inducible factor 1α (HIF1α) onto the CUL1 promoter and induced CUL1 expression in CRC cells. CUL1 coupled with RBX1, SKP1, and FBXL1 to assemble the SCFFBXL1 complex in CRC biopsies and cells. The SCFFBXL1 E3 ligase specifically ubiquitinated and degraded the MEN1 tumor suppressor. Knockdown of HIF1α or SCFFBXL1 members, or blockage of SCFFBXL1 by two inhibitors (DT204 and SZLP1-41) caused the accumulation of MEN1 protein and led to a significant decrease in cell proliferation and migration in vitro and tumor growth in vivo. Conclusion The SCFFBXL1 E3 ligase is required for the ubiquitination of MEN1, and disruption of this complex may represent a new therapeutic strategy for the treatment of CRC. PurposeEmerging evidence has shown that SKP1-cullin-1-F-box-protein (SCF) E3 ligases contribute to the pathogenesis of different cancers by mediating the ubiquitination and degradation of tumor suppressors. However, the functions of SCF E3 ligases in the pathogenesis of colorectal cancer (CRC) remain obscure. Materials and MethodsThe cancerous and adjacent noncancerous tissues from CRC patients were collected, and protein levels were analyzed. Lentiviral short hairpin RNA (shRNA) and plasmid transfection were used to knock down and overexpress gene expression in CRC cell lines. Immunoprecipitation (IP), mass spectrometry, and co-IP analyses were used to determine protein interactions and the assembly of the SCF complex. Cell proliferation, migration, and tumor xenograft assays were performed to examine the effects of SCF members on CRC cell growth in vitro and in vivo.ResultsHypoxia activated the docking of hypoxia-inducible factor 1α (HIF1α) onto the CUL1 promoter and induced CUL1 expression in CRC cells. CUL1 coupled with RBX1, SKP1, and FBXL1 to assemble the SCFFBXL1 complex in CRC biopsies and cells. The SCFFBXL1 E3 ligase specifically ubiquitinated and degraded the MEN1 tumor suppressor. Knockdown of HIF1α or SCFFBXL1 members, or blockage of SCFFBXL1 by two inhibitors (DT204 and SZLP1-41) caused the accumulation of MEN1 protein and led to a significant decrease in cell proliferation and migration in vitro and tumor growth in vivo.ConclusionThe SCFFBXL1 E3 ligase is required for the ubiquitination of MEN1, and disruption of this complex may represent a new therapeutic strategy for the treatment of CRC.

Interactive Effect of Bisphenol A (BPA) Exposure with -22G/C Polymorphism in LOX Gene on the Risk of Osteosarcoma

Jia, Jie,Tian, Qing,Liu, Yong,Shao, Zeng-Wu,Yang, Shu-Hua Asian Pacific Journal of Cancer Prevention 2013 Asian Pacific journal of cancer prevention Vol.14 No.6

Background: Osteosarcomas have many established risk factors, both genetic and environmental, but by themselves these explain only part of the total cancer incidence. Bisphenol A (BPA) is an environmental estrogen associated with risk of several kinds of tumour. The lysyl oxidase gene (LOX) may also contribute to risk of tumours including osteosarcomas. Here, we investigated possible interactions of BPA and a LOX polymorphism on the risk of osteosarcoma. Method: The present hospital-based case-control study included 106 cancer patients and 112 controls from a Chinese population. Internal burden of BPA exposure was assessed using high-performance liquid chromatography-mass spectrometry (HPLC-MS) method. Genotypes were determined using PCR-RFLP methods. Results: Compared with those in low BPA exposure group, subjects with BPA more than or equal to median value had significant increased risk of osteosarcoma among subjects who carried GC or CC genotypes. A significant interaction with BPA level and the -22G/C polymorphism was observed for osteosarcoma overall, osteosarcoma affecting knee and osteosarcoma affecting hip, as $P_{forinteraction}$ = 0.036 for osteosarcoma overall; $P_{forinteraction}$ = 0.024 for osteosarcoma affecting knee; and $P_{forinteraction}$ = 0.017 for osteosarcoma affecting hip. Conclusions: The results suggest that BPA exposure interacts with the -22G/C polymorphism of the LOX gene to increase the risk of osteosarcoma.

Risk factors and long-term prognosis of BCLC stage 0/A hepatocellular carcinoma for beyond milan recurrence after hepatectomy: A multicenter observational study

Zi-Han FENG,Ming-Da WANG,Li-Yang SUN,Xiao XU,Qing-Yu KONG,Zi-Xiang CHEN,Yong-Yi ZENG,Timothy M. PAWLIK,Wan Yee LAU,Feng SHEN,Zhong CHEN,Tian YANG 한국간담췌외과학회 2022 Annals of hepato-biliary-pancreatic surgery Vol.26 No.-

Expression of lipid metabolism genes provides new insights into intramuscular fat deposition in Laiwu pigs

Wang, Hui,Wang, Jin,Yang, Dan-dan,Liu, Zong-li,Zeng, Yong-qing,Chen, Wei Asian Australasian Association of Animal Productio 2020 Animal Bioscience Vol.33 No.3

Objective: The objective of this study was to measure the special expression pattern of lipid metabolism genes and investigate the molecular mechanisms underlying intramuscular fat (IMF) deposition in Longissimus dorsi muscle of Laiwu pigs. Methods: Thirty-six pigs (Laiwu n = 18; Duroc×Landrace×Yorkshire n = 18) were used for the measurement of the backfat thickness, marbling score, IMF content, and expression of lipid metabolism genes. Results: Significant correlations were found between IMF content and the mRNA expression of lipid metabolism genes. Of the 14 fat deposition genes measured, fatty acid synthase (FASN) showed the strongest correlation (r = 0.75, p = 0.001) with IMF content, and of the 6 fat removal genes, carnitine palmitoyl transferase 1B (CPT1B) exhibited the greatest negative correlation (r = -0.66, p = 0.003) with IMF content in Laiwu pig. Multiple regression analysis showed that CPT1B, FASN, solute carrier family 27 member 1 (SLC27A1), and fatty acid binding protein 3 (FABP3) contributed 38% of the prediction value for IMF content in Laiwu pigs. Of these four variables, CPT1B had the greatest contribution to IMF content (14%) followed by FASN (11%), SLC27A1 (9%), and FABP3 (4%). Conclusion: Our results indicate that the combined effects of an upregulation in fat deposition genes and downregulation in fat removal genes promotes IMF deposition in Laiwu pigs.

Characterization and differential expression of microRNA in skeletal muscle of Laiwu and Yorkshire pig breeds

Wei Chen,Guo-Feng Fang,Shou-Dong Wang,Hui Wang,Yong-Qing Zeng 한국유전학회 2017 Genes & Genomics Vol.39 No.2

The domestic pig is an important agricultural animal used as a source of meat worldwide. As an important resource, Laiwu pig has some special characteristics compared with Yorkshire pig. It is necessary to identify the differentially expressed microRNAs (miRNAs) in skeletal muscle between two pig breeds. Therefore, we herein performed a comprehensive investigation for miRNA transcriptome of skeletal muscle from the two pig breeds. On average, we obtained approximately 13,493,607 clean reads in Laiwu pig and 12,938,257 clean reads in Yorkshire pig in this study. Totally, 265 known miRNAs were identified, and among these, 229 miRNAs were expressed in all the six pigs. From the 265 known miRNAs, 19 of these miRNAs were found significantly differentially expressed (q\0.05). Of these significantly expressed miRNAs, seven miRNAs were significantly upregulated and 12 miRNAs were down-regulated in Laiwu pig compared with Yorkshire pig. Among the significantly expressed miRNAs, ssc-miR-194-5p has the largest foldchange (6.35-fold). Ten differentially expressed miRNAs selected from high throughput sequencing were confirmed by real-time RT-PCR (RT-qPCR), and the results indicated that the expression patterns were consistent with sequencing results. Our results not only present a comprehensive miRNA transcriptome profile of skeletal muscle between two pig breeds with distinct phenotypes, but also could be useful for investigating the functions of differentially expressed miRNAs associated with the phenotype traits.

Comparative Analysis on Antioxidative Ability of Muscle between Laiwu Pig and Large White

Chen, Wei,Zhu, Hong-Lei,Shi, Yuan,Zhao, Meng-Meng,Wang, Hui,Zeng, Yong-Qing Asian Australasian Association of Animal Productio 2012 Animal Bioscience Vol.25 No.8

This study was conducted to evaluate effects of storage temperatures ($4^{\circ}C$ and $20^{\circ}C$) and pig breeds (Laiwu pig and Large White pig) on the main antioxidative enzymes (superoxide dismutase, catalase, and glutathione peroxidase) activity and lipid oxidation in porcine Longissimus dorsi muscle. Activities of antioxidative enzymes (AOE) decreased slightly during storage, regardless of storage temperatures. Muscle antioxidative enzymes activities stored at $4^{\circ}C$ were higher than that stored at $20^{\circ}C$. Laiwu pig's enzymes activities were significantly (p<0.01) higher than Large White's. The level of malondialdehyde is a direct expression of the grade of lipid oxidation in meat. In our study, the malondialdehyde contents increased after 6 days storage. However, malondialdehyde contents of Laiwu pig were significantly (p<0.01) lower than Large White's. A lower content of malondialdehyde corresponds to a lower oxidation of lipids. These results indicated the muscle antioxidative ability of Laiwu pig was higher than Large White pig. It also implied that antioxidative enzymes were involved in the essentials and deciding mechanisms of meat quality by quenching oxygen free radicals and inhibiting lipid oxidation in muscle.

Longissimus lumborum muscle transcriptome analysis of Laiwu and Yorkshire pigs differing in intramuscular fat content

Wei Chen,Guo‑feng Fang,Shou‑dong Wang,Hui Wang,Yong‑qing Zeng 한국유전학회 2017 Genes & Genomics Vol.39 No.7

The pig provides meat products for human consumption on a large scale. High-throughput sequencing technology provides a powerful approach for the characterization of the muscle transcriptome of pigs. Despite many studies using high-throughput technologies, the functional complexity of porcine muscle transcriptome is not fully elucidated. Therefore, the aim of this study was to gain insight into the longissimus lumborum muscle transcriptome between two pig breeds with distinct phenotypes, Laiwu pig, a Chinese indigenous pig breed, and Yorkshire pig, a Western breed. RNA-seq was applied to sample transcripts of the longissimus lumborum muscle between the two pig breeds to gain insights into wholegenome transcription profiles. For the Laiwu and Yorkshire libraries, we obtained a total of 40,498,476 and 59,072,892 high quality reads, respectively. Moreover, the resulting data set provided expression patterns for many annotated, predicted and novel pig genes. 178 significantly differentially expressed genes were identified between the Laiwu and Yorkshire pigs, with 98 up-regulated and 80 downregulated genes in Laiwu pig compared with Yorkshire pig. Gene expression results of the RNA-seq data were validated by qRT-PCR for twelve genes. Genes that were differentially expressed were involved in lipid metabolism and were more highly expressed in Laiwu pigs. The present study provides a comprehensive view of differences in the muscle transcriptome between two pig phenotypes. These results expand our knowledge of the genes transcribed in the skeletal muscle of two breeds and provide a basis for future research of the molecular mechanisms underlying the phenotypic differences on meat quality.

Characteristics of circRNAs expression profiles in the piglets intestine induced by oxidative stress

Li Zhi-xin,Chen Wei,Qin Ming,Wang Li-xue,Zeng Yong-qing 한국유전학회 2022 Genes & Genomics Vol.44 No.4

Backgroud: Oxidative stress (OS) can affect the expression of key genes and destroy the intestinal structure. However, it is unclear how OS regulates the expression of circular RNAs (circRNAs), microRNAs (miRNAs) and mRNAs. Objective: The aim of this study was to examine the expression of circRNAs, miRNAs and mRNAs exposed to OS. Methods: Piglets were exposed to diquat (DQ), a herbicide, and the activity of antioxidant enzymes and the morphology of the intestine were investigated. We utilized whole transcriptome sequencing to examine the global expression of circRNAs, miRNAs and mRNAs in the jejunum. Results: Compared to controls, 751 circRNAs, 731 miRNAs and 164 mRNAs were differentially expressed in diquat-treated piglets. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that oxidative phosphorylation, RNA degradation and ubiquitin-mediated proteolysis were closely associated with OS. Conclusions: Our results indicated that diquat-induced OS alters the intestinal structure, resulting in the differential expression of circRNAs, miRNAs and mRNAs in the jejunum of piglets. Meanwhile, OS weakened the enzyme antioxidant system in serum of piglets. Our results provide a foundation for further studies on the mechanisms involved in the response to OS in the jejunum.

Role of IFNLR1 gene in PRRSV infection of PAM cells

Ming Qin,Wei Chen,Zhixin Lin,Lixue Wang,Lixia Ma,Jinhong Geng,Yu Zhang,Jing Zhao,Yong-Qing Zeng 대한수의학회 2021 Journal of Veterinary Science Vol.22 No.3

Background: Interferon lambda receptor 1 (IFNLR1) is a type II cytokine receptor that clings to interleukins IL-28A, IL29B, and IL-29 referred to as type III IFNs (IFN-λs). IFN-λs act through the JAK-STAT signaling pathway to exert antiviral effects related to preventing and curing an infection. Although the immune function of IFN-λs in virus invasion has been described, the molecular mechanism of IFNLR1 in that process is unclear. Objectives: The purpose of this study was to elucidate the role of IFNLR1 in the pathogenesis and treatment of porcine reproductive and respiratory syndrome virus (PRRSV). Methods: The effects of IFNLR1 on the proliferation of porcine alveolar macrophages (PAMs) during PRRSV infection were investigated using interference and overexpression methods. Results: In this study, the expressions of the IFNLR1 gene in the liver, large intestine, small intestine, kidney, and lung tissues of Dapulian pigs were significantly higher than those in Landrace pigs. It was determined that porcine IFNLR1 overexpression suppresses PRRSV replication. The qRT-PCR results revealed that overexpression of IFNLR1 upregulated antiviral and IFN-stimulated genes. IFNLR1 overexpression inhibits the proliferation of PAMs and upregulation of p-STAT1. By contrast, knockdown of IFNLR1 expression promotes PAMs proliferation. The G0/G1 phase proportion in IFNLR1-overexpressing cells increased, and the opposite change was observed in IFNLR1-underexpressing cells. After inhibition of the JAK/STAT signaling pathway, the G2/M phase proportion in the IFNLR1-overexpressing cells showed a significant increasing trend. In conclusion, overexpression of IFNLR1 induces activation of the JAK/STAT pathway, thereby inhibiting the proliferation of PAMs infected with PRRSV. Conclusion: Expression of the IFNLR1 gene has an important regulatory role in PRRSV-infected PAMs, indicating it has potential as a molecular target in developing a new strategy for the treatment of PRRSV.