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        Relationship between porcine miR-20a and its putative target low-density lipoprotein receptor based on dual luciferase reporter gene assays

        Yueyun Ding,Shujiao Zhu,Chaodong Wu,Li Qian,DengTao Li,Li Wang,Yuanlang Wang,Wei Zhang,Min Yang,Jian Ding,Xudong Wu,Xiao-Dong Zhang,Yafei Gao,Zongjun Yin 아세아·태평양축산학회 2019 Animal Bioscience Vol.32 No.7

        Objective: Mutations in low-density lipoprotein receptor (LDLR), which encodes a critical protein for cholesterol homeostasis and lipid metabolism in mammals, are involved in cardiometabolic diseases, such as familial hypercholesterolemia in pigs. Whereas microRNAs (miRNAs) can control LDLR regulation, their involvement in circulating cholesterol and lipid levels with respect to cardiometabolic diseases in pigs is unclear. We aimed to identify and analyze LDLR as a potential target gene of SSC-miR-20a. Methods: Bioinformatic analysis predicted that porcine LDLR is a target of SSC-miR-20a. Wild-type and mutant LDLR 3′-untranslated region (UTR) fragments were generated by polymerase chain reaction (PCR) and cloned into the pGL3-Control vector to construct pGL3 Control LDLR wild-3′-UTR and pGL3 Control LDLR mutant-3′-UTR recombinant plasmids, respectively. An miR-20a expression plasmid was constructed by inserting the porcine pre-miR-20a-coding sequence between the HindIII and BamHI sites in pMR-mCherry, and constructs were confirmed by sequencing. HEK293T cells were co-transfected with the miR-20a expression or pMR-mCherry control plasmids and constructs harboring the corresponding 3′-UTR, and relative luciferase activity was determined. The relative expression levels of miR-20a and LDLR mRNA and their correlation in terms of expression levels in porcine liver tissue were analyzed using reverse-transcription quantitative PCR. Results: Gel electrophoresis and sequencing showed that target gene fragments were successfully cloned, and the three recombinant vectors were successfully constructed. Compared to pMR-mCherry, the miR-20a expression vector significantly inhibited wild-type LDLR-3′-UTR-driven (p<0.01), but not mutant LDLR-3′-UTR-driven (p>0.05), luciferase reporter activity. Further, miR-20a and LDLR were expressed at relatively high levels in porcine liver tissues. Pearson correlation analysis revealed that porcine liver miR-20a and LDLR levels were significantly negatively correlated (r = –0.656, p<0.05). Conclusion: LDLR is a potential target of miR-20a, which might directly bind the LDLR 3′-UTR to post-transcriptionally inhibit expression. These results have implications in understanding the pathogenesis and progression of porcine cardiovascular diseases.

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        Disrupted Pathways Associated with Neonatal Sepsis: Combination of Protein-protein Interactions and Pathway Data

        Xin Qiao,Silian Zhu,Shujiao Zhang,Hongmei Dong 한국바이오칩학회 2017 BioChip Journal Vol.11 No.1

        To identify disrupted pathways associated with neonatal sepsis, we performed a research based on the combination of protein-protein interactions (PPIs) and pathway data. Firstly, a total of 23,292 genes, 787,896 PPIs and 1,675 human pathways were obtained, respectively. Then, under the threshold value of false discovery rate (FDR)<0.05 and a delta cut-off value >4.36, a total of 986 differentially expressed genes (DEGs) were identified. In the following, by degree centrality for the objective PPI network, 20 hub genes were obtained. Finally, pathway enrichment analysis and randomization tests indicated that pathways of gene expression, immune system and innate immune system were with remarkable significance in neonatal sepsis. Therefore, in the present study, we presented a novel pathway method, and we successfully identified several pathways in neonatal sepsis, which might be underlying indicators in the detection and treatment of neonatal sepsis.

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