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The Functional Analysis of the Three Different Isoforms of Sheep Nuclear Factor I/B
Zhang Xiaofei1, Song He1, Rong Enguang1, Yang Hua2, Yan Xiaohong1, Li Yumao1, Li Hui1, Wang Ning1*
1College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China;
2Institute of Animal Husbandry and Veterinary, Xinjiang Academy of Agriculture and Reclamation Science, Shihezi 832000, China
2Institute of Animal Husbandry and Veterinary, Xinjiang Academy of Agriculture and Reclamation Science, Shihezi 832000, China
Abstract: The nuclear factor I/B (NFIB) is an important transcription factor and plays important roles in cell proliferation， apoptosis and tissue development. Our previous results revealed that sheep NFIB gene produces three different transcript isoforms and three protein isoforms (NFIB-1， NFIB-2 and NFIB-3). NFIB-1， NFIB-2 and NFIB-3 have a molecular weight of 48 kDa， 43 kDa and 39 kDa， respectively. To date， their functional differences are not clear. In the present study， the transcript-specific primers were designed and used to measure the abundance of the three different NFIB transcript isoforms in various sheep tissues using Real-time PCR. To explore their functional differences， the eukaryotic expression vectors of these three different NFIB isoforms were constructed and transfected into HaCAT cells. The CCK-8， Cell Death Detection ELISA and Annexin V-FITC Apoptosis Detection kits were used to investigate their effects on the proliferation and apoptosis of HaCAT cells.The results of tissue expression profiling showed that the three NFIB isoforms (NFIB-1， NFIB-2 and NFIB-3) were expressed in various sheep tissues including skin， fat， kidney， and their tissue expression patterns were different in different tissues. The results of CCK-8 assay showed that compared with the empty vector， NFIB-1 overexpression promoted cell proliferation (P<0.01); NFIB-2 had no obvious effect on cell proliferation， and NFIB-3 inhibited cell proliferation (P<0.01). The Cell Death Detection ELISA and Annexin V-FITC Apoptosis assays showed that overexpression of any one of the three NFIB isoforms could induce HaCAT cell apoptosis to some extent. By comparison， NFIB-1 and NFIB-3 were more potent to induce apoptosis than NFIB-2 (P<0.01). However， there was no significant difference in promoting apoptosis of HaCAT cells between NFIB-1 and NFIB-3. These results suggested that the three different sheep NFIB isoforms played different roles in cell proliferation and apoptosis.
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