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| Effect of the gene silencing of phosphorus transporters on phosphorus absorption across primary cultured duodenal epithelial cell monolayers of chick embryos |
| LI Ting-ting1*, LU Na2*, SHAO Yu-xin2, ZHANG Li-yang2, LU Lin2, LIU Zong-ping3, LUO Xu-gang1, LIAO Xiu-dong2 |
1 Poultry Mineral Nutrition Laboratory, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R.China
2 Mineral Nutrition Research Division, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China
3 College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, P.R.China |
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摘要
本研究旨在确定II b型钠磷协同转运载体（NaP-IIb）和无机磷转运载体2（PiT2）是否直接参与原代培养肉鸡鸡胚十二指肠上皮细胞磷吸收。针对NaP-IIb和PiT2基因设计小干扰RNA（siRNA）序列，合成并转染至原代培养肉鸡鸡胚十二指肠细胞，通过抑制效率分析筛选出对NaP-IIb和PiT2基因干扰有效的siRNAs，用于后续磷吸收试验。转染有效抑制NaP-IIb或PiT2的siRNA至原代培养肉鸡鸡胚十二指肠上皮细胞，待Transwell培养板上的细胞汇合成单层后，将磷转运载体基因（NaP-IIb或PiT2）沉默细胞或未转染细胞在含有0或0.25 mM磷（以KH2PO4形式引入）的吸收培养基中孵育，以检测十二指肠上皮细胞对磷的吸收。结果表明，si-1372和si-890分别为抑制NaP-IIb和PiT2基因表达的有效siRNA。与无磷组相比，添加磷可显著提高（P=0.065）原代培养肉鸡鸡胚十二指肠上皮细胞PiT2蛋白丰度，并增强（P<0.0001）其磷吸收。另外，NaP-IIb沉默显著降低（P=0.07）原代培养肉鸡鸡胚十二指肠上皮细胞磷吸收，但PiT2沉默对其并无影响（P=0.345）。综上所述，NaP-IIb可能直接参与肉鸡十二指肠上皮细胞磷吸收，而PiT2并未直接参与。
Abstract The aim of the study was to investigate whether phosphorus (P) transporters, type IIb sodium-dependent phosphate cotransporter (NaP-IIb) and inorganic phosphate transporter 2 (PiT2), were directly involved in P absorption across primary cultured duodenal epithelial cell monolayers of chick embryos. The siRNAs against NaP-IIb or PiT2 were designed, synthesized and transfected into primary cultured duodenal epithelial cells of chick embryos. Then, the inhibitory efficiency of siRNAs against NaP-IIb or PiT2 was analyzed, and the most efficacious siRNAs were selected to be used for subsequent P absorption experiments. Briefly, primary cultured duodenal epithelial cells of chick embryos were transfected with either NaP-IIb or PiT2 siRNAs and grown in confluent monolayers on transwell plates. The untransfected or transfected cell monolayers were then incubated in an uptake medium containing 0 or 0.25 mmol L–1 of P as KH2PO4 to measure the P absorption across duodenal epithelial cell monolayers. The results showed that among the siRNAs designed, si-1372 and si-890 were demonstrated to be the most effective in inhibiting the NaP-IIb and PiT2 expressions, respectively. Supplemental P increased (P=0.065) the protein abundance of PiT2 and enhanced (P<0.0001) P absorption in primary cultured duodenal epithelial cell of chick embryos. Furthermore, NaP-IIb silencing decreased (P=0.07) P absorption across duodenal epithelial cell monolayers, while PiT2 silencing had no effect (P=0.345). It is concluded that the NaP-IIb, but not PiT2, might be directly involved in the P absorption of chick duodenal epithelial cells.
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Received: 19 February 2021
Accepted: 15 June 2021
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| Fund: This present work was supported by the Key Program of the National Natural Science Foundation of China (31630073), the National Natural Science Foundation of China (31472116), the National Key R&D Program of China (2017YFD0502200), the China Agriculture Research System of MOF and MARA (CARS-41), and the Agricultural Science and Technology Innovation Program (ASTIP-IAS09). |
| About author: LI Ting-ting, E-mail: d150070@yzu.edu.cn; Correspondence LUO Xu-gang, Tel: +86-514-87976732, E-mail: wlysz@263.net; LIAO Xiu-dong, Tel: +86-10-62820866, E-mail: liaoxd56@163.com
* These authors contributed equally to the present study. |
Cite this article:
LI Ting-ting, LU Na, SHAO Yu-xin, ZHANG Li-yang, LU Lin, LIU Zong-ping, LUO Xu-gang, LIAO Xiu-dong.
2022.
Effect of the gene silencing of phosphorus transporters on phosphorus absorption across primary cultured duodenal epithelial cell monolayers of chick embryos. Journal of Integrative Agriculture, 21(7): 2076-2085.
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