Institute of Oncologic Pathology, Department of Biochemistry and Molecular Biology, Shantou University Medical College, 22 Xinling Road, Shantou, 515041, Guangdong, China.
Department of Pathology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, 510000, Guangdong, China.
Amino Acids. 2021 Aug;53(8):1197-1209. doi: 10.1007/s00726-021-03025-4. Epub 2021 Jul 5.
Riboflavin is an essential micronutrient for normal cellular growth and function. Lack of dietary riboflavin is associated with an increased risk for esophageal squamous cell carcinoma (ESCC). Previous studies have identified that the human riboflavin transporter SLC52A3a isoform (encoded by SLC52A3) plays a prominent role in esophageal cancer cell riboflavin transportation. Furthermore, SLC52A3 gene single nucleotide polymorphisms rs3746804 (T>C, L267P) and rs3746803 (C >T, T278M) are associated with ESCC risk. However, whether SLC52A3a (p.L267P) and (p.T278M) act in riboflavin transportation in esophageal cancer cell remains inconclusive. Here, we constructed the full-length SLC52A3a protein fused to green fluorescent protein (GFP-SLC52A3a-WT and mutants L267P, T278M, and L267P/T278M). It was confirmed by immunofluorescence-based confocal microscopy that SLC52A3a-WT, L267P, T278M, and L267P/T278M expressed in cell membrane, as well as in a variety of intracellular punctate structures. The live cell confocal imaging showed that SLC52A3a-L267P and L267P/T278M increased the intracellular trafficking of SLC52A3a in ESCC cells. Fluorescence recovery after photobleaching of GFP-tagged SLC52A3a meant that intracellular trafficking of SLC52A3a-L267P and L267P/T278M was rapid dynamics process, leading to its stronger ability to transport riboflavin. Taken together, the above results indicated that the rs3746804 (p.L267P) polymorphism promoted intracellular trafficking of SLC52A3a and riboflavin transportation in ESCC cells.
核黄素是正常细胞生长和功能所必需的微量营养素。饮食中缺乏核黄素会增加患食管鳞状细胞癌（ESCC）的风险。先前的研究已经确定，人类核黄素转运蛋白 SLC52A3a 同工型（由 SLC52A3 编码）在食管癌细胞的核黄素转运中起着重要作用。此外，SLC52A3 基因单核苷酸多态性 rs3746804（T>C，L267P）和 rs3746803（C>T，T278M）与 ESCC 风险相关。然而，SLC52A3a（p.L267P）和（p.T278M）是否在食管癌细胞中的核黄素转运中起作用仍不确定。在这里，我们构建了全长 SLC52A3a 蛋白与绿色荧光蛋白（GFP-SLC52A3a-WT 和突变体 L267P、T278M 和 L267P/T278M）融合。通过基于免疫荧光的共焦显微镜证实，SLC52A3a-WT、L267P、T278M 和 L267P/T278M 在细胞膜上以及各种细胞内点状结构中表达。活细胞共焦成像显示 SLC52A3a-L267P 和 L267P/T278M 增加了 ESCC 细胞中 SLC52A3a 的细胞内转运。GFP 标记的 SLC52A3a 的光漂白后荧光恢复意味着 SLC52A3a-L267P 和 L267P/T278M 的细胞内转运是快速动力学过程，导致其更强的转运核黄素能力。综上所述，上述结果表明 rs3746804（p.L267P）多态性促进了 SLC52A3a 的细胞内转运和 ESCC 细胞中的核黄素转运。
