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先天性塩化物下痢症関連SLC26A3 c.392C>G (p.P131R)多型発現細胞モデルの確立とその作用機序の予備的解析
[Establishment of a congenital chloride diarrhea-associated SLC26A3 c.392C>G (p.P131R) polymorphism-expressing cell model and a preliminary analysis of its mechanism of action].
PMID: 31753097
抄録
目的:
先天性塩化物下痢症（CCD）関連SLC26A3 c.392C>G（p.P131R）多型発現細胞モデルを確立し、その生物学的機能を調べる。
OBJECTIVE: To establish a congenital chloride diarrhea (CCD)-associated SLC26A3 c.392C>G (p.P131R) polymorphism-expressing cell model, and to investigate its biological function.
方法:
GenBankにあるSLC26A3遺伝子の配列を用いて、SLC26A3遺伝子の392遺伝子座を特異的に認識できる上流および下流のシングルガイドRNA（sgRNA）を設計し、このsgRNAを酵素消化後にpSpCas9-puroベクターと混合して真核生物組換え発現プラスミド（pSpCas9-SLC26A3）を構築した。Caco-2細胞を組換えプラスミドと合成した一本鎖DNAオリゴヌクレオチド(ssODN)でトランスフェクトし、TaqmanジェノタイピングアッセイとSangerシークエンシングを用いて、Caco-2細胞におけるSLC26A3 c.392C>G(p.P131R)の発現を同定した。野生型Caco-2細胞を正常対照群とし、SLC26A3 c.392C>G (p.P131R)の発現に成功したCaco-2細胞をP131R群とした。両群とも100ng/mLの腫瘍壊死因子-α（TNF-α）で処理した後、正常対照群をTNF-α群、P131R群をTNF-α＋P131R群と命名した。また、上記４群の腸管上皮細胞の単層バリア機能の変化を電気細胞基質インピーダンスセンシング（ECIS）アッセイで評価し、正常対照群とP131R群のSLC26A3タンパク質発現量の変化をウエスタンブロットで測定した。
METHODS: The sequence of the SLC26A3 gene in GenBank was used to design the upstream and downstream single-guide RNA (sgRNA) that could specifically recognize the 392 locus of the SLC26A3 gene, and the sgRNA was mixed with the pSpCas9-puro vector after enzyme digestion to construct an eukaryotic recombinant expression plasmid (pSpCas9-SLC26A3). Caco-2 cells were transfected with the recombinant plasmid and synthesized single-stranded DNA oligonucleotides (ssODNs), and Taqman genotyping assay and Sanger sequencing were used to identify the expression of SLC26A3 c.392C>G (p.P131R) in Caco-2 cells. Wild-type Caco-2 cells were selected as normal control group and the Caco-2 cells with successful expression of SLC26A3 c.392C>G (p.P131R) was selected as P131R group. Both groups were treated with 100 ng/mL tumor necrosis factor-α (TNF-α), and then the normal control group was named as TNF-α group, and the P131R group was named as TNF-α+P131R group. Electric cell-substrate impedance sensing (ECIS) assay was used to evaluate the change in the monolayer barrier function of intestinal epithelial cells in the above four groups, and Western blot was used to measure the change in the expression of SLC26A3 protein in the normal control group and the P131R group.
結果:
真核生物組換え発現プラスミド（pSpCas9-SLC26A3）の構築に成功した。TaqmanジェノタイピングアッセイとSangerシークエンシングの両方で、SLC26A3 c.392C>G（p.P131R）発現のCaco-2細胞モデルの確立に成功したことを確認した。ECISアッセイの結果、P131R群は正常対照群に比べて腸管上皮細胞の単層透過性が有意に増加し（P＜0.05）、同時に100ng/mL TNF-αによる誘導後の細胞膜透過性が有意に増加した（P＜0.05）ことが示された。ウェスタンブロットの結果、P131R群は正常対照群と比較して、SLC26A3タンパク質の発現が有意に低下していた（P=0.001）。
RESULTS: The eukaryotic recombinant expression plasmid (pSpCas9-SLC26A3) was successfully constructed. Both Taqman genotyping assay and Sanger sequencing confirmed the successful establishment of the Caco-2 cell model of SLC26A3 c.392C>G (p.P131R) expression. ECIS assay showed that compared with the normal control group, the P131R group had a significant increase in the monolayer permeability of intestinal epithelial cells (P<0.05), and at the same time, the P131R group had a significantly greater increase in cell membrane permeability after the induction with 100 ng/mL TNF-α (P<0.05). Western blot showed that compared with the normal control group, the P131R group had a significant reduction in the expression of SLC26A3 protein (P=0.001).
結論:
SLC26A3 c.392C>G (p.P131R)は、SLC26A3タンパク質の発現を低下させ、腸管上皮細胞の単層透過性を増加させ、下痢を引き起こす可能性があります。
CONCLUSIONS: SLC26A3 c.392C>G (p.P131R) can reduce the expression of SLC26A3 protein, increase the monolayer permeability of intestinal epithelial cells, and thus lead to diarrhea.