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.
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.
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).
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.
目的: 建立先天性失氯性腹泻（CCD）相关SLC26A3 c.392C > G（p.P131R）突变体细胞模型并初步研究其生物学功能。
方法: 根据GenBank中SLC26A3基因序列，设计特异性识别SLC26A3基因392位点的上下游单导RNA（sgRNA），与酶切后的pSpCas9-puro载体混合构建真核重组表达质粒（pSpCas9-SLC26A3）。将重组质粒和供体DNA模板单链DNA寡核苷酸（ssODN）共转染至Caco-2细胞，采用Taqman基因型分析和Sanger测序鉴定SLC26A3 c.392C > G（p.P131R）在Caco-2细胞中的表达。以野生型Caco-2细胞为正常对照组，以成功表达SLC26A3 c.392C > G（p.P131R）的Caco-2细胞为P131R组，两组细胞分别经100 ng/mL TNF-α诱导并命名为TNF-α组和TNF-α+P131R组，通过电-细胞-基质阻抗传感（ECIS）分析检测上述4组肠上皮细胞单层屏障功能的变化；通过Western blot检测正常对照组和P131R组细胞SLC26A3蛋白的表达变化。
结果: 成功构建了真核重组表达质粒（pSpCas9-SLC26A3）。Taqman基因型分析和Sanger测序均验证SLC26A3 c.392C > G（p.P131R）表达的Caco-2细胞模型构建成功。ECIS分析显示：与正常对照组相比，P131R组肠上皮细胞单层通透性明显增加（P < 0.05）；同时P131R组细胞经TNF-α诱导后的细胞膜通透性增加更为显著（P < 0.05）。Western blot结果显示：与正常对照组相比，P131R组细胞SLC26A3蛋白的相对表达水平显著降低（P=0.001）。
结论: SLC26A3 c.392C > G（p.P131R）可引起SLC26A3蛋白表达下降，从而增加肠上皮细胞单层通透性，引起相关腹泻的发生。