Zhang Ni-Ni, Guo Hong-Wei, Lin Yan, Zhang Wei, Zhang Wei, Wang Bao-Xi, Jiang Xun
Department of Pediatrics, Tangdu Hospital, Air Force Military Medical University/The Fourth Military Medical University, Xi'an 710038, China.
Zhongguo Dang Dai Er Ke Za Zhi. 2019 Nov;21(11):1131-1137. doi: 10.7499/j.issn.1008-8830.2019.11.014.
To establish a congenital chloride diarrhea (CCD)-associated SLC26A3 c.392C>G (p.P131R) polymorphism-expressing cell model, and to investigate its biological function.
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.
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).
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.
建立先天性氯化物腹泻（CCD）相关的SLC26A3基因c.392C>G（p.P131R）多态性表达细胞模型，并研究其生物学功能。
利用GenBank中SLC26A3基因序列设计能特异性识别SLC26A3基因392位点的上下游单导向RNA（sgRNA），酶切后与pSpCas9-puro载体混合构建真核重组表达质粒（pSpCas9-SLC26A3）。将重组质粒和合成的单链DNA寡核苷酸（ssODNs）转染Caco-2细胞，采用Taqman基因分型检测法和Sanger测序法鉴定SLC26A3基因c.392C>G（p.P131R）在Caco-2细胞中的表达情况。选取野生型Caco-2细胞作为正常对照组，将成功表达SLC26A3基因c.392C>G（p.P131R）的Caco-2细胞作为P131R组。两组均用100 ng/mL肿瘤坏死因子-α（TNF-α）处理，正常对照组命名为TNF-α组，P131R组命名为TNF-α+P131R组。采用电阻抗细胞传感（ECIS）检测法评估上述四组肠上皮细胞单层屏障功能的变化，采用蛋白质免疫印迹法检测正常对照组和P131R组中SLC26A3蛋白表达的变化。
成功构建真核重组表达质粒（pSpCas9-SLC26A3）。Taqman基因分型检测法和Sanger测序法均证实成功建立了SLC26A3基因c.392C>G（p.P131R）表达的Caco-2细胞模型。ECIS检测法显示，与正常对照组相比，P131R组肠上皮细胞单层通透性显著增加（P<0.05），同时，100 ng/mL TNF-α诱导后，P131R组细胞膜通透性增加更显著（P<0.05）。蛋白质免疫印迹法显示，与正常对照组相比，P131R组SLC26A3蛋白表达显著降低（P=0.001）。
SLC26A3基因c.392C>G（p.P131R）可降低SLC26A3蛋白表达，增加肠上皮细胞单层通透性，从而导致腹泻。
