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

Abstract:

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.