MicroRNA-regulated flounder CLDN4 functions in anti-bacterial immunity

CLDN4 belongs to a multi-transmembrane protein family of claudins, which mainly functions in cell-cell adhesion and migration. MicroRNAs (miRNAs) are important post-transcriptional regulating factors that participate in broad biological process including immunity. Through high-throughput RNA sequencing strategy, a flounder miRNA, miR-29-x, was identified to be responsible to both bacteria and virus. In this study, we explored the regulatory mechanism and function of miR-29-x and its target gene of flounder CLDN4 (named PoCLDN4). We proved that miR-29-x could interact with the 3'UTR of PoCLDN4 and negatively regulate its expression. PoCLDN4 located on cell membrane, while the depletion of extracellular loop E2 abolished the membrane localization of this protein. E3 could bind different bacteria, and mutation of the amino acids of ¹³E and ¹⁸E enhanced this capacity, while mutation of ¹⁰L abolish this capacity. Further study revealed the bacteria killing effect of E3 and verified ¹⁰L as a key factor. These results identified the interaction between miR-29-x and PoCLDN4, and unraveled the function as well as the molecular basis of flounder CLDN4 in anti-bacterial immunity.