miR-2c Targets Notch2 and FAM166B and Regulates Biomineralization-Associated and Cellular Regulatory Processes in the Pearl Oyster Pinctada fucata martensii

MicroRNA-2c (miR-2c) has previously been implicated in shell formation in the pearl oyster Pinctada fucata martensii, but its broader downstream regulatory network remains unclear. Here, mantle tissues following in vivo miR-2c overexpression (M2, n = 3), inhibition (I2, n = 3), and negative-control treatment (NC, n = 4) were subjected to RNA sequencing. A total of 121, 82, and 242 differentially expressed genes were identified in the NC versus M2, NC versus I2, and I2 versus M2 comparisons, respectively. Functional enrichment of the combined DEG set highlighted biomineralization-associated processes, including chitin and amino-sugar metabolism and extracellular matrix organization, together with broader cellular regulatory pathways. FAM166B and Notch2 were identified as additional direct targets of miR-2c through reciprocal expression responses and wild-type/mutant dual-luciferase assays; a descriptive Western blot pattern for Notch2 was consistent with this regulatory relationship. The previously established LncMPEG1–miR-2c regulatory relationship was also extended to these targets. These findings broaden the known miR-2c regulatory network and identify miR-2c, Notch2, and FAM166B as candidate regulatory loci for future functional and population-level genetic studies. However, these transcriptomic responses should be interpreted within the context of the experimental design, including the use of a single shared tank and the selection of strongly responding individuals for RNA-seq; further replicated experiments are required to determine the generality of these responses.