Abstract: Objective To verify the function of three genes obtained from Whole-Exome Sequencing (WES) in vitro, in order to lay the theoretical foundation for pituitary stalk interruption syndrome etiology research. Methods The cell division cycle protein 27 (CDC27) siRNA, neurofibromatosis type 1 (NF1) gene siRNA, ubiquitin specific peptidase 9, X-linked (USP9X) siRNA were transfected into GH3 cells respectively and every gene was set up in experiment group, negative control (NC) group and blank control group. The real-time polymerase chain reaction (RT-PCR) was adopted to screen expressions of mRNA of each gene to determine the maximum knockdown efficiency at first, then western bolt (WB) were utilized for secondary screening to determine the optimal gene at the protein levels. Cell counting kit-8 (CCK-8) was used to detect cell proliferation and morphology, wound healing assay for cell migration, enzyme linked immunosorbent assay (ELISA) for hormone secretion ability, and WB for expression levels of pit-octunc class 1 homeobox 1 (POU1F1) and homeobox protein prophet of Pit-1(PROP1). Results 1) CDC27 could be knocked down by siRNA-117 and siRNA-1968 at both protein and mRNA levels, while NF1 and UPS9X could only be knocked down at mRNA level, thus causing a halt to follow-up experiments; 2) CDC27 silencing had no significant effect on the proliferation and morphology of GH3 cells (P> 0.05); 3) CDC27 silencing inhibited cell migration (P< 0.05); 4) CDC27 silencing inhibited GH and PRL secretion significantly (all P< 0.05), but no significant effect was found on TSH (P> 0.05); 5) CDC27 silencing inhibited both POU1F1 and PROP1 expression significantly (all P< 0.05). Conclusion CDC27 silencing suppresses GH3 cell migration and GH and PRL secretion.