Verification Experiments In Vitro On Three Likely Causative Genes Of PSIS Obtained From Whole-Exome Sequencing

Objective:To investigate the verification function in vitro of three likely causative genes of PSIS obtained from Whole-Exome Sequencing(WES),in order to lay the theoretical foundation for pituitary stalk interruption syndrome etiology research in vivo.Methods:The three likely causative genes were screened from one child who was diagnosed with pituitary stalk interruption syndrome combined with central precocious puberty and her parents(treated as control cases,totally three ones)by using WES.After all the siRNAs of three genes were transfected into GH3 cell,every gene was set up in experiment groups,negative control(NC)group and Blank group.The real-time polymerase chain reaction(RT-PCR)was adopted to screen for expressions of mRNA of each gene to determine the maximum knockdown efficiency at first,then western blot(WB)were utilized for secondary screening to determine the optimal gene by detecting the protein levels.Cell counting kit-8(CCK-8)analyzed cell proliferation and morphology,wound healing assay analyzed cell migration,enzyme linked immunosorbent assay(ELISA)analyzed hormone secretion ability,WB analyzed pit-oct-unc class 1 homeobox 1(POU1F1)and homeobox protein prophet of Pit-1(PROP 1),as well.Results:1)the three likely causative genes were division cycle protein 27(CDC27)siRNA,neurofibromatosis type 1(NF1)gene siRNA,ubiquitin specific peptidase 9,X-linked(USP9X),respectively;2)CDC27 could be knocked down by siRNA-117 and siRNA-1968,and the inhibitory efficiency was optimal,while NF1 and UPS9X could only be knocked down at mRNA level but without achieving the knockdown of protein level,thus causing a halt to follow-up experiments;3)CDC27 silencing had no significant effect on the proliferation and morphology of cells(P>0.05);4)CDC27 silencing inhibited cell migration(P<0.05);5)CDC27 silencing inhibited POU1F1 and PROP1 expression significantly both(P<0.05);6)CDC27 silencing inhibited GH and PRL secretion significantly(P<0.05),but no significant effect on TSH(P>0.05).Conclusion:WES could successfully screen three mutation genes from the child,CDC27 silencing suppressed GH3 cell migration,GH and PRL secretion,and down-regulated the expression of POU1F1 and PROP1.