GATA3 gene expression is significantly lower in children with a combination of VSD and pulmonary arterial hypertension than in those with PAH, so low GATA3 gene expression may be associated with VSD occurrence. The hsa-miR-222-3p miRNA is involved in the regulation of the target gene ZFPM2, also known as transcription factor GATA4, another member of the GATA family. FOG2 is essential in heart development and can specifically interact with the GATA4 gene, inhibiting GATA4-dependent transcription. FOG2 knockout mice die from heart defects on day E13.5 of the embryonic period, hence FOG2 may be associated with the occurrence of VSD. Another of the four target genes predicted in our study, HAND1, is an important member of the HAND family. As a transcription factor regulating heart development, HAND2 is closely associated with ventricular muscle cell differentiation, the conduction bundle, the cardiac outflow tract, and epicardial and As a new research area, miRNA regulatory mechanisms need to be explored Spirosendan further. It is Gomisin-N generally considered that miRNA regulates gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Interestingly lower expression of NOTCH1 and GATA3 is associated with VSD and yet we report reduced miRNA expression, which would in a simplistic model be expected to increase expression of these genes. It has also been suggested that miRNA may also function to induce gene expression for example, miR-373 and its premature hairpin, induced E-cadherin and CSDC2 expression by targeting specific sites in gene promoters. In that case the target genes would not have been identified in this study. While these miRNA target genes suggest that the miRNAs may be involved in the development of VSD, it is worth considering that their altered levels may also be related to secondary consequences of the VSD. This is suggested because VSDs develop early in the embryo, so the subjects of this study with an average age around one year will already have developed their VSD. Nevertheless, whatever the reason for their altered levels in the VSD patients these miRNAs may prove important as diagnostic markers for VSD.