Transcriptomic prediction of pig Leg1s functions in HepG2 cells

The newly identified liver-enriched gene 1 (Leg1) encodes a protein with characteristic Domain of Unknown Function 781 (DUF781/LEG1) domain constituting a protein family with only one member. Functional study in zebrafish suggested that Leg1 genes were involved in the liver development, while the platypus MLP homolog that was enriched in mammary gland and milk acts as an anti-bacterial substance. However, no functional study on eutherian Leg1s has been published at present. Thus, we first report here a functional prediction research in a cellular model. As previously reported, eutherian Leg1s could be classified into three paralogous groups. Pig has all three Leg1 genes, while human and mouse only have retained Leg1a. Hence, pig is an ideal model to study the gene function. RNA-seq was then performed by overexpression of pig Leg1s and platypus MLP in the HepG2 cells. Enrichment analysis showed that pLeg1a and pLeg1b might be of little function in the liver cell; however, pLeg1c was probably involved in the ER stress response and protein folding. Additionally, gene set enrichment analysis revealed that platypus MLP has anti-bacterial activity confirming the functional study in the platypus. Therefore, our study, from the transcriptomic perspective of view, concluded that the mammalian Leg1s have different functions in the liver cells due to subfunctionalization of the paralogous genes. Two batches of experiments were conducted in series. In the first batch, the HepG2 cells were transfected with pLeg1a, pLeg1b expression plasmids and an empty vector as control; the second batch involved the experiment using pLeg1c plasmid and the empty vector.