Ingenuity pathway analysis of differentially expressed genes involved in signaling pathways and molecular networks in RhoE gene-edited cardiomyocytes

RhoE/Rnd3 is an atypical member of the Rho super-family of proteins, However, the global biological function profile of this protein remains unsolved. In the present study, a RhoE-knockout H9C2 cardiomyocyte cell line was established using CRISPR/Cas9 technology, following which differentially expressed genes (DEGs) between the knockout and wild-type cell lines were screened using whole genome expression gene chips. A total of 829 DEGs, including 417 upregulated and 412 downregulated, were identified using the threshold of fold changes ≥1.2 and P<0.05. Using the ingenuity pathways analysis system with a threshold of -Log (P-value)>2, 67 canonical pathways were found to be enriched. Many of the detected signaling pathways, including that of oncostatin M signaling, were found to be associated with the inflammatory response. Subsequent disease and function analysis indicated that apart from cardiovascular disease and development function, RhoE may also be involved in other diseases and function, including organismal survival, cancer, organismal injury and abnormalities, cell-to-cell signaling and interaction, and molecular transport. In addition, 885 upstream regulators were enriched, including 59 molecules that were predicated to be strongly activated (Z-score >2) and 60 molecules that were predicated to be significantly inhibited (Z-scores <-2). In particular, 33 regulatory effects and 25 networks were revealed to be associated with the DEGs. Among them, the most significant regulatory effects were 'adhesion of endothelial cells' and 'recruitment of myeloid cells' and the top network was 'neurological disease', 'hereditary disorder, organismal injury and abnormalities'. In conclusion, the present study successfully edited the RhoE gene in H9C2 cells using CRISPR/Cas9 technology and subsequently analyzed the enriched DEGs along with their associated canonical signaling pathways, diseases and functions classification, upstream regulatory molecules, regulatory effects and interaction networks. The results of the present study should facilitate the discovery of the global biological and functional properties of RhoE and provide new insights into role of RhoE in human diseases, especially those in the cardiovascular system.