Small RNA deep sequencing revealed microRNAs’ involvement in modulating cellular senescence and immortalization state

Unlike rodent cells, spontaneous immortalization of avian cells and human cells is a very rare event. According to patent publications and current literature, there are no more than 4 spontaneously immortalized chicken embryo fibroblast (CEF) cell lines established up to date. One of those cell lines is ADOL (Avian Disease and Oncology Laboratory) ZS-1 cell line, which was established by continuous passaging of the CEFs derived from the specific pathogen free (SPF) 0.TVB*S1 (commonly known as rapid feathering susceptible or RFS) genetic line of chickens. The RFS genetic line of chickens was developed and has been maintained on the SPF chicken farm of USDA-ARS facility, ADOL, in East Lansing, Michigan, which is known as one of a few lines of chickens that are free of any known avian endogenous virus genes. To explore potential roles that epigenetic factors may play in modulating cellular senescence processes and spontaneous immortalization state, total RNAs extracted from samples of the RFS primary CEFs, RFS CEFs reached the 21st passage, and the ZS-1 cells were subjected to small RNA sequencing. Collectively, a total of 531 miRNAs was identified in the 3 types of samples. In contrast to the primary CEF samples, 50 miRNAs were identified with significantly differential expression only in the 21st passage samples; a different subset of 63 differentially expressed miRNAs was identified only in the ZS-1 samples; the majority of differentially expressed miRNAs identified in both the 21st passage CEF and the ZS-1 samples were more or less directionally consistent. Gene Ontology analysis results suggested that the epigenetic factor, miRNAs, plays a role in modulating the cellular senescence and spontaneous immortalization processes through various bioprocesses and key pathways including ErbB and MAPK signaling pathways. These findings provided the experimental and bioinformatic evidence for a better understanding on the epigenetic factor of miRNAs in association with cellular senescence and spontaneous immortalization process in avian cells.