The RNA sequencing analysis of corylin ameliorating cellular senescence in HUVECs

Purpose: To understand the mechanism by which corylin ameliorates cellular senescence in HUVECs, we used RNA sequencing to analyze transcriptome differences. Methods: RNA was collected from HUVECs and extracted by TRIzol (T9424, Sigma, USA). The extracted RNA samples were sent to Genomics (Taipei, Taiwan) for analysis, and a library was constructed. Briefly, after quality control of raw reads, mRNA was purified using reverse transcriptase and a random primer to synthesize single-strand cDNA, and dUTP was used in place of dTTP to generate double-stranded cDNA. A single ‘A’ nucleotide was added to the 3’ end of ds cDNAs. Then, multiple indexing adapters were ligated to the 5’ and 3’ ends of ds cDNA. PCR was used to selectively amplify the DNA fragments with adapters on both ends. The library was validated on an Agilent 2100 Bioanalyzer and Real-Time PCR System. Result: We found 433 shared transcripts between S/Y and S+C/S. By comparing the KEGG pathway annotation, we found shared pathways between the two groups associated with cellular senescence, the cell cycle, DNA replication, and the p53 signaling pathway. We next decoded the transcriptome differences in those pathways. Numerous transcripts were shifted in S/Y, such as a reduction in cyclin protein, replication factor, DNA helicase, cell proliferation signals, and an increase in SASP, indicating that HUVECs are in the senescent state. Furthermore, we found that the transcriptome pattern of senescent cells treated with corylin was similar to that of young cells. Most importantly, we found that eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) downregulated and Rho upregulated with corylin treatment, which are the downstream mTOR1 and mTOR2 pathways respectively. Additionally, we found that corylin decreased mTOR1 and p70S6k phosphorylation in U2OS cells . Taken together, these data reveal that corylin ameliorates cellular senescence. Conclusions: By comparing the KEGG pathway annotation, we found shared pathways between the two groups associated with cellular senescence, the cell cycle, DNA replication, and the p53 signaling pathway. We next decoded the transcriptome differences in those pathways. Numerous transcripts were shifted in S/Y, such as a reduction in cyclin protein, replication factor, DNA helicase, cell proliferation signals, and an increase in SASP, indicating that HUVECs are in the senescent state. RNA sequencing to analyze transcriptome differences in three different groups: proliferating cells (Y), senescent cells (S), senescent cells with corylin treatment (S+C)