Author:
Sun Li,Wan Jun,Sun Bin,Tian Qiuyan,Li Mei,Xu Li-Xiao,Feng Chen-Xi,Tong Xiao,Feng Xing,Yang Xiaofeng,Ding Xin
Abstract
AbstractThe purpose of this study was to identify the target genes of tcon_00044595, elucidate its activation site, and provide novel insights into the pathogenesis and treatment of neonatal hypoxic–ischemic brain damage (HIBD). Through homologous blast analysis, we identified predicted target sequences in the neighboring regions of the long non-coding RNA (lncRNA) tcon_00044595, suggesting that limd1 is its target gene. Starbase was utilized to identify potential candidate microRNAs associated with the lncRNA. The interaction between the candidate microRNAs and limd1 was investigated and validated using various experimental methods including in vitro cell culture, cell transfection, dual fluorescence reporter detection system, and real-time PCR. Homology alignment analysis revealed that the lncRNA tcon_00044595 exhibited a 246 bp homologous sequence at the 3' end of the adjacent limd1 gene, with a conservation rate of 68%. Analysis conducted on Starbase online identified three potential microRNA candidates: miR-3471, miR-883a-5p, and miR-214-3p. Intracellular expression of the limd1 gene was significantly down-regulated upon transfection with miR-3471, while the other two microRNAs did not produce noticeable effects. Luciferase reporter assays identified two interaction sites (UTR-1, UTR-2) between miR-3471 and the limd1 3ʹUTR, with UTR-1 exhibiting a strong influence. Further CCK8 assay indicated a protective role of miR-3471 during low oxygen stroke in HIBD. The potential regulatory relationship between lncRNA (tcon_00044595), miR-3471, and the target gene limd1 suggests their involvement in the occurrence and development of HIBD, providing new insights for investigating the underlying mechanisms and exploring targeted therapeutic approaches for HIBD.
Funder
National Natural Science Foundation of China
Natural Science Foundation of Jiangsu Province
Jiangsu Province's Key Provincial Talents Program
The Training Program Foundation for health talents of Gusu
Science and Technology Program of Suzhou
Publisher
Springer Science and Business Media LLC