Abstract:

Objective To explore regulation of long non⁃coding RNA（lncRNA）SOS1⁃IT1 on hepatocellular carcinoma（HCC）cells and the underlying molecular mechanism. Methods Human HCC cell line QGY ⁃ 7703 was served as a cell model. CCK⁃8 assay was performed to detect cell viability，and EdU assay was applied to detect DNA replication activity. The potential miRNA binding sites（miRNA response elements，MREs）were predicted by bioinformatics analysis. The eukaryotic expression plasmid pcDNA3. 1（+）was used in ectopic expres⁃ sions of SOS1⁃IT1，SOS1⁃MRE and miR⁃124. The MREs were cloned into the downstream of an enhanced green fluorescent protein（EGFP）coding region to form fluorescent reporter plasmids. Fluorescent reporter gene assay was performed to detect the specific binding and regulation of miR⁃124 to the MREs. Results In human HCC cell line QGY⁃7703，overexpression of lncRNA SOS1⁃IT1 led to increase cell viability［（1.184 ± 0.114）vs.（0.928 ± 0.104），P < 0.05］and DNA replication［（0.625 ± 0.013）vs. 0.206 ± 0.016），P < 0.05］，while suppression of SOS1⁃IT1 resulted in decreased cell viability［（0.648 ± 0.062）vs.（0.870 ± 0.091），P < 0.05］and DNA replication ［（0.126 ± 0.022）vs. 0.271 ± 0.033，P < 0.05］. Bioinformatics analysis showed that SOS1 mRNA contained three and SOS1⁃IT1 contained one potential miR⁃124 reaction elements（MREs）. Fluorescent reporter gene assay indicated that the first two MREs within SOS1 mRNA and the MRE within SOS1⁃IT1 were the functional binding sites of miR ⁃124. QGY ⁃7703 cells were transfected with the fluorescent reporter plasmid containing SOS1⁃MREs，alongwith SOS1⁃IT1⁃MRE expression plasmid，and an increase in fluorescent intensity was observed［（3.68 ± 0.315）vs. （2.71± 0.180），P < 0.05］. Further overexpression of miR⁃124 led to a fallback of the fluorescence intensity［（1.09 ± 0.143）vs.（4.04 ± 0.079），P < 0.05］. A change in fluorescence intensity did not occur if any of the MREs was mutated［（2.57 ± 0.244）vs.（2.71 ± 0.180）and（2.66 ± 0.20）vs.（2.88 ± 0.169），P > 0.05］. Conclusions lncRNA SOS1⁃ IT1 competitively binds endogenous miR ⁃124 with its host gene SOS1. SOS1⁃ IT1 promotes SOS1 expression and enhances HCC cell viability and DNA replication by the competing endogenous RNA（ceRNA）mechanism.

Key words:

hepatocellular carcinoma, long non ?coding RNA, microRNA, competing endogenous RNA, intron