[miR-497 suppresses proliferation of human cervical carcinoma HeLa cells by targeting cyclin E1].

OBJECTIVE

To evaluate the effect of miR-497 on proliferation of human cervical carcinoma HeLa cells and target relationship between miR-497 and cyclin E1 (CCNE1).

METHODS

Pre-miR-497 sequences were synthesized and cloned into pcDNATM6.2-GW to construct recombinant plasmid pcDNATM6.2-GW-pre-miR-497 and identified by real-time quantitative PCR (qRT-PCR). In addition, sequences of the wild-type CCNE1 (WT-CCNE1) and mutant CCNE1 (MT-CCNE1) were respectively cloned into pmirGLO vectors. MTT assay was used to explore the impact of miR-497 on the proliferation of HeLa cells. Furthermore, the target effect of miR-497 on the CCNE1 was identified by dual-luciferase reporter assay system, qRT-PCR and Western blotting.

RESULTS

The recombinant plasmids pcDNATM6.2-GW-pre-miR-497 and pmirGLO-WT-CCNE1, pmirGLO-MT-CCNE1 were successfully constructed, and the miR-497 expression level in HeLa cells transfected with pre-miR-497 was significantly higher than that in the neg-miR group (P<0.05). MTT assay showed that miR-497 could significantly inhibit the proliferation of HeLa cells (P<0.05). A remarkable reduction of luciferase activities of WT-CCNE1 reporter was observed in HeLa cells with pre-miR-497 transfection (P<0.01), and the mRNA and protein expression levels of CCNE1 were down-regulated in HeLa cells transfected with pre-miR-497 (P<0.05).

CONCLUSION

Over-expressed miR-497 in HeLa cells could suppress cell proliferation by targeting CCNE1.