Background & objective: Notch signal transduction pathway mediates cell differentiation and proliferation. Its dysfunction is supposed to be involved in tumorigenesis and development. This study was to construct a course recombination enzyme(CRE)-dependent short hairpin RNA (shRNA) expression plasmid targeting Notch1, and investigate its effect on proliferation of cervical cancer cell line HeLa.
Methods: RNA interfering vectors pSico and pSicoR were used to construct CRE-dependent shRNA expression plasmids targeting GAPDH and Notch1; pBS185-CRE was used as an expression vector of CRE. HeLa cells were divided into 4 groups and transfected with pSico, pSico/CRE, pSicoR, and pSicoR/CRE, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were carried out to assess the efficiency of RNA interference (RNAi), and intracellular Notch signal level was tested by CBF-1 reporter plasmid. The proliferation of HeLa cells after CRE-dependent Notch1 RNAi was detected by MTS assay.
Results: After transfection of pSico (R)-GAPDH and pSico (R)-Notch1, CRE-dependent green fluorescent cells were detected; Notch1 expression was inhibited; intracellular Notch1 signal level was decreased; the proliferation of HeLa cells was suppressed.
Conclusion: RNAi of Notch1 mediated by CRE-dependent shRNA expression plasmid can down-regulate intracellular Notch1 signal level and suppress the proliferation of HeLa cells.