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.
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.
Authors: Miguel Aste-Amézaga; Ningyan Zhang; Janet E Lineberger; Beth A Arnold; Timothy J Toner; Mingcheng Gu; Lingyi Huang; Salvatore Vitelli; Kim T Vo; Peter Haytko; Jing Zhang Zhao; Frederic Baleydier; Sarah L'Heureux; Hongfang Wang; Wendy R Gordon; Elizabeth Thoryk; Marie Blanke Andrawes; Kittichoat Tiyanont; Kimberly Stegmaier; Giovanni Roti; Kenneth N Ross; Laura L Franlin; Hui Wang; Fubao Wang; Michael Chastain; Andrew J Bett; Laurent P Audoly; Jon C Aster; Stephen C Blacklow; Hans E Huber Journal: PLoS One Date: 2010-02-08 Impact factor: 3.240