[Effect of CRE-dependent RNA interference targeting Notch1 on proliferation of cervical cancer cell line HeLa].
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.


CONCLUSIONS
RNAi of Notch1 mediated by CRE-dependent shRNA expression plasmid can down-regulate intracellular Notch1 signal level and suppress the proliferation of HeLa cells.
Yu
Hai
H
Research Center for Human Gene Therapy, Department of Biochemistry and Molecular Biology, School of Medicine, Shanghai Jiaotong University, Shanghai, PR China.
Huang
Sheng-Lin
SL
Zhao
Xiao-Ping
XP
Lu
Jian
J
Qian
Guan-Xiang
GX
Ge
Sheng-Fang
SF
D049109:Cell Proliferation; D005260:Female; D005822:Genetic Vectors; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D019426:Integrases / Q000235:genetics; D010957:Plasmids; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
C497761:NOTCH1 protein, human; D034741:RNA, Small Interfering; D051881:Receptor, Notch1; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); C045073:Cre recombinase; D019426:Integrases