[Targeted down-regulation of p53 gene expression by individual antisense RNA in vitro].
OBJECTIVE
To investigate the effect of specific blockage of mutant p53 gene by individualized antisense RNA in vitro.


METHODS
Mutation status of p53 in human breast cancer cell lines was determined by immunocytochemical staining, PCR-SSCP and sequencing. Single strand antisense transcription system targeting specific p53 mutation site (mt-p53) was constructed, and corresponding antisense RNA was prepared. The hybridization of antisense RNA with its corresponding mt-p53 gene was confirmed by in-situ hybridization. Human breast cancer cells were transfected with antisense RNA by cationic liposome-mediated method. Time course of effects of antisense RNA was investigated by immunocytochemical staining and cell growth inhibiting assay. Expression of mt-p53 protein was examined by Western blot. Cell proliferation was evaluated by MTT assay and cell cycle distribution was determined by flow cytometry (FCM). Apoptosis was determined by TUNEL assay.


RESULTS
Mutation of p53 exon 8 was found in MDA-MB-231 cells and antisense transcription system (pGEM3zf (+/-) p53exon8) was then constructed successfully. In transfected MDA-MB-231 cells, hybridization signals were observed in cytoplasm. Fourth-eight hours after transfection, the antisense RNA (ASp53exon8'RNA) had a significant retarding effect on p53 related proliferation inhibition, along with a decrease of p53 protein expression.


CONCLUSIONS
ASp53exon8'RNA specifically blocks mt-p53 gene expression, resulting in an inhibition of MDA-MB-231 cell proliferation. Such an approach may be used as a therapeutic option against human malignancy.
Wang
Ya-hong
YH
Department of Breast Pathology and Research, Tianjin Medical University Cancer Hospital, China.
Sun
Yu-lan
YL
Xu
Shao-feng
SF
Zhang
Yuan-yuan
YY
Zhang
Lin
L
Zhang
Bin
B
Feng
Yu-mei
YM
Niu
Rui-fang
RF
Fu
Li
L
D017209:Apoptosis; D001483:Base Sequence; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003062:Codon; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D016372:RNA, Antisense; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism
D003062:Codon; D016372:RNA, Antisense; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53