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Published on: August 12, 2015
[Targeted down-regulation of p53 gene expression by individual antisense RNA in vitro]
Ya-hong Wang1, Yu-lan Sun, Shao-feng Xu
1Department of Breast Pathology and Research, Tianjin Medical University Cancer Hospital, China.
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.
Insights
This study shows that antisense RNA targeting mutant p53 (mt-p53) gene effectively inhibits breast cancer cell proliferation. This specific gene silencing offers a potential therapeutic strategy for human malignancies.
Area of Science:
- Molecular biology
- Cancer genetics
- RNA therapeutics
Context:
- The p53 tumor suppressor gene is frequently mutated in human cancers, including breast cancer.
- Targeting mutant p53 (mt-p53) offers a potential strategy for cancer therapy.
- Developing specific inhibitors for mt-p53 is crucial for effective treatment.
Purpose:
- To investigate the efficacy of individualized antisense RNA in specifically blocking mutant p53 gene expression in vitro.
- To assess the impact of mt-p53 gene blockage on human breast cancer cell proliferation and survival.
Summary:
- Researchers identified a p53 mutation in exon 8 of MDA-MB-231 breast cancer cells.
- An antisense RNA (ASp53exon8'RNA) was designed to target and hybridize with the specific mt-p53 sequence.
- Transfection with ASp53exon8'RNA significantly reduced mt-p53 protein expression and inhibited MDA-MB-231 cell proliferation.
Impact:
- ASp53exon8'RNA demonstrates specific gene silencing of mt-p53, leading to reduced cancer cell proliferation.
- This antisense RNA approach presents a promising therapeutic avenue for treating human malignancies driven by mutant p53.
- Further research into RNA-based therapies targeting specific gene mutations could revolutionize cancer treatment.
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