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Published on: October 27, 2014
Knocking down Wnt9a mRNA levels increases cellular proliferation
Yang Xiang1, Ge Lin, Qianjun Zhang
1Institute of Human Reproduction and Stem Cell Engineering, Central South University, 88 Xiangya Road, Changsha, Hunan, 410078, China. xiangyang397@hotmail.com
Abstract:
Wnts are secreted lipid-modified signaling proteins. Activation of Wnt signalling in many tissues has also been associated with cancer. In many eukaryotes, expression of nuclear-encoded mRNA can be strongly inhibited by the presence of a small double-stranded RNA corresponding to exon sequences in the mRNA. In this study we used pAVU6+27 vectors, which have SalI and XbaI clone sites, to construct the siRNA expression vectors for human Wnt9a. Two kinds of small interfering RNA inserts were designed, synthesized and visually tested for efficacy by in situ hybridization, the results demonstrated that in the cells, transfected with U6+27 cassettes with anti-Wnt9a hairpin siRNA inserts, dramatically reduced Wnt9a signals were observed as compared to the untransfected cells. The results of flow cytometry analysis showed that the cell proliferation was promoted after lowering expression of the human Wnt9a in MCF-7 cells by RNAi, but was inhibited after over-expression of human Wnt9a. These results suggests the expression level of human Wnt9a in MCF-7 that breast cancer may play a role in adjusting the rate of cellular proliferation.
Insights
Researchers used RNA interference (RNAi) to investigate human Wnt9a in breast cancer cells. Lowering Wnt9a expression promoted cell proliferation, while over-expression inhibited it, suggesting Wnt9a
Area of Science:
- Molecular biology
- Cell biology
- Cancer research
Background:
- Wnt signaling proteins are involved in tissue development and are implicated in various cancers.
- RNA interference (RNAi) is a mechanism to inhibit gene expression using small double-stranded RNA.
- Human Wnt9a's role in breast cancer proliferation requires further elucidation.
Purpose of the Study:
- To construct siRNA expression vectors for human Wnt9a.
- To investigate the effect of Wnt9a expression levels on MCF-7 breast cancer cell proliferation.
Main Methods:
- Construction of siRNA expression vectors using pAVU6+27 vectors.
- Synthesis and in situ hybridization of anti-Wnt9a hairpin siRNA inserts.
- Flow cytometry analysis to assess cell proliferation rates.
Main Results:
- Transfection with anti-Wnt9a siRNA significantly reduced Wnt9a signals in cells.
- Reduced Wnt9a expression in MCF-7 cells promoted cellular proliferation.
- Over-expression of Wnt9a inhibited proliferation in MCF-7 cells.
Conclusions:
- Human Wnt9a expression levels influence the proliferation rate of MCF-7 breast cancer cells.
- Wnt9a plays a role in regulating cellular proliferation within breast cancer contexts.
- RNAi is an effective method for modulating Wnt9a expression and studying its cellular effects.
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