Cyclin D1 Gene Silencing by siRNA in Ex Vivo Human Tissue Cultures
Ornella Piazza1, Ilaria Russo, Sabrina Bochicchio
1Department of Medicine, Salerno University, 84081 Baronissi, Via Allende, Italy.
Background:
Short interfering RNAs (siRNAs) are double-stranded RNA molecules able to specifically targeting genes products responsible for human diseases. Cyclin D1 (CyD1) is a cell cycleregulatory molecule, up-regulated at sites of inflammation in several tissues. CyD1 is a very interesting potential target in lung and colon inflammatory diseases.
Objective:
The aim of this paper was testing CyD1 expression in human lung and colon tissues after the application of an inflammatory stimulus, and verifying its gene silencing by using siRNA for CyD1 (siCyD1).
Method:
Colon and pulmonary biopsies were treated with siCyD1 by using two different transfection carriers: a) invivofectamine and b) ad hoc produced nanoliposomes. After 24 hours of incubation with nanoliposomes encapsulating siRNA or invivofectamine-CyD1siRNA, in presence or absence of ECLPS, we analysed the protein expression of CyD1 through Western-Blotting.
Results:
After EC-LPS treatment, in both colon and pulmonary biopsies, an overexpression of CyD1was found (about 64% and 40% respectively). Invivofectamine-CyD1 siRNA reduced the expression of CyD1 approximately by 46% compared to the basal condition, and by around 65% compared to EC-LPS treated colon samples. In lung, following in vivo fectamine siRNA silencing in the presence of EC-LPS, no reduction was observed. Ad hoc nanoliposomes were able to enter colon and lung tissues, but CyD1 silencing was reported in 2 colon samples out of 4 and no efficacy was demonstrated in the only lung sample we studied.
Conclusion:
The silencing of Cyclin D1 expression in vitro "organ culture" model is possible. Our preliminary results encourage further investigations, using different siRNA concentrations delivered by nanoliposomes.
Insights
Short interfering RNA (siRNA) successfully silenced Cyclin D1 (CyD1) in colon and lung tissues, demonstrating potential for treating inflammatory diseases. Nanoliposomes showed promise, warranting further investigation for effective delivery.
Area of Science:
- Molecular Biology
- RNA Interference
- Inflammation Research
Background:
- Short interfering RNAs (siRNAs) target disease-related genes.
- Cyclin D1 (CyD1), a cell cycle regulator, is upregulated in inflammation.
- CyD1 is a potential therapeutic target for inflammatory lung and colon diseases.
Purpose of the Study:
- To assess CyD1 expression in human lung and colon tissues post-inflammation.
- To evaluate gene silencing of CyD1 using siRNA (siCyD1).
Main Methods:
- Human colon and pulmonary biopsies were treated with siCyD1 via invivofectamine or nanoliposomes.
- Epithelial cell-derived lipopolysaccharide (EC-LPS) induced inflammation.
- Western-Blotting analyzed CyD1 protein expression.
Main Results:
- EC-LPS treatment overexpressed CyD1 in colon (64%) and lung (40%) tissues.
- Invivofectamine-siCyD1 reduced CyD1 by ~46% in basal conditions and ~65% in colon.
- Nanoliposomes showed partial CyD1 silencing in colon but not in lung tissue.
Conclusions:
- Cyclin D1 gene silencing is achievable in an in vitro organ culture model.
- Preliminary findings support further research into nanoliposome-delivered siRNAs for CyD1.
- Optimizing siRNA concentration and nanoliposome formulations is recommended.
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