Abstract
We investigated the effects of RNA interference-mediated silencing of the c-myc gene on celluar proliferation and apoptosis in human colon cancer HT-29 cells in vitro and in vivo. A small interfering RNA (siRNA) targeting c-myc was designed, the DNA template was synthesized, and the siRNA was obtained by in vitro transcription. After siRNA transfection into HT-29 and human neuroblastoma IMR-32 cells with Lipofectamine 2000™, the proliferation of the HT-29 and IMR-32 cells was assessed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetry, and Hoechst 33258 staining was used to observe cell apoptosis. Following gene transfer to HT-29 cells, the expression of c-myc mRNA was examined via reverse transcription polymerase chain reaction, and the level of the protein via Western blot assay. Growth curves were constructed and in vivo experiments were performed on nude mice to assess the effects of c-myc silencing on tumor growth. The c-myc expression in the tumor tissue was measured by reverse transcription polymerase chain reaction and subsequently by immunohistochemistry. Our paper demonstrates that the delivery of siRNA directed against c-myc not only efficiently down-regulated the expression of c-myc, inhibited the proliferation of HT-29 cells and induced apoptosis in vitro, but also suppressed the growth of colon cancer cells in vivo.
Key words: c-myc, Cell proliferation, HT-29, siRNA
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Abbreviations used
- DMEM
Dulbecco’s modified Eagle’s medium
- FBS
fetal bovine serum
- GAPDH
glyceraldehyde-3-phosphate dehydrogenase
- MTT
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
- PBS
phosphate-buffered saline
- RT-PCR
reverse transcription polymerase chain reaction
- SDS
sodium dodecyl sulphate
- siRNA
short interfering RNA
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