Mol. Hum. Reprod. Advance Access originally published online on September 3, 2004
Molecular Human Reproduction 2004 10(10):705-711; doi:10.1093/molehr/gah105
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Silencing lamin A/C in human endometrial stromal cells: a model to investigate endometrial gene function and regulation
1Department of Obstetrics and Gynecology, Stanford University, Stanford, CA 94305, USA and 2Department of Obstetrics and Gynecology, Zagreb University School of Medicine, Zagreb 10000, Croatia
3 To whom correspondence should be addressed at: Center for Research on Women's Health and Reproduction, Division of Reproductive Endocrinology and Infertility, Department of Gynecology and Obstetrics, Stanford University, Stanford, CA 94305-5317 USA. Email: giudice{at}stanford.edu
Silencing of a target mRNA by small interfering RNA (siRNA) has emerged as a new and powerful tool to study gene function, and post-transcriptional gene silencing can now be accomplished with 21–23 nucleotide RNA that mediate sequence-specific mRNA degradation. In the current study we employed lamin A/C siRNA to silence lamin A/C expression in cultured human endometrial stromal cells and investigated downstream cellular markers for proof of concept. Human endometrial stromal cells from three subjects were transfected with lamin A/C siRNA or non-silencing fluorescein-labelled siRNA, and flow cytometric analysis revealed 95–98% transfection efficiency after 6 h of treatment. RT–PCR and quantitative RT–PCR were used to measure mRNA degradation of lamin A/C, and 75–88% silencing was observed 48 h post-transfection. Western blotting and immunocytochemistry confirmed corresponding decrease in lamin A/C protein within 48 h of gene silencing. The downstream effect of lamin A/C silencing was investigated by immunocytochemical analysis of the cellular localization of the protein, emerin, an important component of the nuclear lamina and known to be regulated by lamin expression. Marked displacement of emerin from the nuclear lamina to the cytoplasm was observed when lamin A/C was silenced in human endometrial stromal cells, confirming functional silencing of lamin A/C resulting in a nuclear lamina assembly defect. Silencing target mRNA by siRNA in human endometrial stromal cells can be more broadly applied to investigate the function and regulation of other genes in this cell type, and the methodology and data presented herein strongly support the more widespread use of this powerful tool in endometrial biology research.
Key words: emerin/endometrial stromal cells/laminA/C/RNAi
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