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
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Abstract |
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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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Introduction |
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Gene silencing though RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing (PTGS) mechanism that provides a new approach to study gene function. Initially, RNAi was demonstrated to result in gene knockdown in plants (Dalmay et al., 2000
; Wesley et al., 2001), fungi (Catalanotto et al., 2000) and Caenorhabditis elegans (Fire et al., 1998; Montgomery et al., 1998; Bosher et al., 1999), through introduction of long double-stranded RNA (dsRNA) homologous to the gene of interest. However, until recently, mammalian cells were not susceptible to specific gene silencing because introduction of long dsRNA (>30 nucleotides) resulted in activation of the RNA-dependent protein kinase (PKR) pathway leading to global blockage of mRNA degradation (Williams, 1997; Bass, 2000). The discovery that target mRNA is cleaved in 21–23 nucleotide intervals (Elbashir et al., 2001b) that evade the activation of the PKR pathway led to the successful post-transcriptional gene silencing in mammalian cells. The hypothesis that the 21–23 nucleotide RNA are the mediators of sequence-specific mRNA degradation was proven by the fact that chemically synthesized 21 nucleotide duplexes are capable of guiding target RNA cleavage. This discovery rendered RNA interference a powerful research tool for inducing gene silencing and studying gene function.
In assessing gene silencing, evaluation of a downstream event is an important endpoint to verify gene knockdown. The lamin/emerin system provides an opportunity to evaluate this mechanism in cultured human endometrial stromal cells. Lamins are nuclear filament-type proteins that are major components of the nuclear lamina (Fawcett, 1966). They play an important role in organizing nuclear pore complexes and nuclear envelope assembly. They are associated with numerous proteins in the inner nuclear membrane, including emerin (Clements et al., 2000; Moir et al., 2000; Lopez-Soler et al., 2001; Holaska et al., 2002; Shumaker et al., 2003). There are two types of nuclear lamins, A and B, and in adult mammalian somatic cells there are two major A-type lamins (LMNA): lamin A and lamin C. All mammalian A-type lamins are encoded by a single gene and are splice variants of the same primary transcript (Fisher et al., 1986; McKeon et al., 1986). Lamins A and C are identical for the first 566 amino acids, after which their sequences diverge. Lamin A is a 74 kDa protein and lamin C is 65 kDa protein. Lamin A/C association with the nuclear envelope protein emerin and its importance in nuclear organization have been shown in mouse knockout models. Beyond their structural roles, lamins have essential functions in nuclear activities, such as DNA synthesis, replication, transcription and apoptosis (Maniotis et al., 1997; Manilal et al., 1998a; Shumaker et al., 2003). Emerin is a single-membrane-spanning, serine-rich 254 amino acid protein that, in humans, shows ubiquitous tissue distribution with high expression in skeletal and cardiac muscle (Manilal et al., 1996; Nagano et al., 1996). Recent data show that emerin can appear in four differently phosphorylated forms, three of which may be associated with the cell cycle, showing its possible involvement in controlling cell cycle processes (Ellis et al., 1998; Manilal et al., 1998b; Fairley et al., 2002). Mutations in lamins and emerin have been linked to rare human diseases (laminopathies), affecting skeletal and cardiac muscle, as well as fat, bone and neuronal tissues, or causing premature ageing (Sullivan et al., 1999; Raharjo et al., 2001; Salina et al., 2001). To date, the roles of lamin and emerin in endometrial function or dysfunction remain unclear, although they likely function in determining nuclear size and shape, and perhaps are involved in endometrial cellular replication, DNA synthesis, gene transcription and apoptosis.
Herein, we show how lamin A/C can successfully be silenced in human endometrial stromal cells and that silencing lamin A/C in these cells leads to displacement of emerin from the nuclear lamina to the cytoplasm. This model system may provide a more general approach to study endometrial stromal cell gene regulation and function using siRNA.
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Materials and methods |
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Cell culture
Endometrial biopsies were obtained from normally cycling women after informed consent, under an approved protocol by the Stanford University Committee on the Use of Human Subjects in Medical Research. Biopsies were obtained from subjects aged 39–46 years, who had regular menstrual cycles (28–35 days), were documented not to be pregnant, and had no history of endometriosis. Endometrial stromal cells were isolated in a total of six biopsies. Three samples were used for validation of lamin A/C after silencing treatment at the mRNA level, whereas another three samples were used for validation at the protein level (for western blot and immunocytochemistry). Endometrial tissue was subjected to collagenase/hyaluronidase (Sigma, USA) digestion for 2 h at 37°C. After digestion the stroma was dispersed, whereas the epithelial structures remained mostly intact. Stromal cells were separated from epithelium on a size basis, as previously described (Kirk and Irwin, 1980
). Stromal cells were centrifuged and the resulting pellet was resuspended in 4:1 ratio of Dulbecco's modified Eagle's medium (DMEM)–Ham's F12 (Gibco, USA) and DMEM/10% fetal bovine serum (FBS) (Gibco). The cells were pre-plated in 10 cm standard culture plates for 1 h at 37°C and 9% CO2, and the medium was replaced with high-glucose DMEM/MCDB-105 medium with 10% charcoal-stripped FBS, insulin (5 µg/ml) (Sigma–Aldrich, USA), gentamycin, penicillin and streptomycin, as previously described (Irwin et al., 1991). Endometrial stromal cells were passaged two or three times and plated in 12-well culture plates (Costar, USA) at a density of 1.5 x 105/well.
siRNA and transfection
The siRNA sequence targeting lamin A/C (NM_005572) was generated as described by Elbashir et al. (2001a), and was purchased from Qiagen (USA) together with the fluorescein-labelled non-silencing siRNA. Transfection of lamin A/C siRNA and the non-silencing control was performed using the RNAiFect Transfection reagent (Qiagen) following the manufacturer's instructions. Briefly, 2 days before transfection, cells were plated in 12-well culture plates (Costar) at a density of 
1.5 x 105/well. On the day of transfection cells were 70–80% confluent. Different concentrations of lamin A/C siRNA and non-silencing control were diluted into the culture medium, giving a final volume of 100 ml. Samples were incubated for 20 min for complex formation and were added to 600 ml of the culture medium in each well. For complex formation, different concentrations of RNAiFect were added to the diluted lamin A/C siRNA or control non-silencing fluorescein-labelled siRNA. Four different combinations of siRNA:TransiFect reagent were evaluated at ratios of 1 µg:6 µl, 1 µg:9 µl, 1.5 µg:6 µl and 1.5 µg:9 µl (data not shown). Transfection using 1 µg of siRNA (giving final concentration of 200 nmol/l of siRNA) versus 9 µl of transfect reagent, 48 h post-transfection, was found to be optimal and used for all experiments. Cells were incubated with transfection complexes for 6 h, after which media were replaced with regular culture media. Three independent experiments were performed for each subject in order to obtain silencing efficiency for lamin A/C in endometrial stromal cells compared to cells treated with non-silencing control siRNA.
RNA extraction, RT and PCR
Total RNA from endometrial stromal cells was isolated and purified using RNeasy Mini Kit (Qiagen) following the manufacturer's protocol. RNA integrity was verified by agarose gel electrophoresis/ethidium bromide staining and by OD260/280 absorption ratio >1.95. Total RNA (1 mg) was reverse-transcribed using Omniscript kit (Qiagen) according to the manufacturer's instructions. To observe siRNA-mediated reduction of lamin A/C expression, 30 cycles of PCR were performed (94°C for 30 s, 58°C for 30 s, 72°C for 30 s) using HotStarTaq Master Mix (Qiagen) in Eppendorf Mastercycler Gradient (Eppendorf, Germany). Intron spanning primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (F: 5'-CACAGTCCATGCCATCACTGC-3' and R: 5'-CTCACAGTTGCCATGTAGACC-3') and lamin A/C (F: 5'-TGGAGATGATCCCTTGCTGA-3' and R: 5'-GCATGGCCACTTCTTCCCA-3') were designed from public databases and synthesized at the Stanford University School of Medicine Protein and Nuclear Acid (PAN) Facility.
Quantitative RT–PCR
Lamin A/C silencing efficiency was measured by quantitative PCR using human lamin A/C QuantiTect Hs_LMNA Assay (Qiagen) and QuantiTect Hs_GAPDH Assay (Qiagen) as a normalizer. Real-time PCR was performed in triplicate in 25 ml using the QuantiTect Probe PCR Kit (Qiagen) following the manufacturer's instructions and carried out in the Mx4000 Q-PCR system (Stratagene, USA). The thermal cycling conditions included an initial activation step at 95°C for 15 min, followed by 40 cycles of denaturation, annealing and amplification (94°C for 15 s, 56°C for 30 s, 76°C for 30 s). Fluorescence data collection was performed during the annealing step. For standard curve construction, lamin A/C and GAPDH RT–PCR products were cloned by A–T cloning into the pDive cloning vector (Qiagen). One nanogram of pDrive vector with cloned lamin A/C or GAPDH Gene Expression Assays corresponds to 2.4 x 108 copies of the respective target. Standard curves for lamin A/C and GAPDH were obtained from six 10-fold dilutions (2.4 x 107, 2.4 x 106, 2.4 x 105, 2.4 x 104, 2.4 x 103 and 2.4 x 102). The efficiencies of amplification (E) for lamin A/C and GAPDH were calculated according to the equation: E = 10[–1/slope] –1 and ranged from 101 to 103%. Representative standard curves with respective amplification plots for lamin A/C and GAPDH are presented in Figure 1. For each sample, the amount of target (lamin A/C) and normalizer (GAPDH) was determined from their respective standard curves. The percentage of silencing was calculated from expressed differences between lamin A/C-silenced samples and the non-silencing control.
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Rn).Flow cytometry and fluorescence microscopy
To estimate the transfection efficiency, endometrial stromal cells from three subjects were treated in triplicate with 200 nmol/l of non-silencing fluorescein-labelled siRNA for 6 h at 37°C and 9% CO2. Cells were then washed three times in PBS, trypsinized and resuspended in PBS. Flow cytometry analysis was performed on
104 cells/sample at Stanford's Fluorescence Activated Cell Sorting (FACS) facility. To measure background fluorescence, endometrial stromal cells that were treated with transfection reagent alone were used. Stromal cells cultured on fibronectin-coated culture slides (BD Biosciences, USA) until 70–80% confluent were treated with 200 nmol/l of non-silencing fluorescein-labelled siRNA for 6 h at 37°C and 9% CO2 and subjected to fluorescence microscopy analysis. Visualization of fluorescently labelled siRNA was performed using Zeiss Axioskop 2 plus microscope (Carl Zeiss, Inc., Germany) and images were taken with Zeiss AxioCam Hc.
Western blotting
For western blot analysis, endometrial stromal cells were plated in 12-well culture plates (Costar) and transfected with 200 nmol/l of lamin A/C siRNA or with 200 nmol/l of non-silencing control siRNA. After 48 h of treatment, cells were washed with phosphate-buffered saline (PBS), lysed in lysis buffer (Santa Cruz Biotechnology, Inc., USA), centrifuged for 10 min at 16 000 xg, at 4°C, and proteins were denaturated by boiling for 5 min in Laemmli buffer (Santa Cruz Biotechnology, Inc.). Protein samples were subjected to 10% SDS–polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane (Schleicher & Schuell Bioscience, Germany) by electroblotting. Membranes were then placed in blocking buffer (5% non-fat dry milk in TBST: 10 mmol/l Tris, pH 7.5, 100 mmol/l NaCl and 0.1% Tween-20) for 1 h. Polyclonal lamin A/C (Santa Cruz Biotechnology, Inc.)-specific antibody was used at the 1:400 dilution. After 3 x 30 min washes in TBS-T, membranes were incubated with secondary antibody for 1 h. Bound antibodies were detected using ECL Plus chemiluminescent detection system (Amersham, UK) and exposed to X-ray films (Eastman Kodak, USA). The western blot was stripped and re-probed with ß-actin antibody (Santa Cruz Biotechnology, Inc.) to check for equal loading of the protein.
Immunofluorescence microscopy
For immunofluorescence analysis, stromal cells were cultured on fibronectin-coated culture slides (BD Biosciences) until 70–80% confluent, and then treated for 48 h with lamin A/C siRNA or non-silencing siRNA. Cells were washed three times in PBS, fixed for 5 min in cold methanol and incubated for 30 min in 10% goat serum (Santa Cruz Biotechnology, Inc.) for lamin A/C staining or horse serum (Santa Cruz Biotechnology, Inc.) for emerin staining. After blocking, cells were washed in PBS and incubated for 60 min with primary rabbit polyclonal lamin A/C antibody (Santa Cruz Biotechnology, Inc.) diluted 1:200 in PBS and 1.5% goat serum followed by goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated antibody (Vector Laboratories, Inc., USA) for 40 min in the dark. Mouse monoclonal anti-emerin antibody (Santa Cruz Biotechnology, Inc.) diluted 1:200 in PBS and 1.5% horse serum followed by horse anti-mouse FITC-conjugated antibody (Vector Laboratories, Inc., USA) was use to stain emerin. Cells were washed three to five times in PBS. Mounting medium (Vector Laboratories) was added prior to covering with a cover slip and sealing. Samples were analysed by fluorescence microscopy under appropriate illumination with a Leica SP2 AOBS confocal microscope (Leica Microsystems, Germany).
Statistical analysis
Each treatment, which represents the normalized repeated measures of expression data from all relevant experiments, was tested for significant effect compared to the control by repeated-measures analysis of variance (P<0.001), with post hoc analysis using the conservative Bonferroni method. Statistical analysis was performed using SYSTAT Version 10.2.01 (SYSTAT Software Inc., USA).
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Results |
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Regulation of lamin A/C mRNA degradation in endometrial stromal cells transfected with lamin A/C siRNA, compared to the cells treated with non-silencing siRNA, was quantified using real-time RT–PCR. Individual experiments were repeated a minimum of three times to evaluate the degree of variation. Consistent theshold values for triplicates within the same sample and among the three compared samples were observed. Real-time RT–PCR data obtained in stromal cells 48 h post-transfection with lamin A/C siRNA, compared to a non-silencing siRNA treatment, showed significant reduction of lamin A/C mRNA expression (Figure 2). There was 75 to 88% reduction in lamin A/C mRNA expression in the three tested samples, demonstrating that cultured endometrial stromal cells obtained from human subjects can have mRNA successfully degraded using RNA interference.
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RT–PCR performed using different primers for lamin A/C and GAPDH (Figure 3) confirmed the real-time PCR data. Lamin A/C expression was markedly decreased in cells that were treated with lamin A/C siRNA compared to cells treated with non-silencing siRNA. The integrity and relative amounts of these mRNA were confirmed using glyceraldehyde 3-phosphate dehydrogenase as a constitutively expressed marker.
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The reduction of lamin A/C, after silencing treatment, at the protein level was confirmed using immunocytochemistry and western blot analysis (Figure 4). By both techniques, in three tested subjects, lamin A/C protein expression was specifically decreased in cells that were treated with lamin A/C siRNA (Figure 4A and C, lane 1) compared to cells treated with non-silencing siRNA (Figure 4B and C, lane 2) 48 h post transfection. Some distortion of the nuclei was observed in cells treated with lamin A/C siRNA (data not shown). Lamin A and lamin C are produced by alternative splicing in the 3' region and are presented in equal amounts in the lamina, giving two specific bands on the western blot (lamin A at 74 kDa and lamin C at 65 kDa) (Figure 4C).
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Transfection efficiency was measured by flow cytometry using cells that were treated with non-silencing fluorescein-labelled siRNA for 6 h, compared to cells treated with the transfection reagent alone for the same period of time. A representative histogram of fluorescence of treated endometrial stromal cells from one subject's cells is shown in Figure 5A. In this sample, 94% of cells treated with non-silencing fluorescein-labelled siRNA had high levels of fluorescence compared to 3% of controls. Analysis of cells from three different subjects in triplicate consistently showed that 95–98% of the cells were fluorescent following transfection with non-silencing fluorescein-labelled siRNA, compared to a background fluorescence of
0.3–5% in cells treated with the transfection reagent alone. This demonstrated intake of fluorescently labelled siRNA by the majority of stromal cells and confirmed their high ability to be transfected by siRNA. To estimate the transfection efficiency, the percentage of background fluorescence was subtracted from the percentage of fluorescent cells in each sample. Figure 5B represents the means of the transfection efficiencies for samples from each of the three different subjects, performed in triplicate. Fluorescence microscopy also showed that the majority of stromal cells treated with non-silencing fluorescein-labelled RNA exhibit cytoplasmic fluorescence (Figure 5C) as a measure for siRNA intake and high transfection efficiency, confirming the results of the flow cytometry analysis.
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For further confirmation of lamin A/C silencing in endometrial stromal cells, immunofluorescence staining of the nuclear protein, emerin, was performed (Figure 6). In the endometrial stromal cells transfected with non-silencing siRNA, 48 h post-transfection emerin was clearly localized to the nuclear lamina (Figure 6A). In contrast, in the endometrial stromal cells 48 h post-transfection with lamin A/C siRNA, displacement of emerin from the nuclear lamina to the cytoplasmic endoplasmatic reticulum with some emerin still present in the nuclear lamina was observed (Figure 6B).
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Discussion |
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In this paper we describe the feasibility of silencing a specific gene, lamin A/C, in human endometrial stromal cells in culture, with
75–88% efficiency. This efficiency closely approximates to the silencing efficiency obtained in other cells (HeLa, 3T3 cells, rat fibroblasts) (Sullivan et al., 1999; Elbashir et al., 2001a; Harborth et al., 2001; Raharjo et al., 2001). Herein, the new and highly sensitive, reproducible and quantitative method for efficient and rapid determination of silencing efficiency is based on real-time quantitative analysis of PCR amplification. QuantiTect Assays, used herein to monitor silencing efficiency, are sequence-specific fluorescently labelled probes with a minor groove binding and superbases (modified bases) that lead to increased stability and specificity of the PCR reaction. They demonstrate the suitability of this approach to study gene silencing effects that can be substantiated by the reproducibility and consistency of the quantitative data obtained for individual samples and standard points.
In this study, in addition to validating gene silencing by measuring its degradation of mRNA as well as decreased protein levels and monitoring transfection efficiency by flow cytometry and fluorescence microscopy, we also investigated the downstream effects of the silenced gene with regard to subcellular mechanisms and molecular components. Silencing lamin A/C in human endometrial stromal cells extends the previous characterization of lamin A/C silencing in HeLa cells and mouse SW3T3 cells (Harborth et al., 2003) and data obtained with the lamin A/C knockout mouse (Sullivan et al., 1999; Raharjo et al., 2001). Ultrastructural studies of cultured fibroblasts from lamin A/C knockout mice have shown altered distribution of emerin (Sullivan et al., 1999). Wild-type mouse embryonic fibroblasts (MEF) showed emerin to be concentrated within the nuclear envelope, whereas MEF from lamin A knockout (lmna–/–) mice showed a decrease in emerin and its distribution in the peripheral endoplasmatic reticulum. Transfection of lmna–/– MEF with human lamin A cDNA corrected localization of the emerin to the nuclear envelope, suggesting that lamin A expression is required for correct localization of emerin (Sullivan et al., 1999; Holt et al., 2003). In HeLa cells it has been shown that silencing of lamin A/C leads to the displacement of the nuclear inner protein emerin from the nuclear lamina to the cytoplasm (Elbashir et al., 2002). Inducing a human leukaemia cell line (HL-60) to differentiate into granulocytes causes loss of lamin A/C and leads to emerin displacement from the nuclear lamina to the cytoplasm. When these cells are treated with a phorbol ester (TPA), they differentiate into macrophages, and lamin A/C is expressed again, and emerin localizes to the nuclear envelope (Olins et al., 2001). In our experiments, endometrial stromal cells appear to be functional and viable although a distorted nuclear shape was observed in some of the lamin A/C-silenced cells compared to non-silenced controls, consistent with partial loss of emerin from the nuclear envelope. Intermediate levels of emerin delocalization from the nuclear lamina to the cytoplasm (with some emerin still remaining in the nuclear lamina) are likely due to non-complete silencing of lamin A/C or a long half-life of the emerin after lamin A/C has been silenced.
Lamins were originally proposed to support the nuclear envelope and provide anchoring sites for chomatin. It is postulated that expression of lamin A during differentiation may induce chromatin reorganization and also alter gene expression though its interactions with emerin, DNA-binding protein BAF and DNA complexes (reviewed in Goldman et al., 2002). Cells derived from Lmna–/– mice have misshapen nuclei and obvious ultrastructural damage. Distorted nuclear shape had also been demonstrated in fibroblasts from lipodystrophic patients with the mutation in lamin A/C gene (Vigouroux et al., 2001). Nuclear mechanics in cells derived from Lmna–/– mice are defective with nuclei that display increased deformation and fragility under strain (Lammerding et al., 2004). Data obtained from these experiments suggested that structural changes and altered gene regulation can be linked to lamin A/C dysfunction. Therefore, by analogy, we hypothesize a role for lamin A/C and emerin in human endometrium in maintaining cellular chomatin reorganization and stable nuclear mechanics. Indeed, we observed misshapen nuclei in cells treated with lamin A/C siRNA, supporting this possible function for lamin A/C and emerin in endometrial stromal cells. We did not, however, investigate chomatin reorganization in endometrial stromal cells, a subject for future investigation in our laboratory.
Silencing lamin A/C can be used to study the relationship between lamins and inner nuclear membrane proteins such as emerin and their role in disease aetiology. It can also be used as a model to study lamin–emerin molecular interactions in maintaining nuclear membrane stability and to further evaluate lamin–emerin interactions and their involvement in cell cycle regulation and regulation of gene expression. These are interesting, but not yet explained, mechanisms in human endometrial stromal cells.
This paper presents the first demonstration of how silencing of lamin A/C in human endometrial stromal cells can serve as a model for studying endometrial gene expression, regulation and function by using small interfering RNA. Silencing lamin A/C with the resulting emerin displacement from the nuclear membrane to the cytoplasm can also be a model for optimizing silencing efficiency in other cell types. Herein, we present a straightforward and efficient way to transfect with siRNA and to monitor downstream biologically relevant effects of silencing in cultured human endometrial cells.
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Acknowledgements |
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We would like to thank Drs You-Qiang Su, Nihar R. Nayak, Michael T. Overgaard and Nelson L. Jumbe for their helpful discussions. This work is supported by the National Institutes of Health (NIH) Specialized Cooperative Centers Program in Reproduction Research [National Institute of Child Health and Human Development HD 31398-07 (L.C.G.)].
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Submitted on June 2, 2004; resubmitted on August 10, 2004; accepted on August 17, 2004.
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