Gene silencing in alveolar type II cells using cell-specific promoter in vitro and in vivo

Abstract

RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing process. Although it is widely used in the loss-of-function studies, none of the current RNAi technologies can achieve cell-specific gene silencing. The lack of cell specificity limits its usage in vivo. Here, we report a cell-specific RNAi system using an alveolar epithelial type II cell-specific promoter--the surfactant protein C (SP-C) promoter. We show that the SP-C-driven small hairpin RNAs specifically depress the expression of the exogenous reporter (enhanced green fluorescent protein) and endogenous genes (lamin A/C and annexin A2) in alveolar type II cells, but not other lung cells, using cell and organ culture in vitro as well as in vivo. The present study provides an efficient strategy in silencing a gene in one type of cell without interfering with other cell systems, and may have a significant impact on RNAi therapy.

Figure 1

SP-C-driven shRNA silences exogenous EGFP expression in type II cells, but not in L2 cells. (A) A schematic illustration of the human SP-C promoter-driven shRNA adenoviral vector (Ad/SP-C-shRNA). The shRNA contains a sense strand (S), a 9 nt loop (L) and an antisense strand (AS), followed by TT and a 66 bp of mA. (B and C): Effect of siRNA expressed from SP-C promoter constructs on EGFP expression on primary culture of alveolar type II cells (B) and L2 cells (C). The cells were infected with Ad/CMV-EGFP (a and d), Ad/CMV-EGFP and the siRNA control, Ad/SP-C-shCon (b and e), and Ad/CMV-EGFP and Ad/SP-C-shEGFP (c and f). After 5 days, the cells were examined with a fluorescence microscope (a–c), and the nuclei stained with DAPI to visualize the cells in the fields (d–f). Scale bar, 20 μm. mRNA (D) and protein (E) levels of EGFP in type II and L2 cells as determined by RT–PCR and western blotting. β-actin was used as a loading control. Upper panels show a representative gel or blot. Lane 1, Ad/CMV-EGFP; lane 2, Ad/CMV-EGFP and Ad/SP-C-shCon; lane 3, Ad/CMV-EGFP and Ad/SP-C-shEGFP. Lower panels, mRNA and protein levels were quantitated after being normalized with β-actin signals. The results are expressed as a percentage of control (Ad/CMV-EGFP). Data shown are means ± SE (n = 3). The asterisk denotes a P of <0.01versus Ad/CMV-EGFP group.

Figure 2

Analysis of shRNAs transcripts driven by the SP-C promoter in type II cells. Type II cells cultured on an air–liquid model were infected with Ad/SP-C-shCon (lane 1) or Ad/SP-C-shEGFP (lane 2) adenovirus for 5 days. After isolation of total RNA with TRI reagents, 20 μg of RNA was analyzed by northern blot on a 15% polyacrylamide–urea gel. The blot was hybridized with ³²P-labeled sense shEGFP oligonucleotides (top panel). After being stripped, the same blot was re-probed by ³²P-labeled sense control (shCon) oligonucleotides (middle panel). RNA size markers are indicated on the left side of the gel. The hairpin RNAs were labeled on the right side. RNA quality were shown by the ethidum bromide staining of the 28S and 18S RNA (bottom panel).

Figure 3

Specific silencing of EGFP and lamin A/C by SP-C-driven-shRNA in a lung cell mixture. (A) EGFP, the mixed lung cells were infected with Ad/CMV-EGFP plus Ad/SP-C-shCon or Ad/SP-C-shEGFP adenoviruses for 5 days. (a and e) EGFP fluorescence; (b and f) immunostaining with anti-SP-C antibodies to identify type II cells. (B) lamin A/C, the mixed lung cells were infected with Ad/SP-C-shCon or Ad/SP-C-shLamin adenovirus for 5 days. The cells were double-labeled with anti-lamin A/C (a and e) to monitor the lamin A/C expression and anti-SP-C (b and f) antibodies to identify type II cells. Arrows, type II cells; asterisks, non-type-II cells. Scale bar, 20 μm. (C) Quantitation of EGFP (top) or lamin A/C (bottom) silencing in Ad/SP-C-shEGFP- or Ad/SP-C-shLamin-treated type II cells: Cell counting was performed based on the number of type II cells (red) with or without EGPF or lamin A/C expression (green). Eighty to hundred type II cells were counted for each slide. The results were expressed as a percentage of control (Ad/CMV-EGFP/SP-C-shCon or Ad/SP-C-shCon). Data are means ± SE (n = 3). The asterisk denotes a P of <0.01 versus control group.

Figure 4

Adenovirus-mediated annexin A2 gene silencing by SP-C-driven shRNA in in vitro lung organ culture. (A) Purified adenoviruses expressing annexin A2 shRNA or the control shRNA under the control of the SP-C promoter (Ad/SP-C-shAII or Ad/SP-C-shCon) were used to infect in vitro lung organ culture. After 5 days, paraffin-embedded sections from cultured lung organs were double-immunostained with anti-annexin A2 (a and e) and anti-SP-C (b and f) antibodies. The merged images and bright fields are shown in (c and g) and (d and h), respectively. The upper right corners show the enlarged images. Scale bar, 20 μm. (B) Quantitation of annexin A2 silencing in type II cells: Cell counting was performed based on the number of type II cells (red) with or without EGPF or lamin A/C expression (green). Eighty to hundred type II cells were counted for each slide. The results were expressed as a percentage of control (Ad/SP-C-shCon). Data shown are means ± SE (n = 3).

Figure 5

Adenovirus-mediated annexin A2 gene silencing by SP-C-driven shRNA in rat lungs. Purified adenoviruses (5 × 10¹¹ particles) expressing annexin A2 shRNA or the control shRNA under the control of SP-C promoter (Ad/SP-C-shAII or Ad/SP-C-shCon) were directly delivered into rat lungs. After 5 days, paraffin-embedded sections from infused lung were double-immunostained with anti-annexin A2 (a and e) and anti-SP-C (b and f) antibodies. The merged images and bright fields are shown in (c and g) and (d and h), respectively. The upper right corners show the enlarged images. Scale bar, 20 μm.