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Inhibition of proliferation of rabbit lens epithelial cells by S-phase kinase-interacting protein 2 targeting small interfering RNA.

Su Y, Wang F, Yan Q, Teng Y, Cui H - Mol. Vis. (2010)

Bottom Line: Cell viability was measured using the tetrazolium reduction (3-(4,5-dimethylthiazolyl-2-)-2,5-diphenyltetrazoliumbromide [MTT]) assay.Skp2 siRNA transfected cells showed significantly less 59-bromodeoxyuridine- and proliferating cell nuclear antigen-positive staining compared with control cells.Skp2 siRNA inhibits cell proliferation and decreases cell viability of rLECs in vitro by suppression of p27(kip1) downregulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, First Clinic College of Harbin Medical University, Harbin, China.

ABSTRACT

Purpose: Improper proliferation of lens epithelial cells is causally related to posterior capsule opacification. In the present study, we investigated whether small interfering RNA (siRNA)-mediated gene silencing of S-phase kinase-interacting protein 2 (Skp2) can be employed to inhibit rabbit lens epithelial cell (rLEC) proliferation by increasing the p27(kip1) level.

Methods: A plasmid containing Skp2 siRNA was used to decrease the high constitutive level of Skp2 protein in rLECs, which can lead to consequent degradation of p27(kip1). Protein expression of Skp2 and p27(kip1) was detected by immunocytochemistry and western blot. Cell viability was measured using the tetrazolium reduction (3-(4,5-dimethylthiazolyl-2-)-2,5-diphenyltetrazoliumbromide [MTT]) assay. Cell proliferation was assayed by cell counts, immunocytochemistry, and western blot by using antibodies against proliferating cell nuclear antigen.

Results: Immunocytochemistry and western blot showed a decreased level of Skp2 and increased level of p27(kip1) in cells transfected with pSkp2 siRNA but not in vehicle transfection and uninfected cells. MTT assay showed that cell viability significantly declined in rLECs transfected with Skp2 siRNA. Skp2 siRNA transfected cells showed significantly less 59-bromodeoxyuridine- and proliferating cell nuclear antigen-positive staining compared with control cells.

Conclusions: Skp2 siRNA inhibits cell proliferation and decreases cell viability of rLECs in vitro by suppression of p27(kip1) downregulation. Our findings suggest that siRNA-mediated gene silencing of Skp2 can be a novel gene therapy for posterior capsule opacification induced by LEC abnormal proliferation.

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Downregulation of S phase-kinase-interacting protein 2 protein by small interfering ribonucleic acid in rabbit len epithelial cells in vitro. Immunocytochemistry staining (one representative of three experiments) indicated high constitutive levels of Skp2 protein (indicated by arrow) in the nucleolus of rLECs transfected with pSuppressor vehicle (B) or without transfection (C). Transfection with Skp2 siRNA dramatically decreased the expression of Skp2 protein in rLECs (A). Scale bar is equal to 40 μm. After 48 h of transfection and 2 weeks of treatment with G418, several stable transfectant cells were cloned. Western blot analysis (one representative of three experiments) showed that high constitutive Skp2 expression can be detected in rLECs of the vehicle control group and blank control group (D). However, little expression of Skp2 was detected in the experimental group, indicating transfection with Skp2 siRNA can inhibit expression of Skp2 in rLECs in vitro (D). GAPDH served as loading control.
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f2: Downregulation of S phase-kinase-interacting protein 2 protein by small interfering ribonucleic acid in rabbit len epithelial cells in vitro. Immunocytochemistry staining (one representative of three experiments) indicated high constitutive levels of Skp2 protein (indicated by arrow) in the nucleolus of rLECs transfected with pSuppressor vehicle (B) or without transfection (C). Transfection with Skp2 siRNA dramatically decreased the expression of Skp2 protein in rLECs (A). Scale bar is equal to 40 μm. After 48 h of transfection and 2 weeks of treatment with G418, several stable transfectant cells were cloned. Western blot analysis (one representative of three experiments) showed that high constitutive Skp2 expression can be detected in rLECs of the vehicle control group and blank control group (D). However, little expression of Skp2 was detected in the experimental group, indicating transfection with Skp2 siRNA can inhibit expression of Skp2 in rLECs in vitro (D). GAPDH served as loading control.

Mentions: After 48 h of transfection and 2 weeks of treatment with G418, we cloned several stable transfectant cells. Immunofluorescence staining demonstrated high constitutive expression of Skp2 protein in the nucleolus of rLECs transfected with pSuppressor vehicle (Figure 2B) or without transfection (Figure 2C). Transfection with Skp2 siRNA dramatically decreased the expression of Skp2 protein in cells of rLEC (Figure 2A). Each clone was also screened for expression of Skp2 by western blot analysis. Skp2 expression can be detected in rLECs of the vehicle control group and the blank control group (Figure 2D, lanes 2 and 3, respectively). However, there was little expression of Skp2 in the experimental group, which indicated that transfection with Skp2 siRNA can inhibit expression of Skp2 in rLECs in vitro (Figure 2D, lane 1).


Inhibition of proliferation of rabbit lens epithelial cells by S-phase kinase-interacting protein 2 targeting small interfering RNA.

Su Y, Wang F, Yan Q, Teng Y, Cui H - Mol. Vis. (2010)

Downregulation of S phase-kinase-interacting protein 2 protein by small interfering ribonucleic acid in rabbit len epithelial cells in vitro. Immunocytochemistry staining (one representative of three experiments) indicated high constitutive levels of Skp2 protein (indicated by arrow) in the nucleolus of rLECs transfected with pSuppressor vehicle (B) or without transfection (C). Transfection with Skp2 siRNA dramatically decreased the expression of Skp2 protein in rLECs (A). Scale bar is equal to 40 μm. After 48 h of transfection and 2 weeks of treatment with G418, several stable transfectant cells were cloned. Western blot analysis (one representative of three experiments) showed that high constitutive Skp2 expression can be detected in rLECs of the vehicle control group and blank control group (D). However, little expression of Skp2 was detected in the experimental group, indicating transfection with Skp2 siRNA can inhibit expression of Skp2 in rLECs in vitro (D). GAPDH served as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875256&req=5

f2: Downregulation of S phase-kinase-interacting protein 2 protein by small interfering ribonucleic acid in rabbit len epithelial cells in vitro. Immunocytochemistry staining (one representative of three experiments) indicated high constitutive levels of Skp2 protein (indicated by arrow) in the nucleolus of rLECs transfected with pSuppressor vehicle (B) or without transfection (C). Transfection with Skp2 siRNA dramatically decreased the expression of Skp2 protein in rLECs (A). Scale bar is equal to 40 μm. After 48 h of transfection and 2 weeks of treatment with G418, several stable transfectant cells were cloned. Western blot analysis (one representative of three experiments) showed that high constitutive Skp2 expression can be detected in rLECs of the vehicle control group and blank control group (D). However, little expression of Skp2 was detected in the experimental group, indicating transfection with Skp2 siRNA can inhibit expression of Skp2 in rLECs in vitro (D). GAPDH served as loading control.
Mentions: After 48 h of transfection and 2 weeks of treatment with G418, we cloned several stable transfectant cells. Immunofluorescence staining demonstrated high constitutive expression of Skp2 protein in the nucleolus of rLECs transfected with pSuppressor vehicle (Figure 2B) or without transfection (Figure 2C). Transfection with Skp2 siRNA dramatically decreased the expression of Skp2 protein in cells of rLEC (Figure 2A). Each clone was also screened for expression of Skp2 by western blot analysis. Skp2 expression can be detected in rLECs of the vehicle control group and the blank control group (Figure 2D, lanes 2 and 3, respectively). However, there was little expression of Skp2 in the experimental group, which indicated that transfection with Skp2 siRNA can inhibit expression of Skp2 in rLECs in vitro (Figure 2D, lane 1).

Bottom Line: Cell viability was measured using the tetrazolium reduction (3-(4,5-dimethylthiazolyl-2-)-2,5-diphenyltetrazoliumbromide [MTT]) assay.Skp2 siRNA transfected cells showed significantly less 59-bromodeoxyuridine- and proliferating cell nuclear antigen-positive staining compared with control cells.Skp2 siRNA inhibits cell proliferation and decreases cell viability of rLECs in vitro by suppression of p27(kip1) downregulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, First Clinic College of Harbin Medical University, Harbin, China.

ABSTRACT

Purpose: Improper proliferation of lens epithelial cells is causally related to posterior capsule opacification. In the present study, we investigated whether small interfering RNA (siRNA)-mediated gene silencing of S-phase kinase-interacting protein 2 (Skp2) can be employed to inhibit rabbit lens epithelial cell (rLEC) proliferation by increasing the p27(kip1) level.

Methods: A plasmid containing Skp2 siRNA was used to decrease the high constitutive level of Skp2 protein in rLECs, which can lead to consequent degradation of p27(kip1). Protein expression of Skp2 and p27(kip1) was detected by immunocytochemistry and western blot. Cell viability was measured using the tetrazolium reduction (3-(4,5-dimethylthiazolyl-2-)-2,5-diphenyltetrazoliumbromide [MTT]) assay. Cell proliferation was assayed by cell counts, immunocytochemistry, and western blot by using antibodies against proliferating cell nuclear antigen.

Results: Immunocytochemistry and western blot showed a decreased level of Skp2 and increased level of p27(kip1) in cells transfected with pSkp2 siRNA but not in vehicle transfection and uninfected cells. MTT assay showed that cell viability significantly declined in rLECs transfected with Skp2 siRNA. Skp2 siRNA transfected cells showed significantly less 59-bromodeoxyuridine- and proliferating cell nuclear antigen-positive staining compared with control cells.

Conclusions: Skp2 siRNA inhibits cell proliferation and decreases cell viability of rLECs in vitro by suppression of p27(kip1) downregulation. Our findings suggest that siRNA-mediated gene silencing of Skp2 can be a novel gene therapy for posterior capsule opacification induced by LEC abnormal proliferation.

Show MeSH
Related in: MedlinePlus