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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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Related in: MedlinePlus

S phase-kinase-interacting protein 2 protein by small interfering ribonucleic acid induced p27 kinase inhibition protein 1in rabbit lens epithelial cells in vitro. Immunofluorescence staining indicated the expression of p27kip1 dramatically increased in rLECs transfected with Skp2 siRNA (C) when compared with rLECs transfected with pSuppressor vehicle (A) or without transfection (B). The scale bar is equal to 40 μm. Western blot (one representative of three experiments) showed that the expression of p27kip1 dramatically increased in rLECs transfected with Skp2 siRNA (D) when compared with rLECs transfected with pSuppressor vehicle (D) or without transfection (D). GAPDH served as the loading control.
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f3: S phase-kinase-interacting protein 2 protein by small interfering ribonucleic acid induced p27 kinase inhibition protein 1in rabbit lens epithelial cells in vitro. Immunofluorescence staining indicated the expression of p27kip1 dramatically increased in rLECs transfected with Skp2 siRNA (C) when compared with rLECs transfected with pSuppressor vehicle (A) or without transfection (B). The scale bar is equal to 40 μm. Western blot (one representative of three experiments) showed that the expression of p27kip1 dramatically increased in rLECs transfected with Skp2 siRNA (D) when compared with rLECs transfected with pSuppressor vehicle (D) or without transfection (D). GAPDH served as the loading control.

Mentions: Skp2 is required for the ubiquitination and consequent degradation of p27kip1 [2]. Immunofluorescence staining demonstrated little expression of p27 protein in the rLECs transfected with pSuppressor vehicle (Figure 3A) or without transfection (Figure 3B). Transfection with Skp2 siRNA increased the expression of p27kip1 protein in rLECs (Figure 3C) in vitro. Western blot analysis in our study demonstrated that p27kip1 expression of rLECs in the experimental group increased (Figure 3D, lane 3) when expression of Skp2 decreased (Figure 2). Upregulation of p27kip1 made it possible for us to investigate the effect of p27kip1 inhibition on the proliferation of rLECs.


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)

S phase-kinase-interacting protein 2 protein by small interfering ribonucleic acid induced p27 kinase inhibition protein 1in rabbit lens epithelial cells in vitro. Immunofluorescence staining indicated the expression of p27kip1 dramatically increased in rLECs transfected with Skp2 siRNA (C) when compared with rLECs transfected with pSuppressor vehicle (A) or without transfection (B). The scale bar is equal to 40 μm. Western blot (one representative of three experiments) showed that the expression of p27kip1 dramatically increased in rLECs transfected with Skp2 siRNA (D) when compared with rLECs transfected with pSuppressor vehicle (D) or without transfection (D). GAPDH served as the loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: S phase-kinase-interacting protein 2 protein by small interfering ribonucleic acid induced p27 kinase inhibition protein 1in rabbit lens epithelial cells in vitro. Immunofluorescence staining indicated the expression of p27kip1 dramatically increased in rLECs transfected with Skp2 siRNA (C) when compared with rLECs transfected with pSuppressor vehicle (A) or without transfection (B). The scale bar is equal to 40 μm. Western blot (one representative of three experiments) showed that the expression of p27kip1 dramatically increased in rLECs transfected with Skp2 siRNA (D) when compared with rLECs transfected with pSuppressor vehicle (D) or without transfection (D). GAPDH served as the loading control.
Mentions: Skp2 is required for the ubiquitination and consequent degradation of p27kip1 [2]. Immunofluorescence staining demonstrated little expression of p27 protein in the rLECs transfected with pSuppressor vehicle (Figure 3A) or without transfection (Figure 3B). Transfection with Skp2 siRNA increased the expression of p27kip1 protein in rLECs (Figure 3C) in vitro. Western blot analysis in our study demonstrated that p27kip1 expression of rLECs in the experimental group increased (Figure 3D, lane 3) when expression of Skp2 decreased (Figure 2). Upregulation of p27kip1 made it possible for us to investigate the effect of p27kip1 inhibition on the proliferation of rLECs.

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