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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

Cell viability assay by 3-(4,5-dimethylthiazolyl-2-)-2,5-diphenyltetrazoliumbromide (MTT). After transfection with Skp2 siRNA, cell viability was significantly decreased in cultured rLECs at days 6 and 10 compared with the vehicle control and blank control. The asterisk indicates p<0.05 versus the vehicle and blank controls, while the double asterisk indicates p<0.01 versus the vehicle and blank controls (n=32).
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f4: Cell viability assay by 3-(4,5-dimethylthiazolyl-2-)-2,5-diphenyltetrazoliumbromide (MTT). After transfection with Skp2 siRNA, cell viability was significantly decreased in cultured rLECs at days 6 and 10 compared with the vehicle control and blank control. The asterisk indicates p<0.05 versus the vehicle and blank controls, while the double asterisk indicates p<0.01 versus the vehicle and blank controls (n=32).

Mentions: As shown in Figure 4, cell viability did not show any significant differences after vehicle transfection compared with the blank control group, indicating that the plasmid did not influence cell metabolism. However, cell viability in Skp2 siRNA transfected rLECs (experimental group) significantly decreased at 6 and 10 days after transfection when compared with the vehicle control and blank control.


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)

Cell viability assay by 3-(4,5-dimethylthiazolyl-2-)-2,5-diphenyltetrazoliumbromide (MTT). After transfection with Skp2 siRNA, cell viability was significantly decreased in cultured rLECs at days 6 and 10 compared with the vehicle control and blank control. The asterisk indicates p<0.05 versus the vehicle and blank controls, while the double asterisk indicates p<0.01 versus the vehicle and blank controls (n=32).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Cell viability assay by 3-(4,5-dimethylthiazolyl-2-)-2,5-diphenyltetrazoliumbromide (MTT). After transfection with Skp2 siRNA, cell viability was significantly decreased in cultured rLECs at days 6 and 10 compared with the vehicle control and blank control. The asterisk indicates p<0.05 versus the vehicle and blank controls, while the double asterisk indicates p<0.01 versus the vehicle and blank controls (n=32).
Mentions: As shown in Figure 4, cell viability did not show any significant differences after vehicle transfection compared with the blank control group, indicating that the plasmid did not influence cell metabolism. However, cell viability in Skp2 siRNA transfected rLECs (experimental group) significantly decreased at 6 and 10 days after transfection when compared with the vehicle control and blank control.

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