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Downregulation of lentivirus-mediated ILK RNAi on tractional force generation in human retinal Müller cells.

Zheng YP, Liu H, Zeng H, Xiong L, Feng ZH, Sun NX - Acta Pharmacol. Sin. (2009)

Bottom Line: Significant decreases in ILK mRNA and protein expression were detected in Müller cells carrying lentiviral ILK-shRNA vector.Lentivirus-mediated ILK RNAi decreased cell migration and contractile force generation by inhibiting alpha-SMA stress fiber formation in human retinal Müller cells.This tool might be useful to treat ocular fibroproliferative diseases associated with transdifferentiated Müller cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, 2nd Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, China.

ABSTRACT

Aim: To investigate the effect of lentivirus-mediated integrin-linked kinase (ILK) RNA interference (RNAi) on human retinal Müller cells transdifferentiation into contractile myofibroblasts.

Methods: A lentiviral vector expressing ILK-specific shRNA was constructed and introduced into cultured retinal Müller cells. Silencing of the ILK gene was identified by real time RT-PCR and Western blot. The Müller cell phenotype change was confirmed by immunodetection of alpha-smooth muscle actin (alpha-SMA) stress fiber formation. The generation of tractional force was assessed using a tissue culture assay with cells incubated in three-dimensional collagen gels; cell migration was determined by the Boyden chamber method, using 10% FBS as a chemotactic factor.

Results: Significant decreases in ILK mRNA and protein expression were detected in Müller cells carrying lentiviral ILK-shRNA vector. Cells treated with anti-ILK siRNA showed less alpha-SMA stress fiber formation under hypoxic conditions or cell subcultivation. Lentiviral ILK-shRNA vector transfection also significantly reduced cell migration and cell-mediated gel contraction.

Conclusion: Lentivirus-mediated ILK RNAi decreased cell migration and contractile force generation by inhibiting alpha-SMA stress fiber formation in human retinal Müller cells. This tool might be useful to treat ocular fibroproliferative diseases associated with transdifferentiated Müller cells.

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Design of lentiviral siRNAs. (A) Core target recognition sequences of siRNAs from human ILK cDNA. (B) Schematic gene map of the pGCSIL-GFP viral vector into which the siRNA sequences derived from (A) were cloned.
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fig1: Design of lentiviral siRNAs. (A) Core target recognition sequences of siRNAs from human ILK cDNA. (B) Schematic gene map of the pGCSIL-GFP viral vector into which the siRNA sequences derived from (A) were cloned.

Mentions: Four different ILK-specific target sequences (Figure 1A) were chosen according to online siRNA tools provided by Invitrogen (http://www.invitrogen.com/rnai), using the ILK reference sequence (Gene Bank Accession No NM_004517.2). Double-stranded DNA containing the interference sequences were synthesized according to the structure of a pGCSIL-GFP viral vector (Figure 1B, Gikai gene company, Shanghai, China), and then inserted into linearized vector. All the constructs were cloned and sequenced to confirm their structure. The positive clones were identified as lentiviral vectors that expressed human ILK short hairpin RNA (shRNA), thereafter designated pGCSIL/ILK-A, pGCSIL/ILK-B, pGCSIL/ILK-C, and pGCSIL/ILK-D, respectively. The four lentiviral vectors were transfected into HEK 293 cells separately to evaluate their RNA interference effects and the pGCSIL/ILK-A (sequence: 5′-CGAAGCTCAACGAGAATCA-3′) induced the highest levels of downregulation. So, pGCSIL/ILK-A vector and Viral packaging system (Gikai gene company, Shanghai, China, containing an optimized mixture of two packaging plasmids: pHelper 1.0 vector and pHelper 2.0 vector) were cotransfected into 293 cells to replicate competent lentivirus. Viral supernatant was harvested 48 h after transfection, filtered through a 0.45-mm cellulose acetate filter and frozen at −70 °C. The lentivirus(LV) containing the human ILK shRNA-expressing cassette (pGCSIL/ILK-A) was used as a positive control for lentivirus production and denoted as ILK-RNAi-LV in the next experiments. The pGCSIL/U6 mock vector was also packaged and used as negative control, denoted as NC-GFP-LV. Viral concentrations were determined by serial dilutions of the concentrated vector stocks in 293 cells in 96-well plates. The number of green fluorescent protein (GFP)-positive cells was measured 4 d post-transduction under microscopy. The titers averaged to 8×109 TU/mL.


Downregulation of lentivirus-mediated ILK RNAi on tractional force generation in human retinal Müller cells.

Zheng YP, Liu H, Zeng H, Xiong L, Feng ZH, Sun NX - Acta Pharmacol. Sin. (2009)

Design of lentiviral siRNAs. (A) Core target recognition sequences of siRNAs from human ILK cDNA. (B) Schematic gene map of the pGCSIL-GFP viral vector into which the siRNA sequences derived from (A) were cloned.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4007506&req=5

fig1: Design of lentiviral siRNAs. (A) Core target recognition sequences of siRNAs from human ILK cDNA. (B) Schematic gene map of the pGCSIL-GFP viral vector into which the siRNA sequences derived from (A) were cloned.
Mentions: Four different ILK-specific target sequences (Figure 1A) were chosen according to online siRNA tools provided by Invitrogen (http://www.invitrogen.com/rnai), using the ILK reference sequence (Gene Bank Accession No NM_004517.2). Double-stranded DNA containing the interference sequences were synthesized according to the structure of a pGCSIL-GFP viral vector (Figure 1B, Gikai gene company, Shanghai, China), and then inserted into linearized vector. All the constructs were cloned and sequenced to confirm their structure. The positive clones were identified as lentiviral vectors that expressed human ILK short hairpin RNA (shRNA), thereafter designated pGCSIL/ILK-A, pGCSIL/ILK-B, pGCSIL/ILK-C, and pGCSIL/ILK-D, respectively. The four lentiviral vectors were transfected into HEK 293 cells separately to evaluate their RNA interference effects and the pGCSIL/ILK-A (sequence: 5′-CGAAGCTCAACGAGAATCA-3′) induced the highest levels of downregulation. So, pGCSIL/ILK-A vector and Viral packaging system (Gikai gene company, Shanghai, China, containing an optimized mixture of two packaging plasmids: pHelper 1.0 vector and pHelper 2.0 vector) were cotransfected into 293 cells to replicate competent lentivirus. Viral supernatant was harvested 48 h after transfection, filtered through a 0.45-mm cellulose acetate filter and frozen at −70 °C. The lentivirus(LV) containing the human ILK shRNA-expressing cassette (pGCSIL/ILK-A) was used as a positive control for lentivirus production and denoted as ILK-RNAi-LV in the next experiments. The pGCSIL/U6 mock vector was also packaged and used as negative control, denoted as NC-GFP-LV. Viral concentrations were determined by serial dilutions of the concentrated vector stocks in 293 cells in 96-well plates. The number of green fluorescent protein (GFP)-positive cells was measured 4 d post-transduction under microscopy. The titers averaged to 8×109 TU/mL.

Bottom Line: Significant decreases in ILK mRNA and protein expression were detected in Müller cells carrying lentiviral ILK-shRNA vector.Lentivirus-mediated ILK RNAi decreased cell migration and contractile force generation by inhibiting alpha-SMA stress fiber formation in human retinal Müller cells.This tool might be useful to treat ocular fibroproliferative diseases associated with transdifferentiated Müller cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, 2nd Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, China.

ABSTRACT

Aim: To investigate the effect of lentivirus-mediated integrin-linked kinase (ILK) RNA interference (RNAi) on human retinal Müller cells transdifferentiation into contractile myofibroblasts.

Methods: A lentiviral vector expressing ILK-specific shRNA was constructed and introduced into cultured retinal Müller cells. Silencing of the ILK gene was identified by real time RT-PCR and Western blot. The Müller cell phenotype change was confirmed by immunodetection of alpha-smooth muscle actin (alpha-SMA) stress fiber formation. The generation of tractional force was assessed using a tissue culture assay with cells incubated in three-dimensional collagen gels; cell migration was determined by the Boyden chamber method, using 10% FBS as a chemotactic factor.

Results: Significant decreases in ILK mRNA and protein expression were detected in Müller cells carrying lentiviral ILK-shRNA vector. Cells treated with anti-ILK siRNA showed less alpha-SMA stress fiber formation under hypoxic conditions or cell subcultivation. Lentiviral ILK-shRNA vector transfection also significantly reduced cell migration and cell-mediated gel contraction.

Conclusion: Lentivirus-mediated ILK RNAi decreased cell migration and contractile force generation by inhibiting alpha-SMA stress fiber formation in human retinal Müller cells. This tool might be useful to treat ocular fibroproliferative diseases associated with transdifferentiated Müller cells.

Show MeSH
Related in: MedlinePlus