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Clusterin silencing inhibits proliferation and reduces invasion in human laryngeal squamous carcinoma cells.

Wang Q, Cao W, Su Q, Liu Z, Zhang L - World J Surg Oncol (2014)

Bottom Line: RT-PCR and Western blot were used to detect the effect of siRNA transfection on sCLU mRNA and sCLU protein expression.It showed that siRNA-mediated down-regulation of sCLU expression in Hep-2 cells significantly inhibited cell proliferation and promoted apoptosis in vitro.Furthermore, siRNA-mediated down-regulation of sCLU expression decreases in vitro cell migration and invasion ability.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology, the Affiliated Hospital of Qingdao University, Qingdao 266003, China. zhanglinmzs@126.com.

ABSTRACT

Background: Clusterin is, in its major form, a secreted heterodimeric disulfide-linked glycoprotein (sCLU), which plays important roles in cell survival and death. In laryngeal squamous cell carcinomas (LSCC), sCLU is up-regulated and its expression is related to the invasiveness of these tumors. The purpose of this study was to explore the inhibiting role of sCLU gene silence in the invasive ability and growth of Hep-2 human laryngeal squamous carcinoma cells (Hep-2) by transfection of short hairpin RNA expression plasmids against sCLU (sCLU-shRNA) (in vivo) or small interference RNA (sCLU-siRNA) (in vitro).

Methods: sCLU-siRNA and the control siRNA were transfected into Hep-2 cells using Lipofectamine 2000. RT-PCR and Western blot were used to detect the effect of siRNA transfection on sCLU mRNA and sCLU protein expression. The invasive activity of sCLU-siRNA-transfected Hep-2 cells was measured with the modified Boyden chamber assay and wound healing assay. The effects of sCLU-siRNA on cell proliferation were evaluated by MTT assay. Apoptosis was measured by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-staining methods. We next evaluated the effects of sCLU silencing by sCLU-shRNA transfection in vivo on tumor growth and metastatic properties to the lung. Terminal deoxytransferase-mediated dUTP nick end labeling (TUNEL) staining was used to observe the apoptosis in the xenografts.

Results: It showed that siRNA-mediated down-regulation of sCLU expression in Hep-2 cells significantly inhibited cell proliferation and promoted apoptosis in vitro. Furthermore, siRNA-mediated down-regulation of sCLU expression decreases in vitro cell migration and invasion ability. In vivo, the average volume of tumors in the sCLU-shRNA transfected group was significantly lower than in the control group (P<0.01), and the significant apoptosis detected with TUNEL was indicated in the sCLU-shRNA transfected groups (P<0.05). Significantly, we found that sCLU-shRNA could exert marked inhibition of the lung metastasis of Hep-2 cells in nude mice in vivo.

Conclusions: sCLU gene silence can inhibit invasion and growth of LSCC. sCLU may provide a potential therapeutic target against human LSCC.

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Related in: MedlinePlus

Effect of sCLU-siRNA transfection on mRNA and protein expressions of sCLU. A. Western immunoblot analysis of sCLU expression in Hep-2 cells transfected with sCLU-siRNA. Blots were re-probed with β-actin antibody to analyze the equal loading of proteins. B. Semiquantitative RT-PCR analysis shows mRNA expression of sCLU in Hep-2 cells transfected with sCLU-siRNA. Scrambled siRNA was used as a control in parallel. β-actin was used as an internal control. This is a representative example with mean densitometric values from triplicate blots. *, P <0.05 versus control siRNA transfectants. sCLU, Clusterin; siRNA, small interference RNA.
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Figure 1: Effect of sCLU-siRNA transfection on mRNA and protein expressions of sCLU. A. Western immunoblot analysis of sCLU expression in Hep-2 cells transfected with sCLU-siRNA. Blots were re-probed with β-actin antibody to analyze the equal loading of proteins. B. Semiquantitative RT-PCR analysis shows mRNA expression of sCLU in Hep-2 cells transfected with sCLU-siRNA. Scrambled siRNA was used as a control in parallel. β-actin was used as an internal control. This is a representative example with mean densitometric values from triplicate blots. *, P <0.05 versus control siRNA transfectants. sCLU, Clusterin; siRNA, small interference RNA.

Mentions: sCLU-siRNA and control siRNA were transiently transfected into Hep-2 cells by use of Lipofectin 2000 for 48 h. The cells were collected and protein extracts were made from the cytoplasm. Protein levels of sCLU were analyzed by Western blot. As shown in Figure 1A, sCLU protein was overexpressed in the Hep-2 cells, in the sCLU-siRNA transfected Hep-2 cells, and sCLU protein was completely inhibited; however, control siRNA transfection did not induce a significant change in expression of sCLU in the Hep-2 cells. A sCLU mRNA assay by RT-PCR has the same results as shown above (Figure 1B).


Clusterin silencing inhibits proliferation and reduces invasion in human laryngeal squamous carcinoma cells.

Wang Q, Cao W, Su Q, Liu Z, Zhang L - World J Surg Oncol (2014)

Effect of sCLU-siRNA transfection on mRNA and protein expressions of sCLU. A. Western immunoblot analysis of sCLU expression in Hep-2 cells transfected with sCLU-siRNA. Blots were re-probed with β-actin antibody to analyze the equal loading of proteins. B. Semiquantitative RT-PCR analysis shows mRNA expression of sCLU in Hep-2 cells transfected with sCLU-siRNA. Scrambled siRNA was used as a control in parallel. β-actin was used as an internal control. This is a representative example with mean densitometric values from triplicate blots. *, P <0.05 versus control siRNA transfectants. sCLU, Clusterin; siRNA, small interference RNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4016627&req=5

Figure 1: Effect of sCLU-siRNA transfection on mRNA and protein expressions of sCLU. A. Western immunoblot analysis of sCLU expression in Hep-2 cells transfected with sCLU-siRNA. Blots were re-probed with β-actin antibody to analyze the equal loading of proteins. B. Semiquantitative RT-PCR analysis shows mRNA expression of sCLU in Hep-2 cells transfected with sCLU-siRNA. Scrambled siRNA was used as a control in parallel. β-actin was used as an internal control. This is a representative example with mean densitometric values from triplicate blots. *, P <0.05 versus control siRNA transfectants. sCLU, Clusterin; siRNA, small interference RNA.
Mentions: sCLU-siRNA and control siRNA were transiently transfected into Hep-2 cells by use of Lipofectin 2000 for 48 h. The cells were collected and protein extracts were made from the cytoplasm. Protein levels of sCLU were analyzed by Western blot. As shown in Figure 1A, sCLU protein was overexpressed in the Hep-2 cells, in the sCLU-siRNA transfected Hep-2 cells, and sCLU protein was completely inhibited; however, control siRNA transfection did not induce a significant change in expression of sCLU in the Hep-2 cells. A sCLU mRNA assay by RT-PCR has the same results as shown above (Figure 1B).

Bottom Line: RT-PCR and Western blot were used to detect the effect of siRNA transfection on sCLU mRNA and sCLU protein expression.It showed that siRNA-mediated down-regulation of sCLU expression in Hep-2 cells significantly inhibited cell proliferation and promoted apoptosis in vitro.Furthermore, siRNA-mediated down-regulation of sCLU expression decreases in vitro cell migration and invasion ability.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology, the Affiliated Hospital of Qingdao University, Qingdao 266003, China. zhanglinmzs@126.com.

ABSTRACT

Background: Clusterin is, in its major form, a secreted heterodimeric disulfide-linked glycoprotein (sCLU), which plays important roles in cell survival and death. In laryngeal squamous cell carcinomas (LSCC), sCLU is up-regulated and its expression is related to the invasiveness of these tumors. The purpose of this study was to explore the inhibiting role of sCLU gene silence in the invasive ability and growth of Hep-2 human laryngeal squamous carcinoma cells (Hep-2) by transfection of short hairpin RNA expression plasmids against sCLU (sCLU-shRNA) (in vivo) or small interference RNA (sCLU-siRNA) (in vitro).

Methods: sCLU-siRNA and the control siRNA were transfected into Hep-2 cells using Lipofectamine 2000. RT-PCR and Western blot were used to detect the effect of siRNA transfection on sCLU mRNA and sCLU protein expression. The invasive activity of sCLU-siRNA-transfected Hep-2 cells was measured with the modified Boyden chamber assay and wound healing assay. The effects of sCLU-siRNA on cell proliferation were evaluated by MTT assay. Apoptosis was measured by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-staining methods. We next evaluated the effects of sCLU silencing by sCLU-shRNA transfection in vivo on tumor growth and metastatic properties to the lung. Terminal deoxytransferase-mediated dUTP nick end labeling (TUNEL) staining was used to observe the apoptosis in the xenografts.

Results: It showed that siRNA-mediated down-regulation of sCLU expression in Hep-2 cells significantly inhibited cell proliferation and promoted apoptosis in vitro. Furthermore, siRNA-mediated down-regulation of sCLU expression decreases in vitro cell migration and invasion ability. In vivo, the average volume of tumors in the sCLU-shRNA transfected group was significantly lower than in the control group (P<0.01), and the significant apoptosis detected with TUNEL was indicated in the sCLU-shRNA transfected groups (P<0.05). Significantly, we found that sCLU-shRNA could exert marked inhibition of the lung metastasis of Hep-2 cells in nude mice in vivo.

Conclusions: sCLU gene silence can inhibit invasion and growth of LSCC. sCLU may provide a potential therapeutic target against human LSCC.

Show MeSH
Related in: MedlinePlus