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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

The growth curve of subcutaneous tumors. Tumor growth was strongly suppressed following sCLU shRNA transfection compared with control shRNA and control. Asterisks indicate P < 0.01. sCLU, Clusterin; shRNA, short hairpin RNA.
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Figure 4: The growth curve of subcutaneous tumors. Tumor growth was strongly suppressed following sCLU shRNA transfection compared with control shRNA and control. Asterisks indicate P < 0.01. sCLU, Clusterin; shRNA, short hairpin RNA.

Mentions: As shown in Figure 4, on the 21st day, the average tumor volume in each group was as follows: controls, 213.5 ± 10.7 mm3; control shRNA, 200.0 ± 9.6 mm3; and sCLU shRNA, 71.3 ± 7.8 mm3. The tumor size in group sCLU shRNA mice was significantly smaller compared to the control, with a tumor inhibition ratio of 72.3%. Statistical analysis revealed no significant difference between the negative control shRNA and the control group (P > 0.05), thereby suggesting that the control plasmids had no obvious side effect on tumor growth.


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)

The growth curve of subcutaneous tumors. Tumor growth was strongly suppressed following sCLU shRNA transfection compared with control shRNA and control. Asterisks indicate P < 0.01. sCLU, Clusterin; shRNA, short hairpin RNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: The growth curve of subcutaneous tumors. Tumor growth was strongly suppressed following sCLU shRNA transfection compared with control shRNA and control. Asterisks indicate P < 0.01. sCLU, Clusterin; shRNA, short hairpin RNA.
Mentions: As shown in Figure 4, on the 21st day, the average tumor volume in each group was as follows: controls, 213.5 ± 10.7 mm3; control shRNA, 200.0 ± 9.6 mm3; and sCLU shRNA, 71.3 ± 7.8 mm3. The tumor size in group sCLU shRNA mice was significantly smaller compared to the control, with a tumor inhibition ratio of 72.3%. Statistical analysis revealed no significant difference between the negative control shRNA and the control group (P > 0.05), thereby suggesting that the control plasmids had no obvious side effect on tumor growth.

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