Clusterin silencing inhibits proliferation and reduces invasion in human laryngeal squamous carcinoma cells.
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.
Qianjin
Q
Wang
Weiyan
W
Cao
Quancai
Q
Su
Zimin
Z
Liu
Department of Anesthesiology, the Affiliated Hospital of Qingdao University, Qingdao 266003, China. zhanglinmzs@126.com.
Lin
L
Zhang
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D002448:Cell Adhesion; D002465:Cell Movement; D049109:Cell Proliferation; D051152:Clusterin / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D005260:Female; D020868:Gene Silencing; D006801:Humans; D007822:Laryngeal Neoplasms / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D008175:Lung Neoplasms / Q000378:metabolism / Q000517:prevention & control* / Q000556:secondary; D051379:Mice; D016513:Mice, SCID; D009361:Neoplasm Invasiveness; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D014945:Wound Healing
C495242:CLU protein, human; D051152:Clusterin; D012333:RNA, Messenger; D034741:RNA, Small Interfering