Suppression of tumorigenesis and metastasis of hepatocellular carcinoma by shRNA interference targeting on homeoprotein Six1.
We previously demonstrated that the overexpression of homeoprotein Six1 in hepatocellular carcinoma (HCC) patients is associated with venous infiltration, advanced pathologic tumor metastasis (pTNM) stage and poor overall survival rate (Ng et al. Br J Cancer 2006;95:1050-5). In this study, short hairpin RNA (shRNA) interference approach was used to suppress the expression of Six1 in a metastatic HCC cell line MHCC97L. Stable transfectant MHCC97L-shSix1 carrying Six1-specific shRNA plasmid was established to downregulate Six1 expression to about 40% when compared with MHCC97L-Control. In vitro functional assays demonstrated that the growth rate and proliferation ability of MHCC97L-shSix1 cells were markedly decreased. Moreover, significant decrease of cell motility and invasiveness were observed in MHCC97L-shSix1 cells. Data from in vivo xenograft tumorigenesis model demonstrated that the size of tumor in MHCC97L-shSix1 group was dramatically reduced. Experimental and spontaneous metastasis models indicated that targeting Six1 suppression noticeably reduced the pulmonary metastasis in MHCC97L-shSix1 group. To identify Six1-regulated targets, cDNA microarray was employed to compare the expression profiles of MHCC97L-Control and MHCC97L-shSix1 cells. Twenty-eight downregulated and 24 upregulated genes with known functions were identified in MHCC97L-shSix1. The functions of these target genes are involved in diverse biological activities. Our data suggest that Six1 may be involved in regulation of proliferation and invasiveness of HCC; thus targeting suppression of Six1 is a viable option for treating HCC patients.
Ng
Kevin T P
KT
Department of Surgery, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China.
Lee
Terence K W
TK
Cheng
Qiao
Q
Wo
Jana Y H
JY
Sun
Chris K W
CK
Guo
Dong-Yong
DY
Lim
Zophia X
ZX
Lo
Chung-Mau
CM
Poon
Ronnie T P
RT
Fan
Sheung-Tat
ST
Man
Kwan
K
D000818:Animals; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000517:prevention & control* / Q000556:secondary; D002448:Cell Adhesion; D002453:Cell Cycle; D002465:Cell Movement; D049109:Cell Proliferation; D003114:Colony-Forming Units Assay; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D018398:Homeodomain Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics / Q000517:prevention & control* / Q000556:secondary; D008114:Liver Neoplasms, Experimental / Q000235:genetics / Q000473:pathology / Q000517:prevention & control*; D051379:Mice; D008819:Mice, Nude; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013160:Splenic Neoplasms / Q000235:genetics / Q000517:prevention & control* / Q000556:secondary; D014945:Wound Healing
D014408:Biomarkers, Tumor; D018398:Homeodomain Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C490947:Six1 protein, mouse