Inhibitory effects of adenovirus mediated tandem expression of RhoA and RhoC shRNAs in HCT116 cells.
BACKGROUND
RhoA and RhoC are deregulated by over expression in many human tumors, including colorectal cancer. Some reports show that they play a pivotal role in the carcinogenesis, tumor development and infiltration metastasis. In this study, for the first time we constructed recombinant adenovirus to investigate the inhibitory effects of RhoA and RhoC shRNAs in tandem expression on the cell proliferation and invasion of colorectal cancer HCT116 cells.


METHODS
The recombinant adenovirus carrying RhoA and RhoC shRNAs in tandem expression was transfected into HCT116. The mRNA transcription and protein expressions of RhoA and RhoC were examined by RT-FQPCR and Western blot respectively. Cellular proliferation inhibitory activity was determined by methyl thiazolyl tetrazolium (MTT) assay and invasive and migrating potential was detected through in vitro Matrigel coated invasion and migration assay.


RESULTS
Both mRNA and proteins Levels of RhoA and RhoC were significantly reduced in HCT116 cells transfected with Ad-A1+A2+C1+C2 than those in Ad-HK group and control one. The relative RhoA and RhoC mRNA transcriptions were decreased to 40% and 36% (P < 0.05), while proteins expression reducing 42% and 35%, respectively (P < 0.05). Growth curves analysis showed that alive cell number in the Ad-A1+A2+C1+C2 group was lower than others in the third to sixth day and transwell chamber analysis showed that migration/invasion activity was significantly suppressed in Ad-A1+A2+C1+C2 group.


CONCLUSIONS
Our results indicate recombinant adenovirus carrying RhoA and RhoC shRNAs in tandem expression may inhibit the growth and invasion of HCT116 cells. Application of such vector to inhibit one or more genes may be a new method to cancer therapy.
Liu
Xiang-Ping
XP
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, PR China.
Wang
Hai-Bo
HB
Yang
Kun
K
Sui
Ai-Hua
AH
Shi
Qiang
Q
Qu
Shen
S
D000256:Adenoviridae / Q000235:genetics*; D001483:Base Sequence; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008969:Molecular Sequence Data; D009361:Neoplasm Invasiveness; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D034622:RNA Interference; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D000075024:rhoC GTP-Binding Protein
C066690:RHOC protein, human; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D000075024:rhoC GTP-Binding Protein