title: Silencing of RhoA and RhoC expression by RNA interference suppresses human colorectal carcinoma growth in vivo.
abstract: BACKGROUND
  RhoA and RhoC have been proved to be over-expressed in many solid cancers, including colorectal cancer. The reduction of RhoA and RhoC expression by RNA interference (RNAi) resulted growth inhibition of cancer cells. The present study was to evaluate the effect of silencing of RhoA and RhoC expression by RNAi on growth of human colorectal carcinoma (CRC) in tumor-bearing nude mice in vivo.
  
  
  METHODS
  To establish HCT116 cell transplantable model, the nude mice were subcutaneously inoculated with 1.0 × 10(7) HCT116 cells and kept growing till the tumor xenografts reached 5-7 mm in diameter. Then the mice were randomly assigned to three groups(seven mice in each group): (1) normal saline(NS) group, (2)replication-defective recombinant adenovirus carrying the negative control shRNA (Ad-HK) group and (3)replication-defective recombinant adenovirus carrying the 4-tandem linked RhoA and RhoC shRNAs (Ad-RhoA-RhoC) group. Ad-HK (4 × 10(8) pfu, 30 ul/mouse), Ad-RhoA-RhoC (4 × 10(8) pfu, 30 ul/mouse) or PBS (30 ul/mouse) was injected intratumorally four times once every other day. The weight and volumes of tumor xenografts were recorded. The levels of RhoA and RhoC mRNA transcripts and proteins in tumor xenografts were detected by reverse quantitative transcription polymerase chain reaction (QRT-PCR) and immunohistochemical staining respectively. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect the death of cells.
  
  
  RESULTS
  The xenografts in mice could be seen at 5th day from the implantation of HCT116 cells and all had reached 5-7 mm in size at 9th day. After injection intratumorally, the growth speed of tumor xenografts in Ad-RhoA-RhoC group was significantly delayed compared with those in NS and Ad-HK group(P < 0.05). The results of QRT-PCR showed that mRNA levels of RhoA and RhoC reduced more in Ad-RhoA-RhoC group than those in NS and Ad-HK group. The relative RhoA and RhoC mRNA transcripts were decreased to 48% and 43% respectively (P < 0.05). Immunohistochemical analyses of tumor xenograft sections also revealed the decreased RhoA and RhoC expression in Ad-RhoA-RhoC group. TUNEL assay also showed higher death of tumor xenograft tissue cells in Ad-RhoA-RhoC group.
  
  
  CONCLUSIONS
  Recombinant adenovirus mediated RhoA and RhoC shRNA in tandem linked expression may inhibit the growth of human colorectal tumor xenografts in vivo. These results indicate that RhoA and RhoC might be potential targets for gene therapy in colorectal cancer.
authors.affiliation: Department of General Surgery, Affiliated Hospital of Qingdao University Medical College, Qingdao, 266003, China.
authors.forename: Haibo
authors.initials: H
authors.lastname: Wang
authors.forename: Gang
authors.initials: G
authors.lastname: Zhao
authors.forename: Xiangping
authors.initials: X
authors.lastname: Liu
authors.forename: Aihua
authors.initials: A
authors.lastname: Sui
authors.forename: Kun
authors.initials: K
authors.lastname: Yang
authors.forename: Ruyong
authors.initials: R
authors.lastname: Yao
authors.forename: Zongbao
authors.initials: Z
authors.lastname: Wang
authors.forename: Qiang
authors.initials: Q
authors.lastname: Shi
mesh_terms: D000818:Animals; D016923:Cell Death; D015179:Colorectal Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D015972:Gene Expression Regulation, Neoplastic; D015316:Genetic Therapy / Q000379:methods*; D045325:HCT116 Cells; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D047368:Tumor Burden; D023041:Xenograft Model Antitumor Assays; D020741:rho GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000235:genetics* / Q000378:metabolism; D000075024:rhoC GTP-Binding Protein
chemical_list: D012333:RNA, Messenger; C066737:RHOA protein, human; C066690:RHOC protein, human; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D000075024:rhoC GTP-Binding Protein