ShRNA-mediated gene silencing of beta-catenin inhibits growth of human colon cancer cells.
OBJECTIVE
To observe the gene silencing mediated by the specific shRNA targeted against beta-catenin and its effect on cell proliferation and cycle distribution in the human colon cancer cell line Colo205.


METHODS
Two shRNA plasmid vectors against beta-catenin were constructed and transfected into Colo205 cells with Lipofectamine2000. The down-regulations of beta-catenin, c-myc and cyclinD1 expressions were detected by RT-PCR and western blot analysis. The cell proliferation inhibitions were determined by MTT assay and soft agar colony formation assay. The effect of these two beta-catenin shRNAs on cell cycle distribution and apoptosis was examined by flow cytometry.


RESULTS
These two shRNA vectors targeted against beta-catenin efficiently suppressed the expression of beta-catenin and its down stream genes, c-myc and cyclinD1. The expression inhibition rates were around 40%-50% either at the mRNA or at the protein level. The shRNA-mediated gene silencing of beta-catenin resulted in significant inhibition of cell growth both on the culture plates and in the soft agar. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis at 72 h post transfection due to gene silencing.


CONCLUSIONS
These specific shRNAs targeted against beta-catenin could have a gene silencing effect and block the WNT signaling pathway. They could inhibit cell growth, increase apoptosis, and induce cell cycle arrest in Colo205 cells. ShRNA interference against beta-catenin is of potential value in gene therapy of colon cancer.
Huang
Wen-Sheng
WS
Surgery Department, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China.
Wang
Jian-Ping
JP
Wang
Ting
T
Fang
Jie-Yu
JY
Lan
Ping
P
Ma
Jin-Ping
JP
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D017209:Apoptosis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D010957:Plasmids / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*
C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; D051153:Wnt Proteins; D051176:beta Catenin; D019938:Cyclin D1; D012313:RNA