Stable RNA interference of hexokinase II gene inhibits human colon cancer LoVo cell growth in vitro and in vivo.
The purpose of this study is to investigate the effect of silencing hexokinase II (HK II) gene with RNA interference (RNAi) technique on colon cancer LoVo cell proliferation in vitro and in vivo. A short hairpin RNA (shRNA) eukaryotic expression vector against HK II gene was constructed, named as plasmid pGenesil-1-HK II, and transfected into LoVo cells. The expression of HK II gene was detected by RT-PCR and Western blot analysis respectively. Then, tumor colony formation was observed, and cell cycle was also assessed by flow cytometry and the contents of intracellular adenosine triphosphate (ATP) by high performance liquid chromatography (HPLC). Furthermore, LoVo cells were injected subcutaneously into nude mice. After a 4-week follow-up period, the sizes and weights of tumors were measured. Moreover, the expression of Ki67 protein was observed by immunohistochemical technique, and cell apoptosis by terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL). Consequently, the expression of HK II gene was efficiently blocked by RNAi. Downregulation of HK II gene expression significantly suppressed cloning efficiency and cell cycle of LoVo cell in vitro and tumor growth in vivo. Compared with untransfected LoVo cells, LoVo cells transfected with pGenesil-1-HK II plasmids showed significant decrease in the cellular ATP contents and Ki67 expression, and obvious increase in the apoptosis indexes. Our results suggest that HK II gene can act as a crucial therapeutic target for slowing colon cancer growth.
Peng
Qiuping
Q
Department of Oncology, Southwest Hospital, Third Military Medical University, Chongqing, China.
Zhou
Qi
Q
Zhou
Jinming
J
Zhong
Daping
D
Pan
Feng
F
Liang
Houjie
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D006019:Glycolysis; D006593:Hexokinase / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008819:Mice, Nude; D049268:Positron-Emission Tomography / Q000379:methods; D034622:RNA Interference
D000255:Adenosine Triphosphate; D006593:Hexokinase