RNA interference-mediated silencing of the Hsp70 gene inhibits human gastric cancer cell growth and induces apoptosis in vitro and in vivo.
OBJECTIVE
The role of heat shock protein (HSP) 70 in gastric cancer has been extensively examined in many studies for the past decade. It has been demonstrated that over-expression of Hsp70 might play important role in malignant transformation and maintenance of malignant phenotypes. Therefore, silencing the Hsp70 gene could be applicable in molecular therapies of human gastric cancer. Herein, we designed a small interfering RNA targeting Hsp70 to knock down its expression and investigated its effect on cell proliferation and apoptosis in a gastric cancer cell line.


METHODS
Two plasmids (phsp1-siRNA, phsp2-siRNA), along with a negative control (phsp3-siRNA), were created using a genic recombination technique. BGC823 cell lines were used to perform experiments. Western blotting and RT-PCR were used to detect Hsp70 expression in vitro and in vivo. Cell morphology was observed under light microscope. Cell cycle and apoptosis were analyzed by flow cytometry and acridine orange/ethidium bromide double stain, and cell proliferative activity was measured by alamarblue assay. In all experiments, a negative control served as a baseline measure.


RESULTS
We successfully constructed phsps-siRNA plasmids and transfected them into BGC823 cells. RT-PCR and western blotting revealed that the expression of Hsp70 was down-regulated in transfection groups compared with the control group. Flow cytometric analysis indicated that less S-phase fraction accumulated in small interfering RNA transfected cells than in parental cells and the cells transfected with empty vector.


CONCLUSIONS
Our results demonstrated that RNAi against Hsp70 could effectively knock down gene expression, inhibit growth of cancer cells, induce cell cycle arrest and increase cell apoptosis in vitro and in vivo. Hsp70 might serve as a therapeutic target for human gastric cancer.
Xiang
Ting-Xiu
TX
Experiment Research Center, The First Affiliated Hospital, Chongqing Medical University, Chongqing, China. Xiang68733201@yahoo.com.cn
Li
Yan
Y
Jiang
Zheng
Z
Huang
Ai-Long
AL
Luo
Cong
C
Zhan
Bin
B
Wang
Pi-Long
PL
Tao
Xiao-Hong
XH
D017209:Apoptosis / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010957:Plasmids; D034622:RNA Interference; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics* / Q000473:pathology*
D018840:HSP70 Heat-Shock Proteins; D034741:RNA, Small Interfering