[siRNA-mediated silencing of ClC-2 gene inhibits proliferation of human U-87 glioma cells].
OBJECTIVE
Small interfering RNA (siRNA), which has been used to inhibit mammalian gene expression, is demonstrated to be an effective tool for gene function investigation. The aim of the present study was to observe the effect of siRNA, which was designed to target volume-regulated chloride channel ClC-2 gene, on the proliferation of a human glioma cell line U-87.


METHODS
Two recombinant eukaryotic CIC-2 siRNA expression vectors were designed and constructed. Sequences were identified and confirmed by restrictive endonuclease digestion and DNA sequencing. The empty vector, pSUPER. puro, and two recombinant plasmids, pSUPER. puro-shRNA-ClC-21 and pSUPER. puro-shRNA-ClC-22, were transfected into U-87 cells using Lipofectamine2000 (Groups: PP0, PP1 and PP2, respectively). ClC-2 mRNA expression was detected by reverse transcription polymerase chain reaction (RT-PCR); the cellular proliferation rate was determined by MTT assay; the cell cycle was measured by flow cytometry (FCM); and the cell colony formation rate was measured by plate colony formation assay.


RESULTS
The DNA fragments encoding siRNA targeting ClC-2-gene were successfully connected onto pSUPER. puro vector. ClC-2 mRNA expression and the cell growth rate in PP1 and PP2 groups were significantly inhibited compared to those in PP0 and control groups. Meanwhile, cell cycle was arrested in G1 phase and the percentage of G1 phase cells were increased by about 30.24% in PP1 and 18.04% in PP2 vs. in control and PP0 groups, P < 0.05. Moreover, the cell colony formation rates were statistically decreased in siRNA treated groups, which were (11.0+/-1.0)% in PP1 and (20+/-3.1)% in PP2 vs. (46.5+/-1.6)% in control and (47.5+/-2.8)% in PP0 groups (P<0.01).


CONCLUSIONS
These results demonstrate that CIC-2 siRNA could inhibit the cell proliferation of a human glioma cell line U-87, thus ClC-2 gene may be used as a novel target for the suppression of the growth of human malignant glioma cells.
Yang
Xiang-Yun
XY
Department of Physiology, Xioan, Shaanxi, 710032, P. R. China.
Lai
Xiao-Gang
XG
Zhang
Yong
Y
Pei
Jian-Ming
JM
Yang
An-Gang
AG
Zhou
Shi-Sheng
SS
D001932:Brain Neoplasms / Q000378:metabolism / Q000473:pathology*; D000090007:CLC-2 Chloride Channels; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018118:Chloride Channels / Q000096:biosynthesis / Q000235:genetics*; D020868:Gene Silencing; D005822:Genetic Vectors; D005910:Glioma / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection
D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; D012333:RNA, Messenger; D034741:RNA, Small Interfering