[siRNA-mediated silencing of ClC-2 gene inhibits proliferation of human U-87 glioma cells].

BACKGROUND & 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). CONCLUSION: 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.

Involvement of anion channel(s) in the modulation of the transient outward K(+) channel in rat ventricular myocytes.

The cardiac Ca(2+)-independent transient outward K(+) current (I(to)), a major repolarizing ionic current, is markedly affected by Cl(-) substitution and anion channel blockers. We reexplored the mechanism of the action of anions on I(to) by using whole cell patch-clamp in single isolated rat cardiac ventricular myocytes. The transient outward current was sensitive to blockade by 4-aminopyridine (4-AP) and was abolished by Cs(+) substitution for intracellular K(+). Replacement of most of the extracellular Cl(-) with less permeant anions, aspartate (Asp(-)) and glutamate (Glu(-)), markedly suppressed the current. Removal of external Na(+) or stabilization of F-actin with phalloidin did not significantly affect the inhibitory action of less permeant anions on I(to). In contrast, the permeant Cl(-) substitute Br(-) did not markedly affect the current, whereas F(-) substitution for Cl(-) induced a slight inhibition. The I(to) elicited during Br(-) substitution for Cl(-) was also sensitive to blockade by 4-AP. The ability of Cl(-) substitutes to induce rightward shifts of the steady-state inactivation curve of I(to) was in the following sequence: NO(3)(-) > Cl(-) approximately Br(-) > gluconate(-) > Glu(-) > Asp(-). Depolymerization of actin filaments with cytochalasin D (CytD) induced an effect on the steady-state inactivation of I(to) similar to that of less permeant anions. Fluorescent phalloidin staining experiments revealed that CytD-pretreatment significantly decreased the intensity of FITC-phalloidin staining of F-actin, whereas Asp(-) substitution for Cl(-) was without significant effect on the intensity. These results suggest that the I(to) channel is modulated by anion channel(s), in which the actin cytoskeleton may be implicated.