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Silencing of voltage-gated potassium channel KV9.3 inhibits proliferation in human colon and lung carcinoma cells.

Lee JH, Park JW, Byun JK, Kim HK, Ryu PD, Lee SY, Kim DY - Oncotarget (2015)

Bottom Line: We confirmed the expression of KV9.3 mRNA in HCT15 and A549 cells and showed that silencing KV9.3 using small interfering RNA caused G0/G1 cell cycle arrest and alterations in cell cycle regulatory proteins in both HCT15 and A549 cells without affecting apoptosis.We further found that Sp1 bound to this region and showed that the Sp1 inhibitor, mithramycin A, induced a concentration-dependent decrease in KV9.3 expression.Taken together, these data suggest that knockdown of KV9.3 inhibits proliferation in colon carcinoma and lung adenocarcinoma cell lines and may be regulated by Sp1.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Veterinary Pathology, Seoul National University, Seoul, Korea.

ABSTRACT
Voltage-gated potassium (Kv) channels are known to be involved in cancer development and cancer cell proliferation. KV9.3, an electronically silent subunit, forms heterotetramers with KV2.1 in excitable cells and modulates its electrophysiological properties. However, the role of KV9.3 alone in non-excitable cancer cells has not been studied. Here, we evaluated the effect of silencing KV9.3 on cancer cell proliferation in HCT15 colon carcinoma cells and A549 lung adenocarcinoma cells. We confirmed the expression of KV9.3 mRNA in HCT15 and A549 cells and showed that silencing KV9.3 using small interfering RNA caused G0/G1 cell cycle arrest and alterations in cell cycle regulatory proteins in both HCT15 and A549 cells without affecting apoptosis. Also, stable knockdown of KV9.3 expression using short-hairpin RNA inhibited tumor growth in SCID mouse xenograft model. Using a bioinformatics approach, we identified Sp1 binding sites in the promoter region of the gene encoding KV9.3. We further found that Sp1 bound to this region and showed that the Sp1 inhibitor, mithramycin A, induced a concentration-dependent decrease in KV9.3 expression. Taken together, these data suggest that knockdown of KV9.3 inhibits proliferation in colon carcinoma and lung adenocarcinoma cell lines and may be regulated by Sp1.

No MeSH data available.


Related in: MedlinePlus

Inhibition of K9.3 gene expression by Sp1 inhibitor, mithramycin AA. Sp1 binds to KV9.3 promoter region. ChIP assay was performed with the anti-Sp1 or nonspecific rabbit (negative control) antibody. The GC rich region in the KV9.3 promoter region was amplified by RT-PCR. B. Inhibition of Sp1 by mithramycin A reduces Kv9.3 expression in HCT15 and A549 cells. The cells were treated with mithramycin A (100 nM, 250 nM) for 24 h and KV9.3 mRNA expression level was measured by real-time PCR. Each bar represents the mean ± S.E.M. (n=4, two independent experiments, **P < 0.01, ***P < 0.001 by the Student's t-test versus control group).
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Figure 7: Inhibition of K9.3 gene expression by Sp1 inhibitor, mithramycin AA. Sp1 binds to KV9.3 promoter region. ChIP assay was performed with the anti-Sp1 or nonspecific rabbit (negative control) antibody. The GC rich region in the KV9.3 promoter region was amplified by RT-PCR. B. Inhibition of Sp1 by mithramycin A reduces Kv9.3 expression in HCT15 and A549 cells. The cells were treated with mithramycin A (100 nM, 250 nM) for 24 h and KV9.3 mRNA expression level was measured by real-time PCR. Each bar represents the mean ± S.E.M. (n=4, two independent experiments, **P < 0.01, ***P < 0.001 by the Student's t-test versus control group).

Mentions: We searched for possible transcription factor binding sites in the promoter region of the KCNS3 gene encoding KV9.3 using the TFSEARCH program and found several possible Sp1 binding sites (G-C rich regions). To determine if Sp1 binds to the promoter region of KCNS3, we performed ChIP assay using a Sp1 antibody. These assays revealed that Sp1 bound to a predicted G-C rich binding site in both cell lines (Fig. 7A). To assess the functional consequences of this Sp1 binding, we tested whether inhibition of Sp1 with mithramycin A affected the expression of KV9.3. In both cells lines, mithramycin A significantly reduced the expression level of KV9.3 in a concentration-dependent manner (Fig. 7B). In HCT15 cells, 100 and 250 nM mithramycin A decreased KV9.3 expression by 0.43-fold and 0.24-fold relative to that in controls, respectively, whereas in A549 cells, the corresponding values were 0.24-fold and 0.16-fold (n = 4, two independent experiments).


Silencing of voltage-gated potassium channel KV9.3 inhibits proliferation in human colon and lung carcinoma cells.

Lee JH, Park JW, Byun JK, Kim HK, Ryu PD, Lee SY, Kim DY - Oncotarget (2015)

Inhibition of K9.3 gene expression by Sp1 inhibitor, mithramycin AA. Sp1 binds to KV9.3 promoter region. ChIP assay was performed with the anti-Sp1 or nonspecific rabbit (negative control) antibody. The GC rich region in the KV9.3 promoter region was amplified by RT-PCR. B. Inhibition of Sp1 by mithramycin A reduces Kv9.3 expression in HCT15 and A549 cells. The cells were treated with mithramycin A (100 nM, 250 nM) for 24 h and KV9.3 mRNA expression level was measured by real-time PCR. Each bar represents the mean ± S.E.M. (n=4, two independent experiments, **P < 0.01, ***P < 0.001 by the Student's t-test versus control group).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4480740&req=5

Figure 7: Inhibition of K9.3 gene expression by Sp1 inhibitor, mithramycin AA. Sp1 binds to KV9.3 promoter region. ChIP assay was performed with the anti-Sp1 or nonspecific rabbit (negative control) antibody. The GC rich region in the KV9.3 promoter region was amplified by RT-PCR. B. Inhibition of Sp1 by mithramycin A reduces Kv9.3 expression in HCT15 and A549 cells. The cells were treated with mithramycin A (100 nM, 250 nM) for 24 h and KV9.3 mRNA expression level was measured by real-time PCR. Each bar represents the mean ± S.E.M. (n=4, two independent experiments, **P < 0.01, ***P < 0.001 by the Student's t-test versus control group).
Mentions: We searched for possible transcription factor binding sites in the promoter region of the KCNS3 gene encoding KV9.3 using the TFSEARCH program and found several possible Sp1 binding sites (G-C rich regions). To determine if Sp1 binds to the promoter region of KCNS3, we performed ChIP assay using a Sp1 antibody. These assays revealed that Sp1 bound to a predicted G-C rich binding site in both cell lines (Fig. 7A). To assess the functional consequences of this Sp1 binding, we tested whether inhibition of Sp1 with mithramycin A affected the expression of KV9.3. In both cells lines, mithramycin A significantly reduced the expression level of KV9.3 in a concentration-dependent manner (Fig. 7B). In HCT15 cells, 100 and 250 nM mithramycin A decreased KV9.3 expression by 0.43-fold and 0.24-fold relative to that in controls, respectively, whereas in A549 cells, the corresponding values were 0.24-fold and 0.16-fold (n = 4, two independent experiments).

Bottom Line: We confirmed the expression of KV9.3 mRNA in HCT15 and A549 cells and showed that silencing KV9.3 using small interfering RNA caused G0/G1 cell cycle arrest and alterations in cell cycle regulatory proteins in both HCT15 and A549 cells without affecting apoptosis.We further found that Sp1 bound to this region and showed that the Sp1 inhibitor, mithramycin A, induced a concentration-dependent decrease in KV9.3 expression.Taken together, these data suggest that knockdown of KV9.3 inhibits proliferation in colon carcinoma and lung adenocarcinoma cell lines and may be regulated by Sp1.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Veterinary Pathology, Seoul National University, Seoul, Korea.

ABSTRACT
Voltage-gated potassium (Kv) channels are known to be involved in cancer development and cancer cell proliferation. KV9.3, an electronically silent subunit, forms heterotetramers with KV2.1 in excitable cells and modulates its electrophysiological properties. However, the role of KV9.3 alone in non-excitable cancer cells has not been studied. Here, we evaluated the effect of silencing KV9.3 on cancer cell proliferation in HCT15 colon carcinoma cells and A549 lung adenocarcinoma cells. We confirmed the expression of KV9.3 mRNA in HCT15 and A549 cells and showed that silencing KV9.3 using small interfering RNA caused G0/G1 cell cycle arrest and alterations in cell cycle regulatory proteins in both HCT15 and A549 cells without affecting apoptosis. Also, stable knockdown of KV9.3 expression using short-hairpin RNA inhibited tumor growth in SCID mouse xenograft model. Using a bioinformatics approach, we identified Sp1 binding sites in the promoter region of the gene encoding KV9.3. We further found that Sp1 bound to this region and showed that the Sp1 inhibitor, mithramycin A, induced a concentration-dependent decrease in KV9.3 expression. Taken together, these data suggest that knockdown of KV9.3 inhibits proliferation in colon carcinoma and lung adenocarcinoma cell lines and may be regulated by Sp1.

No MeSH data available.


Related in: MedlinePlus