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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

siRNA-K9.3 reduces cell viability of HCT15 and A549 cellsA. Decreased expression of KV9.3 mRNA by KV9.3 siRNA treatment in HCT15 and A549 cells. The cells were harvested 48 h after KV9.3 siRNA or negative control RNA transfection. Real-time PCR was performed for 45 cycles to quantify the KV9.3 mRNA level. Each bar represents the mean ± S.E.M. (n=4, ***P < 0.001 by the Student's t-test versus negative control RNA treated group, NC: negative control) B. KV9.3 knockdown decreases cell viability of HCT15 and A549 cells. Cell viability was measured by MTT assay 72 h after KV9.3 siRNA transfection. Each bar represents the mean ± S.E.M. (n=9, three independent experiments, ***P < 0.001 by the Student's t-test versus negative control RNA treated group).
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Figure 2: siRNA-K9.3 reduces cell viability of HCT15 and A549 cellsA. Decreased expression of KV9.3 mRNA by KV9.3 siRNA treatment in HCT15 and A549 cells. The cells were harvested 48 h after KV9.3 siRNA or negative control RNA transfection. Real-time PCR was performed for 45 cycles to quantify the KV9.3 mRNA level. Each bar represents the mean ± S.E.M. (n=4, ***P < 0.001 by the Student's t-test versus negative control RNA treated group, NC: negative control) B. KV9.3 knockdown decreases cell viability of HCT15 and A549 cells. Cell viability was measured by MTT assay 72 h after KV9.3 siRNA transfection. Each bar represents the mean ± S.E.M. (n=9, three independent experiments, ***P < 0.001 by the Student's t-test versus negative control RNA treated group).

Mentions: To examine the role of KV9.3 in cell viability, we transiently down-regulated KV9.3 using KV9.3 siRNA and performed MTT cell viability assays. siRNA treatment decreased KV9.3 mRNA levels by 75% (n = 4) and 85% (n = 4) in HCT15 and A549 cells, respectively (Fig. 2A). MTT assays revealed that KV9.3 siRNA treatment reduced the viability of HCT15 and A549 cells by 22% and 29%, respectively (n = 9, three independent experiments), compared to treatment with negative control siRNA (Fig. 2B). siRNA-mediated KV9.3 knockdown did not affect KV2.1 mRNA expression levels in HCT15 or A549 cell lines (Supplementary Fig. S2).


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)

siRNA-K9.3 reduces cell viability of HCT15 and A549 cellsA. Decreased expression of KV9.3 mRNA by KV9.3 siRNA treatment in HCT15 and A549 cells. The cells were harvested 48 h after KV9.3 siRNA or negative control RNA transfection. Real-time PCR was performed for 45 cycles to quantify the KV9.3 mRNA level. Each bar represents the mean ± S.E.M. (n=4, ***P < 0.001 by the Student's t-test versus negative control RNA treated group, NC: negative control) B. KV9.3 knockdown decreases cell viability of HCT15 and A549 cells. Cell viability was measured by MTT assay 72 h after KV9.3 siRNA transfection. Each bar represents the mean ± S.E.M. (n=9, three independent experiments, ***P < 0.001 by the Student's t-test versus negative control RNA treated group).
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Related In: Results  -  Collection

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Figure 2: siRNA-K9.3 reduces cell viability of HCT15 and A549 cellsA. Decreased expression of KV9.3 mRNA by KV9.3 siRNA treatment in HCT15 and A549 cells. The cells were harvested 48 h after KV9.3 siRNA or negative control RNA transfection. Real-time PCR was performed for 45 cycles to quantify the KV9.3 mRNA level. Each bar represents the mean ± S.E.M. (n=4, ***P < 0.001 by the Student's t-test versus negative control RNA treated group, NC: negative control) B. KV9.3 knockdown decreases cell viability of HCT15 and A549 cells. Cell viability was measured by MTT assay 72 h after KV9.3 siRNA transfection. Each bar represents the mean ± S.E.M. (n=9, three independent experiments, ***P < 0.001 by the Student's t-test versus negative control RNA treated group).
Mentions: To examine the role of KV9.3 in cell viability, we transiently down-regulated KV9.3 using KV9.3 siRNA and performed MTT cell viability assays. siRNA treatment decreased KV9.3 mRNA levels by 75% (n = 4) and 85% (n = 4) in HCT15 and A549 cells, respectively (Fig. 2A). MTT assays revealed that KV9.3 siRNA treatment reduced the viability of HCT15 and A549 cells by 22% and 29%, respectively (n = 9, three independent experiments), compared to treatment with negative control siRNA (Fig. 2B). siRNA-mediated KV9.3 knockdown did not affect KV2.1 mRNA expression levels in HCT15 or A549 cell lines (Supplementary Fig. S2).

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