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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

K9.3 knockdown changes protein expression level of cell cycle regulatory proteins in HCT15 and A549 cellsThe cells were harvested 72 h after KV9.3 siRNA transfection. Western blot was performed to examine expression of cyclin D3, CDK2, p21, p27. ß-actin was used as a loading control and, the protein expression level was normalized to that of the negative control RNA treatment group. Each bar represents the mean ± S.E.M. (n=3-5, *P < 0.05, **P < 0.01 by the paired Student's t-test versus negative control RNA treated group, NC: negative control RNA, siRNA: KV9.3 siRNA).
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Figure 5: K9.3 knockdown changes protein expression level of cell cycle regulatory proteins in HCT15 and A549 cellsThe cells were harvested 72 h after KV9.3 siRNA transfection. Western blot was performed to examine expression of cyclin D3, CDK2, p21, p27. ß-actin was used as a loading control and, the protein expression level was normalized to that of the negative control RNA treatment group. Each bar represents the mean ± S.E.M. (n=3-5, *P < 0.05, **P < 0.01 by the paired Student's t-test versus negative control RNA treated group, NC: negative control RNA, siRNA: KV9.3 siRNA).

Mentions: Next, we analyzed KV9.3 siRNA-induced changes in the expression of cell cycle regulatory proteins that participate in G1-S transition, examining the relative protein levels of cyclin D3, CDK2, p21, and p27. In HCT15 cells, KV9.3 knockdown significantly decreased cyclin D3 protein levels (0.44-fold relative to controls) and markedly increased p21 (2.7-fold) and p27 (2.9-fold) levels. CDK2 expression showed little change with KV9.3 knockdown in these cells. In A549 cells, KV9.3 knockdown decreased the expression of cyclin D3 and CDK2 protein (0.64-fold and 0.59-fold relative to controls, respectively) and increased the expression of p21 protein 2.41-fold. The expression level of p27 trended higher following KV9.3 knockdown, but this difference did not reach statistical significance (Fig. 5).


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)

K9.3 knockdown changes protein expression level of cell cycle regulatory proteins in HCT15 and A549 cellsThe cells were harvested 72 h after KV9.3 siRNA transfection. Western blot was performed to examine expression of cyclin D3, CDK2, p21, p27. ß-actin was used as a loading control and, the protein expression level was normalized to that of the negative control RNA treatment group. Each bar represents the mean ± S.E.M. (n=3-5, *P < 0.05, **P < 0.01 by the paired Student's t-test versus negative control RNA treated group, NC: negative control RNA, siRNA: KV9.3 siRNA).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480740&req=5

Figure 5: K9.3 knockdown changes protein expression level of cell cycle regulatory proteins in HCT15 and A549 cellsThe cells were harvested 72 h after KV9.3 siRNA transfection. Western blot was performed to examine expression of cyclin D3, CDK2, p21, p27. ß-actin was used as a loading control and, the protein expression level was normalized to that of the negative control RNA treatment group. Each bar represents the mean ± S.E.M. (n=3-5, *P < 0.05, **P < 0.01 by the paired Student's t-test versus negative control RNA treated group, NC: negative control RNA, siRNA: KV9.3 siRNA).
Mentions: Next, we analyzed KV9.3 siRNA-induced changes in the expression of cell cycle regulatory proteins that participate in G1-S transition, examining the relative protein levels of cyclin D3, CDK2, p21, and p27. In HCT15 cells, KV9.3 knockdown significantly decreased cyclin D3 protein levels (0.44-fold relative to controls) and markedly increased p21 (2.7-fold) and p27 (2.9-fold) levels. CDK2 expression showed little change with KV9.3 knockdown in these cells. In A549 cells, KV9.3 knockdown decreased the expression of cyclin D3 and CDK2 protein (0.64-fold and 0.59-fold relative to controls, respectively) and increased the expression of p21 protein 2.41-fold. The expression level of p27 trended higher following KV9.3 knockdown, but this difference did not reach statistical significance (Fig. 5).

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