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Silencing of hERG1 Gene Inhibits Proliferation and Invasion, and Induces Apoptosis in Human Osteosarcoma Cells by Targeting the NF-κB Pathway.

Zeng W, Liu Q, Chen Z, Wu X, Zhong Y, Wu J - J Cancer (2016)

Bottom Line: Knockdown of hERG1 significantly suppressed cellular proliferation and invasion, and induced apoptosis, while inhibition of hERG1 significantly decreased activation of NF-κB.Overall, hERG1 may stimulate nuclear translocation of p65, thus regulating the NF-κB pathway through the activation of the hERG1/beta1 integrin complex and PI3K/AKT signaling.Furthermore, this regulation by hERG1 is, at least in part, through mediation of the NF-κB pathway.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Orthopaedics, the Affiliated Southeast Hospital of Xiamen University, Orthopaedic Center of People's Liberation Army, Zhangzhou, 363000, People's Republic of China.

ABSTRACT
Recently, the human ether à go-go (eag) related gene 1 (hERG1) channel, a member of the voltage-dependent potassium channel (Kv) family, was determined to have a critical role in cancer cell proliferation, invasion, tumorigenesis and apoptosis. However, the expression levels and functions of hERG1 in osteosarcoma cells remain poorly characterized. In this study, hERG1 transcript and protein levels in osteosarcoma cells and tissues were measured using semi-quantitative real time PCR (RT-PCR), Western blot, and immunohistochemistry. The effects of hERG1 knockdown on osteosarcoma cell proliferation, apoptosis and invasion were examined using CCK-8, colony formation, flow cytometry, caspase-3 activity, wound healing and transwell based assays. Furthermore, semi-quantitative RT-PCR, Western blot and a luciferase reporter assay were used to assess the effects of hERG1 inhibition on the nuclear factor-κB (NF-κB) pathway. In addition, the effect of NF-κB p65-siRNA and NF-κB p65 expression on the survival of osteosarcoma cells was investigated. Through this work, a relationship for hERG1 with the NF-κB pathway was identified. Osteosarcoma cells and tissues were found to express high levels of hERG1. Knockdown of hERG1 significantly suppressed cellular proliferation and invasion, and induced apoptosis, while inhibition of hERG1 significantly decreased activation of NF-κB. Overall, hERG1 may stimulate nuclear translocation of p65, thus regulating the NF-κB pathway through the activation of the hERG1/beta1 integrin complex and PI3K/AKT signaling. Taken together, these results demonstrate that hERG1 is necessary for regulation of osteosarcoma cellular proliferation, apoptosis and migration. Furthermore, this regulation by hERG1 is, at least in part, through mediation of the NF-κB pathway.

No MeSH data available.


Related in: MedlinePlus

Knockdown of hERG1 reduces proliferation of osteosarcoma cells. (A) Efficiency of knockdown by hERG1-siRNA was measured by Western blot. (B-E) Proliferation of MG-63 cells transfected with hERG1-siRNA (30 nM) (B and C), or treated with hERG1 inhibitor E-4031 (D) or activator PD 118057 (E) was measured using CCK-8 or colony formation assay (n = 6). (F) Protein expression of hERG1 in HEK293-wt and HEK293-hERG1 cells was detected by Western blot. (G) 1 × 105 HEK293-wt and HEK293-hERG1 cells were cultured for 48 h and the CCK-8 assay was performed. (H) The effects of E-4031 on the proliferation of HEK293-wt and HEK293-hERG1 cells were determined by CCK-8 assay. * P < 0.05, ** P < 0.01, *** P < 0.001.
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Figure 2: Knockdown of hERG1 reduces proliferation of osteosarcoma cells. (A) Efficiency of knockdown by hERG1-siRNA was measured by Western blot. (B-E) Proliferation of MG-63 cells transfected with hERG1-siRNA (30 nM) (B and C), or treated with hERG1 inhibitor E-4031 (D) or activator PD 118057 (E) was measured using CCK-8 or colony formation assay (n = 6). (F) Protein expression of hERG1 in HEK293-wt and HEK293-hERG1 cells was detected by Western blot. (G) 1 × 105 HEK293-wt and HEK293-hERG1 cells were cultured for 48 h and the CCK-8 assay was performed. (H) The effects of E-4031 on the proliferation of HEK293-wt and HEK293-hERG1 cells were determined by CCK-8 assay. * P < 0.05, ** P < 0.01, *** P < 0.001.

Mentions: In order to delineate the role of hERG1 in osteosarcoma tumorigenesis, hERG1 knockdown was performed in MG-63 cells by transfecting in hERG1-siRNA. Following transfection, hERG1-siRNA treated cells had significantly lower hERG1 protein levels than cells given control-siRNA (Fig. 2A), indicating that hERG1-siRNA is an efficient means of hERG1 knockdown. Next, the effects of hERG1 knockdown on MG-63 cellular proliferation and growth were assessed using CCK-8 and colony formation assays following treatment with hERG1-siRNA (30 nM) for 48 h. As shown in Fig. 2B and 2C, hERG1-siRNA transfected cells had a remarkable decrease in proliferation and growth compared to untreated and control-siRNA treated cells. Similarly, proliferation was also attenuated in MG-63 cells incubated for 48 h with E-4031 (10 μM and 20 μM), a hERG1 specific inhibitor (Fig. 2D). Furthermore, incubation for 48 h with PD 118057 (5 μM and 10 μM), a hERG1 activator, resulted in an increase in MG-63 cell proliferation compared to untreated cells (Fig. 2E). Next, hERG1 protein levels in HEK293-hERG1 and HEK293-wt cells were measured by Western blot (Fig. 2F). There was an apparent increase in HEK293-hERG1 cell proliferation compared to the HEK-293wt cells after 48 h of incubation (Fig. 2G). When HEK293-wt and HEK293-hERG1 cells were treated with E-4031 (20 μM) for 48 h, a similar phenotype to MG-63 cells was observed, where the treated HEK293-hERG1 cellular proliferation was inhibited using E-4031 (Fig. 2H). These results indicated that hERG1 plays a critical role in the proliferation of osteosarcoma cells.


Silencing of hERG1 Gene Inhibits Proliferation and Invasion, and Induces Apoptosis in Human Osteosarcoma Cells by Targeting the NF-κB Pathway.

Zeng W, Liu Q, Chen Z, Wu X, Zhong Y, Wu J - J Cancer (2016)

Knockdown of hERG1 reduces proliferation of osteosarcoma cells. (A) Efficiency of knockdown by hERG1-siRNA was measured by Western blot. (B-E) Proliferation of MG-63 cells transfected with hERG1-siRNA (30 nM) (B and C), or treated with hERG1 inhibitor E-4031 (D) or activator PD 118057 (E) was measured using CCK-8 or colony formation assay (n = 6). (F) Protein expression of hERG1 in HEK293-wt and HEK293-hERG1 cells was detected by Western blot. (G) 1 × 105 HEK293-wt and HEK293-hERG1 cells were cultured for 48 h and the CCK-8 assay was performed. (H) The effects of E-4031 on the proliferation of HEK293-wt and HEK293-hERG1 cells were determined by CCK-8 assay. * P < 0.05, ** P < 0.01, *** P < 0.001.
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Figure 2: Knockdown of hERG1 reduces proliferation of osteosarcoma cells. (A) Efficiency of knockdown by hERG1-siRNA was measured by Western blot. (B-E) Proliferation of MG-63 cells transfected with hERG1-siRNA (30 nM) (B and C), or treated with hERG1 inhibitor E-4031 (D) or activator PD 118057 (E) was measured using CCK-8 or colony formation assay (n = 6). (F) Protein expression of hERG1 in HEK293-wt and HEK293-hERG1 cells was detected by Western blot. (G) 1 × 105 HEK293-wt and HEK293-hERG1 cells were cultured for 48 h and the CCK-8 assay was performed. (H) The effects of E-4031 on the proliferation of HEK293-wt and HEK293-hERG1 cells were determined by CCK-8 assay. * P < 0.05, ** P < 0.01, *** P < 0.001.
Mentions: In order to delineate the role of hERG1 in osteosarcoma tumorigenesis, hERG1 knockdown was performed in MG-63 cells by transfecting in hERG1-siRNA. Following transfection, hERG1-siRNA treated cells had significantly lower hERG1 protein levels than cells given control-siRNA (Fig. 2A), indicating that hERG1-siRNA is an efficient means of hERG1 knockdown. Next, the effects of hERG1 knockdown on MG-63 cellular proliferation and growth were assessed using CCK-8 and colony formation assays following treatment with hERG1-siRNA (30 nM) for 48 h. As shown in Fig. 2B and 2C, hERG1-siRNA transfected cells had a remarkable decrease in proliferation and growth compared to untreated and control-siRNA treated cells. Similarly, proliferation was also attenuated in MG-63 cells incubated for 48 h with E-4031 (10 μM and 20 μM), a hERG1 specific inhibitor (Fig. 2D). Furthermore, incubation for 48 h with PD 118057 (5 μM and 10 μM), a hERG1 activator, resulted in an increase in MG-63 cell proliferation compared to untreated cells (Fig. 2E). Next, hERG1 protein levels in HEK293-hERG1 and HEK293-wt cells were measured by Western blot (Fig. 2F). There was an apparent increase in HEK293-hERG1 cell proliferation compared to the HEK-293wt cells after 48 h of incubation (Fig. 2G). When HEK293-wt and HEK293-hERG1 cells were treated with E-4031 (20 μM) for 48 h, a similar phenotype to MG-63 cells was observed, where the treated HEK293-hERG1 cellular proliferation was inhibited using E-4031 (Fig. 2H). These results indicated that hERG1 plays a critical role in the proliferation of osteosarcoma cells.

Bottom Line: Knockdown of hERG1 significantly suppressed cellular proliferation and invasion, and induced apoptosis, while inhibition of hERG1 significantly decreased activation of NF-κB.Overall, hERG1 may stimulate nuclear translocation of p65, thus regulating the NF-κB pathway through the activation of the hERG1/beta1 integrin complex and PI3K/AKT signaling.Furthermore, this regulation by hERG1 is, at least in part, through mediation of the NF-κB pathway.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Orthopaedics, the Affiliated Southeast Hospital of Xiamen University, Orthopaedic Center of People's Liberation Army, Zhangzhou, 363000, People's Republic of China.

ABSTRACT
Recently, the human ether à go-go (eag) related gene 1 (hERG1) channel, a member of the voltage-dependent potassium channel (Kv) family, was determined to have a critical role in cancer cell proliferation, invasion, tumorigenesis and apoptosis. However, the expression levels and functions of hERG1 in osteosarcoma cells remain poorly characterized. In this study, hERG1 transcript and protein levels in osteosarcoma cells and tissues were measured using semi-quantitative real time PCR (RT-PCR), Western blot, and immunohistochemistry. The effects of hERG1 knockdown on osteosarcoma cell proliferation, apoptosis and invasion were examined using CCK-8, colony formation, flow cytometry, caspase-3 activity, wound healing and transwell based assays. Furthermore, semi-quantitative RT-PCR, Western blot and a luciferase reporter assay were used to assess the effects of hERG1 inhibition on the nuclear factor-κB (NF-κB) pathway. In addition, the effect of NF-κB p65-siRNA and NF-κB p65 expression on the survival of osteosarcoma cells was investigated. Through this work, a relationship for hERG1 with the NF-κB pathway was identified. Osteosarcoma cells and tissues were found to express high levels of hERG1. Knockdown of hERG1 significantly suppressed cellular proliferation and invasion, and induced apoptosis, while inhibition of hERG1 significantly decreased activation of NF-κB. Overall, hERG1 may stimulate nuclear translocation of p65, thus regulating the NF-κB pathway through the activation of the hERG1/beta1 integrin complex and PI3K/AKT signaling. Taken together, these results demonstrate that hERG1 is necessary for regulation of osteosarcoma cellular proliferation, apoptosis and migration. Furthermore, this regulation by hERG1 is, at least in part, through mediation of the NF-κB pathway.

No MeSH data available.


Related in: MedlinePlus