The Roles of K+ Channel Interacting Protein 4 in Breast Cancer Progression
Date Issued
2016
Date
2016
Author(s)
Chen, Yen-Hao
Abstract
Purpose: K+ channel interacting protein 4 (KCNIP4) belongs to EF-hand protein superfamily which modulates A type potassium channels and regulates their surface expression. Previous studies have showed that K+ channels are expressed in a variety of cells, including cancer cells. However, the regulatory mechanism of KCNIP4 in breast cancer progression was completely unknown. Method: Human breast cancer cell lines (MCF7, T47D, MDA-MB-361, SkBr-3, and MDA-MB-231) were used as in vitro model to investigate cell invasion and proliferation by KCNIP4 expression plasmid and silenced-KCNIP4 (shKCNIP4) transfection. Boyden chamber assay was performed to check migration and invasion abilities. Proliferation ability was identified by MTT assay and flow cytometry. Methylation PCR and chromatin immunoprecipitation (ChIP) assay were performed to verify the methylation status on ER promoter and H3K4 trimethylation expression in shKCNIP4 transfectants. Mice were orthotopic injection with stable transfected KCNIP4 or vector control in MDA-MB-231 cells to measure metastasis. Results: K+ channel interacting protein 4 was negatively correlated with survival rate in breast cancer patients. KCNIP4 expression was positively correlated with invasion ability in breast cancer cells. Transiently transfected T47D and MDA-MB-231 cells with shKCNIP4 knockdown plasmids significantly reduced invasion ability (P< 0.05) but did not affect proliferation ability. Furthermore, we established shKCNIP4-stable transfectants in MDA-MB-231 cells, and found that silencing KCNIP4 could significantly down-regulated migration and invasion ability (P< 0.05) but did not affect proliferation ability. In mechanically level, silenced-KCNIP4 significantly up-regulated estrogen receptor (ER) in ERα-negative breast cancer cell lines both in mRNA level and protein level, which resulted in re-sensitizing to ER inhibitors treatment. Additionally, we performed high throughput mRNA microarray analysis and identified a downstream effecter, PRMT6. Moreover, transiently transfected PRMT6 expression plasmid could restore invasion ability in shKCNIP4 transfectants (P< 0.05). We also found that silenced-KCNIP4 decreased the methylation status on ERα promoter but overexpressed- PRMT6 in shKCNIP4 transfectants could reverse the effect. In ChIP assay, we verified that silenced-KCNIP4 could up-regulate H3K4 trimethylation expression but overexpressed-PRMT6 in shKCNIP4 transfectants turned down the effect (P< 0.05). Tumor metastasis ability was decreased in orthotopic injection in mammary fat pad with shKCNIP4 transfectants, compared with control mice in vivo.
Subjects
KCNIP4
invasion
estrogen receptor
breast cancer
SDGs
Type
thesis
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