小电导的钙激活钾离子通道1的真核表达及其在乳腺癌细胞增殖中的作用

Eukaryotic Expression of Small Conductance Calcium-gated K^(+)Channels 1 and Its Role in the Proliferation of Breast Cancer Cells

目的构建重组的真核表达质粒pENTER-小电导的钙激活钾离子通道1(small conductance calcium-gated K+channel 1,SK_(Ca)1),并研究其在乳腺癌细胞密歇根癌症基金会7号(Michigan cancer foundation-7,MCF-7)中的表达;观察SK_(Ca)1的亚细胞定位;检测SK_(Ca)1在乳腺癌细胞增殖中的作用。方法从MCF-7细胞中抽提总RNA并逆转录成cDNA;以cDNA为模板扩增SK_(Ca)1基因,通过无缝克隆技术将基因片段克隆至真核表达质粒pENTER中;质粒转染MCF-7细胞后,分别用Western blot和免疫荧光实验检测SK_(Ca)1的表达及亚细胞定位,用细胞计数试剂盒8(cell counting kit-8,CCK-8)实验测定SK_(Ca)1的细胞增殖效应。结果成功构建了带有标签的重组真核表达质粒pENTER-SK_(Ca)1,并在乳腺癌细胞MCF-7中高效表达;SK_(Ca)1主要分布于细胞质和细胞核,并能促进乳腺癌细胞增殖。结论成功制备了研究SK_(Ca)1的必要工具原料并对其分子机制进行了初步探索,为深入研究SK_(Ca)1在乳腺癌细胞中的信号转导作用奠定了基础。

Objective To construct a recombinant eukaryotic expression plasmid pENTER-small conductance calcium-gated K+channel 1(SK_(Ca)1)and to study its expression in breast cancer cells Michigan cancer foundation-7(MCF-7);to observe the subcellular location of SK_(Ca)1 and to detect the role of SK_(Ca)1 in the proliferation of breast cancer cells.Methods Total RNA was extracted from MCF-7 cells and subsequently processed into cDNA;SK_(Ca)1 gene was amplified from cDNA template and cloned into eukaryotic expression plasmid pENTER following dual-enzyme digestion and ligation reactions;after plasmid trasfection into MCF-7,the expression and subcellular location of SK_(Ca)1 were detected by Western blot and immunofluorescence assays respectively.The cell proliferation effects of SKca1 was measured by cell counting kit-8(CCK-8).Results A recombinant eukaryotic expression plasmid pENTER-SK_(Ca)1 with a tag was successfully constructed.It was highly expressed in breast cancer cells MCF-7;SK_(Ca)1 was mainly distributed in cytoplasm and nucleus,and could promote the proliferation of breast cancer cells.Conclusion The necessary tools and materials for the study of SK_(Ca)1 are successfully prepared and the molecular mechanism of SK_(Ca)1 is preliminarily explored,which lay a foundation for further study of SK_(Ca)1 signaling transduction in breast cancer cells.