摘要
目的建立稳定表达E6-AP的前列腺癌细胞系,通过对E6-AP表达调控的研究,探讨E6相关蛋白对前列腺癌细胞增殖与侵袭作用.方法以人前列腺癌细胞株(LNCa P)为基础,将调控质粒pRev Tet-offin和E6-AP表达质粒p GEM-E6-AP转染至LNCa P细胞,建立既能表达E6-AP蛋白又能受DOX调控的LNCa P/E6-AP细胞系.Western bloting检测LNCa P/E6-AP细胞E6-AP蛋白表达及其受DOX调控情况; MTT方法检测细胞增殖及受LY294002抑制情况;基质胶侵袭实验检测细胞浸润迁移能力.结果 LNCa P/E6-AP细胞E6-AP表达量明显高于LNCa P细胞,且可受DOX负向调控;实验组LNCa P/E6-AP细胞的增殖能力明显高于对照组LNCa P细胞,且两组细胞增殖能力均受PI3K抑制剂LY294002的抑制;实验组LNCa P/E6-AP细胞的侵袭能力明显高于对照组LNCa P细胞.结论 E6-AP可以促进前列腺癌细胞的增殖,并增加其侵袭能力; DOX可以调控E6-AP的表达;PI3K抑制剂LY294002可有效抑制前列腺癌细胞的增殖能力.
Objective To establish a prostate cancer cell line stably expressing E6-AP,and to study the proliferation and invasion of E6-associated protein on prostate cancer cells by regulating the expression of E6-AP.Method Based on human prostate cancer cell line(LNCaP),the regulatory plasmid pRevTet-offin and E6-AP expression plasmids p GEM-E6 AP were transfected into LNCaP cells to establish both E6-ap protein and DOX regulation LNCaP/E6-AP cells line. Western blotting was used to detect the expression of E6-AP protein in LNCaP-AP cells and the regulation by DOX. MTT assay was used to detect cell proliferation and inhibition by LY294002; Matrigel invasion assay was used to detect cell infiltration and migration ability. Results The expression of E6-AP in LNCaP/E6-AP cells was significantly higher than that of LNCaP cells,and it was negative by DOX.The proliferation ability of LNCaP/E6-AP cells in experimental group was higher than LNCap cell of control group.The proliferation ability was inhibited by P3 IK inhibitor LY294002,and the invasive ability of LNCaP/E6-AP cells in the experimental group was significantly higher than that in the control group.Conclusion E6-AP can promote proliferation of prostate cancer cells and increase its invasive ability; DOX can regulate the expression of E6-AP,PI3 K inhibitor LY294002 can effectively inhibit the proliferation of prostate cancer cells.
引文
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