E6相关蛋白对前列腺癌细胞增殖与侵袭的作用
|
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.
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
[1]张凯.中国前列腺癌外科治疗专家共识[J].中华外科杂志,2017,10(55):721-724.
[2] Chen W,Zheng R,Baade P D,et al. Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66(2):115-132.
[3] Center M M,Jemal A,Lortet-Tieulent J,et al.International variation in prostate cancer incidence and mortality rates[J].Eur Urol,2012,61(6):1079-1092.
[4] Klotz L,O’Callaghan C,Ding K,et al.Nadir testosterone within first year of androgen-deprivation therapy(ADT)predicts for time to castration-resistant progression:a secondary analysis of the PR-7 trial of intermittent versus continuous ADT[J].J Clin Oncol,2015,33(10):1151-1156.
[5] Wirth M,Tammela T,Cicalese V,et al. Prevention of bone metastases in patients with high-risk nonmetastatic prostate cancer treated with zoledronic acid:Efficacy and safety results of the Zometa European Study(ZEUS)[J]. Eur Urol,2015,67(3):482-491.
[6] Xu Z B,Wang G M,Sun L A,et al. The incidence of prostatic carcinoma in Shanghai[J]. Clinical Medical Journal of China,2003,10(3):344-346.
[7] Culig Z,Klocker H,Bartsch G,et al. Androgen receptors in prostate cancer[J].J Urol,2003,170(1):1363-1369.
[8] Nawaz Z,Lonard D M,Smith C L,et al. The Angelman syndrome-associated protein,E6-AP,is a coactivator for the nuclear hormone receptor superfamily[J]. Mol Cell Biol,1999,19(2):1182-1189.
[9] Smith C L,De Vera D G,Lamb D J,et al.Genetic ablation of the steroid receptor coactivator-ubiquitin ligase,E6-AP results in tissue-selective steroid hormone resistance and effects in reproduction[J]. Mol Cell Biol,2002,22(2):525-535.
[2] Chen W,Zheng R,Baade P D,et al. Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66(2):115-132.
[3] Center M M,Jemal A,Lortet-Tieulent J,et al.International variation in prostate cancer incidence and mortality rates[J].Eur Urol,2012,61(6):1079-1092.
[4] Klotz L,O’Callaghan C,Ding K,et al.Nadir testosterone within first year of androgen-deprivation therapy(ADT)predicts for time to castration-resistant progression:a secondary analysis of the PR-7 trial of intermittent versus continuous ADT[J].J Clin Oncol,2015,33(10):1151-1156.
[5] Wirth M,Tammela T,Cicalese V,et al. Prevention of bone metastases in patients with high-risk nonmetastatic prostate cancer treated with zoledronic acid:Efficacy and safety results of the Zometa European Study(ZEUS)[J]. Eur Urol,2015,67(3):482-491.
[6] Xu Z B,Wang G M,Sun L A,et al. The incidence of prostatic carcinoma in Shanghai[J]. Clinical Medical Journal of China,2003,10(3):344-346.
[7] Culig Z,Klocker H,Bartsch G,et al. Androgen receptors in prostate cancer[J].J Urol,2003,170(1):1363-1369.
[8] Nawaz Z,Lonard D M,Smith C L,et al. The Angelman syndrome-associated protein,E6-AP,is a coactivator for the nuclear hormone receptor superfamily[J]. Mol Cell Biol,1999,19(2):1182-1189.
[9] Smith C L,De Vera D G,Lamb D J,et al.Genetic ablation of the steroid receptor coactivator-ubiquitin ligase,E6-AP results in tissue-selective steroid hormone resistance and effects in reproduction[J]. Mol Cell Biol,2002,22(2):525-535.