下调人质膜型唾液酸酶对前列腺癌PC-3细胞增殖、侵袭能力的影响及其机制
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摘要
目的探讨人质膜型唾液酸酶(Neu3)表达降低对前列腺癌PC-3细胞增殖、迁移、侵袭能力的影响。方法构建p GCsi-Neu3质粒,从Gene Bank中寻找符合Neu3 m RNA特异的靶序列,合成si RNA模板链后与载体相连接,转入感受态细胞,测序鉴定。应用真核细胞转染技术转染前列腺癌PC-3细胞,利用荧光显微镜检测转染效率;细胞增殖实验检测p GCsi-Neu3质粒对细胞增殖的影响;流式细胞术检测细胞凋亡的改变;Transwell迁移、侵袭实验检测细胞迁移、侵袭能力的变化。结果针对Neu3基因设计了si RNA序列并成功构建p GCsi-Neu3质粒,p GCsi-Neu3-3质粒对基因Neu3的抑制最为有效。与对照组比较,p GCsi-Neu3-3质粒可有效抑制肿瘤细胞增殖(P<0.01);p GCsi-Neu3-3质粒可引起前列腺癌PC-3细胞凋亡,凋亡率明显高于空白对照组(MOCK组),差异有统计学意义(P<0.05);p GCsi-Neu3-3质粒可使前列腺癌PC-3细胞增殖细胞核抗原表达明显下降;p GCsiNeu3-3质粒可抑制前列腺癌PC-3细胞迁移、侵袭能力,且与对照组比较,差异有高度统计学意义(P<0.01)。结论针对Neu3基因si RNA表达质粒的构建、鉴定和初始研究,为Neu3对前列腺癌细胞生长、转移发挥作用机制的研究奠定实验基础。
Objective To investigate the influence of Neu3 gene silencing in growth inhibition of PC-3 cell and to elucidate the underlying mechanisms.Methods Three pieces of si RNA sequences on the Neu3 gene were designed and then a recombinant plasmid carrying si RNA was constructed,which was used to transfer PC-3 cells in order to screen the most effective sequence among the three si RNA sequences against Neu3;PC-3 cell transfection p GCsi-Neu3 plasmid,inverted microscope was used to observe the transfection efficiency,determined by MTT test their effects on cell proliferation and flow cytometry was used to detect the change of cell apoptosis;Transwell migration and invasion assay were performed to detect the migration and invasion cells of groups.Results The si RNA sequences targeted to Neu3 gene were designed and their expression vectors were cloned and verified successfully.Compared with control group,p GCsiNeu3-3 plasmid significantly inhibited cell proliferation(P < 0.01);p GCsi-Neu3-3 plasmid significantly promoted the apoptosis of PC-3 cells of prostate cancer compared with the blank control group(Mock group),the difference was statistically significant(P < 0.05).p GCsi-Neu3-3 plasmid could inhibit the migration and invasion assay of PC-3 cells of prostate cancer,compared with the control group,with statistically significant difference(P < 0.01).Conclusion Construction,identification and initial study of Neu3,might lay the foundation of experiment for the study of growth and metastasis mechanism of prostate cancer cell.
Objective To investigate the influence of Neu3 gene silencing in growth inhibition of PC-3 cell and to elucidate the underlying mechanisms.Methods Three pieces of si RNA sequences on the Neu3 gene were designed and then a recombinant plasmid carrying si RNA was constructed,which was used to transfer PC-3 cells in order to screen the most effective sequence among the three si RNA sequences against Neu3;PC-3 cell transfection p GCsi-Neu3 plasmid,inverted microscope was used to observe the transfection efficiency,determined by MTT test their effects on cell proliferation and flow cytometry was used to detect the change of cell apoptosis;Transwell migration and invasion assay were performed to detect the migration and invasion cells of groups.Results The si RNA sequences targeted to Neu3 gene were designed and their expression vectors were cloned and verified successfully.Compared with control group,p GCsiNeu3-3 plasmid significantly inhibited cell proliferation(P < 0.01);p GCsi-Neu3-3 plasmid significantly promoted the apoptosis of PC-3 cells of prostate cancer compared with the blank control group(Mock group),the difference was statistically significant(P < 0.05).p GCsi-Neu3-3 plasmid could inhibit the migration and invasion assay of PC-3 cells of prostate cancer,compared with the control group,with statistically significant difference(P < 0.01).Conclusion Construction,identification and initial study of Neu3,might lay the foundation of experiment for the study of growth and metastasis mechanism of prostate cancer cell.
引文
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[15]Takahashi K,Hosono M,Sato I,et al.Sialidase NEU3contributes neoplastic potential on colon cancer cells as a key modulator of gangliosides by regulating Wnt signaling[J].International Journal of Cancer,2015,137(7):1560-1573.
[16]Mayata M,Kambe M,Tajima O,et al.Membrane sialidase NEU3 is highly expressed in human melanoma cells promoting cell growth with minimal changes in the composition of gangliosides[J].Cancer Sci,2011,102(12):2139-2149.
[17]Takahashi K,Proshin S,Yamaquchi K,et al.Sialidase NEU3 defines invasive potential of human glioblastoma cells by regulating calpain-mediated proteolysis of focal adhesion proteins[J].Biochim Biophys Acta,2017,1861(11):2778-2788.
[18]Shiga K,Takahashi K,Sato I,et al.Up regulation of sialidase NEU3 in head and neck squamous cell carcinoma associated with lymph node metastasis[J].Cancer Sci,2015,106(11):1544-1553.
[19]肖安,彭蓉蓉.ECT 2基因沉默调控人乳腺癌细胞增殖和凋亡及其机制探讨[J].肿瘤,2017,(37):594-603.
[20]房前,杜文亮,王军起,等.转染tbx2 siRNA对人前列腺癌细胞增殖,迁移,侵袭及上皮-间质转化的影响[J].山东医药,2017,57(23):35-38.
[21]Mahler M,Miyachi K,Peebles C,et al.The clinical significance of autoantibodies to the proliferating cell nuclear antigen(PCNA)[J].Autoimmun Rev,2012,11(10):771-775.
[2]Sato H,Niimi A,Yasuhara T,et al.DNA double-strand break repair pathway regulates PD-L1 expression in cancer cells[J].Nat Commun,2017,8(1):1751.
[3]Smith L,Farzan R,Ali S,et al.The responses of cancer cells to PLK1 inhibitors reveal a novel protective role for p53 in maintaining centrosome separation[J].Sci Rep,2017,7(1):16 115.
[4]Arami S,Mandavi M,Rashidi MR,et al.Apoptosis induction activity and molecular docking studies of survivin siRNA carried by Fe3O4-PEG-LAC-chitosan-PEI nanoparticles in MCF-7 human breast cancer cells[J].J Pharm Biomed Anal,2017,142(142):145-154.
[5]Siegel RL,Miller KD,Jemal A.Cancer statistics,2016[J].CA Cancer J Clin,2016,66(1):7-30.
[6]齐金蕾,王黎君,周脉耕,等.1990-2013年中国男性前列腺癌疾病负担分析[J].中华流行病学杂志,2016,37(6):778-782.
[7]Yamamoto K,Takahashi K,Shiozaki K,et al.Potentiation of epidermal growth factor-mediated oncogenic transformation by sialidase NEU3 leading to Src activation[J].PLoS One,2015,10(3):e0120578.
[8]Kawamura S,Sato I,Wada T,et al.Plasma membrane-associated sialidase(NEU3)regulates progression of prostate cancer to androgen-independent growth through modulation of androgen receptor signaling[J].Cell Death Differ,2012,19(1):170-179.
[9]Monti E,Preti A,Rossi E,et al.Cloning and characterization of NEU2,a human gene homologous to rodent soluble sialidases[J].Genomics,1999,57(1):137-143.
[10]Monti E,Preti A,Venerando B,et al.Recent development in mammalian sialidase molecular biology[J].Neurochem Res,2002,27:649-663.
[13]Li X,Zhang L,Shao Y,et al.Effects of a human plasma membrane-associated sialidase siRNA on prostate cancer invasion[J].Biochem Biophys Res Commun,2011,416(3-4):270-276.
[14]Forcella M,Oldani M,Epistolio S,et al.Non-small cell lung cancer(NSCLC),EGFR downstream pathway activation and TKI targeted therapies sensitivity:effect of the plasma membrane-associated NEU3[J].PLoS One,2017,12(10):e0187289.
[15]Takahashi K,Hosono M,Sato I,et al.Sialidase NEU3contributes neoplastic potential on colon cancer cells as a key modulator of gangliosides by regulating Wnt signaling[J].International Journal of Cancer,2015,137(7):1560-1573.
[16]Mayata M,Kambe M,Tajima O,et al.Membrane sialidase NEU3 is highly expressed in human melanoma cells promoting cell growth with minimal changes in the composition of gangliosides[J].Cancer Sci,2011,102(12):2139-2149.
[17]Takahashi K,Proshin S,Yamaquchi K,et al.Sialidase NEU3 defines invasive potential of human glioblastoma cells by regulating calpain-mediated proteolysis of focal adhesion proteins[J].Biochim Biophys Acta,2017,1861(11):2778-2788.
[18]Shiga K,Takahashi K,Sato I,et al.Up regulation of sialidase NEU3 in head and neck squamous cell carcinoma associated with lymph node metastasis[J].Cancer Sci,2015,106(11):1544-1553.
[19]肖安,彭蓉蓉.ECT 2基因沉默调控人乳腺癌细胞增殖和凋亡及其机制探讨[J].肿瘤,2017,(37):594-603.
[20]房前,杜文亮,王军起,等.转染tbx2 siRNA对人前列腺癌细胞增殖,迁移,侵袭及上皮-间质转化的影响[J].山东医药,2017,57(23):35-38.
[21]Mahler M,Miyachi K,Peebles C,et al.The clinical significance of autoantibodies to the proliferating cell nuclear antigen(PCNA)[J].Autoimmun Rev,2012,11(10):771-775.