siRNA特异性沉默ADP核糖基化因子6对前列腺癌PC-3细胞增殖、迁移和侵袭的影响（英文）
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摘要
目的研究ADP核糖基化因子6(Arf6)对雄激素非依赖性前列腺癌PC-3细胞株增殖、迁移和侵袭能力的影响并初步探讨其可能的分子作用机制。方法设计合成3条针对不同靶向区域的Arf6特异性si RNA序列,转染细胞后通过real-time PCR和蛋白质印迹法检测其对Arf6的干扰效果,筛选出干扰效果最佳的si RNA序列;通过噻唑盐(MTT)实验、划痕实验、及transwell细胞迁移和侵袭实验观察si RNA干扰Arf6表达对PC-3细胞增殖、迁移和侵袭的影响;蛋白质印迹法检测AKT、p-AKT、ERK1/2、p-ERK1/2和Rac1蛋白表达水平的变化。结果与空白对照组相比,转染阴性对照序列对PC-3细胞内源性Arf6的m RNA和蛋白表达水平无明显影响,3条si RNA序列均能抑制Arf6的表达,其中si RNA-3对PC-3细胞Arf6表达干扰效果最好,Arf6m RNA和蛋白抑制率分别为(91.88±3.13)%和(86.37±0.57)%。si RNA-3干扰Arf6表达抑制PC-3细胞的增殖,且PC-3细胞体外迁移距离和侵袭细胞数较空白和阴性对照组明显减少(P<0.05)。蛋白质印迹法检测发现转染si RNA-3的PC-3细胞p-ERK1/2和Rac1表达水平明显降低,而AKT、p-AKT和ERK1/2表达水平较对照组差异无统计学意义。结论 si RNA干扰Arf6表达可显著抑制PC-3细胞的增殖、迁移和侵袭能力,其分子作用机制可能与p-ERK1/2和Rac1表达下调相关。
Objective To investigate the effects of silencing ADP-ribosylation factor 6(Arf6) on the proliferation, migration, and invasion of prostate cancer cell line PC- 3 and the possible molecular mechanisms. Methods Three Arf6- specific small interfering RNA(si RNA) were transfected into cultured prostate cancer cell line PC-3. Arf6 expression was examined by realtime PCR and Western blotting. MTT assay, wound healing assay, and Transwell migration and invasion assay were used to observe the effect of Arf6 silencing on the proliferation, migration, and invasion ability of PC- 3 cells. The levels of phosphorylated extracellular signal-regulated kinase 1/2(p-ERK1/2), ERK1/2, p-AKT, AKT and Rac1 were detected by Western blotting. Results Transfection of si RNA- 3 resulted in significantly decreased Arf6 m RNA and protein expression with inhibition rates of(91.88 ± 3.13)% and(86.37 ± 0.57)%, respectively. Arf6 silencing by si RNA- 3 markedly suppressed the proliferation, migration and invasion of PC- 3 cells and reduced the expression levels of p- ERK1/2 and Rac1. Conclusion Silencing of Arf6 efficiently inhibits the proliferation, migration, and invasion of PC- 3 cells in vitro, and the underlying mechanisms may involve the down-regulation of p-ERK1/2 and Rac1.
Objective To investigate the effects of silencing ADP-ribosylation factor 6(Arf6) on the proliferation, migration, and invasion of prostate cancer cell line PC- 3 and the possible molecular mechanisms. Methods Three Arf6- specific small interfering RNA(si RNA) were transfected into cultured prostate cancer cell line PC-3. Arf6 expression was examined by realtime PCR and Western blotting. MTT assay, wound healing assay, and Transwell migration and invasion assay were used to observe the effect of Arf6 silencing on the proliferation, migration, and invasion ability of PC- 3 cells. The levels of phosphorylated extracellular signal-regulated kinase 1/2(p-ERK1/2), ERK1/2, p-AKT, AKT and Rac1 were detected by Western blotting. Results Transfection of si RNA- 3 resulted in significantly decreased Arf6 m RNA and protein expression with inhibition rates of(91.88 ± 3.13)% and(86.37 ± 0.57)%, respectively. Arf6 silencing by si RNA- 3 markedly suppressed the proliferation, migration and invasion of PC- 3 cells and reduced the expression levels of p- ERK1/2 and Rac1. Conclusion Silencing of Arf6 efficiently inhibits the proliferation, migration, and invasion of PC- 3 cells in vitro, and the underlying mechanisms may involve the down-regulation of p-ERK1/2 and Rac1.
引文
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[26]Palacios F,D'Souza-Schorey C.Modulation of Rac1 and ARF6activation during epithelial cell scattering[J].J Biol Chem,2003,278(19):17395-400.
[2]Li H,Zhang Y,Zhang Y,et al.Rsf-1 overexpression in human prostate cancer,implication as a prognostic marker[J].Tumor Biol,2014,35(6):5771-6.
[3]de Bono JS,Oudard S,Ozguroglu M,et al.Prednisone plus cabazitaxel or mitoxantrone for metastatic castration-resistant prostate cancer progressing after docetaxel treatment:a randomised open-label trial[J].Lancet,2010,376(9747):1147-54.
[4]Grant CM,Kyprianou N.Epithelial mesenchymal transition(EMT)in prostate growth and tumor progression[J].Transl Androl Urol,2013,2(3):202-11.
[5]Schweitzer JK,Sedgwick AE,D'Souza-Schorey C.ARF6-mediated endocytic recycling impacts cell movement,cell division and lipid homeostasis[J].Semin Cell Dev Biol,2011,22(1):39-47.
[6]Hongu T,Kanaho Y.Activation machinery of the small GTPase Arf6[J].Adv Biol Regul,2014,54:59-66.
[7]Hu B,Shi B,Jarzynka MJ,et al.ADP-ribosylation factor 6 regulates glioma cell invasion through the IQ-domain GTPase-activating protein 1-Rac1-mediated pathway[J].Cancer Res,2009,69(3):794-801.
[8]Hu Z,Du J,Yang L,et al.GEP100/Arf6 is required for epidermal growth factor-induced ERK/Rac1 signaling and cell migration in human hepatoma Hep G2 cells[J].PLo S One,2012,7(6):e38777.
[9]Li M,Wang J,Ng SS,et al.Adenosine diphosphate-ribosylation factor 6 is required for epidermal growth factor-induced glioblastoma cell proliferation[J].Cancer,2009,115(21):4959-72.
[10]Mccubrey JA,Steelman LS,Chappell WH,et al.Roles of the Raf/MEK/ERK pathway in cell growth,malignant transformation and drug resistance[J].BBA Mol Cell Res,2007,1773(8):1263-84.
[11]Tian Y,Guan Y,Jia Y,et al.Chloride intracellular channel 1regulates prostate cancer cell proliferation and migration through the MAPK/ERK pathway[J].Cancer Biother Radio,2014,29(8):339-44.
[12]Henderson V,Smith B,Burton LJ,et al.Snail promotes cell migration through PI3K/AKT-dependent Rac1 activation as well as PI3K/AKT-independent pathways during prostate cancer progression[J].Cell Adh Migr,2015,9(4):255-64.
[13]Kato T,Kawai K,Egami Y,et al.Rac1-dependent lamellipodial motility in prostate cancer PC-3 cells revealed by optogenetic control of Rac1 activity[J].PLo S One,2014,9(5):e97749.
[14]Chan CH,Jo U,Kohrman A,et al.Posttranslational regulation of Akt in human cancer[J].Cell Biosci,2014,4(1):59.
[15]Wei Q,Costanzi S,Balasubramanian R,et al.A2B adenosine receptor blockade inhibits growth of prostate cancer cells[J].Purinerg Signal,2013,9(2):271-80.
[16]Wei Q,Costanzi S,Liu Q,et al.Activation of the P2Y1 receptor induces apoptosis and inhibits proliferation of prostate cancer cells[J].Biochem Pharmacol,2011,82(4):418-25.
[17]Knizhnik AV,Kovaleva OV,Komelkov AV,et al.Arf6 promotes cell proliferation via the PLD-m TORC1 and p38MAPK pathways[J].JCell Biochem,2012,113(1):360-71.
[18]Taichman RS,Loberg RD,Mehra R,et al.The evolving biology and treatment of prostate cancer[J].J Clin Invest,2007,117(9):2351-61.
[19]Xu R,Zhang Y,Gu L,et al.Arf6 regulates EGF-induced internalization of E-cadherin in breast cancer cells[J].Cancer Cell Int,2015,15(1):11.
[20]Grossmann AH,Yoo JH,Clancy J,et al.The small GTPase ARF6stimulatesβ-catenin transcriptional activity during WNT5A-mediated melanoma invasion and metastasis[J].Sci Signal,2013,6(265):a14.
[21]Muralidharan-Chari V,Hoover H,Clancy J,et al.ADP-ribosylation factor 6 regulates tumorigenic and invasive properties in vivo[J].Cancer Res,2009,69(6):2201-9.
[22]Zhang Y,Du J,Zheng J,et al.EGF-reduced Wnt5a transcription induces epithelial-mesenchymal transition via Arf6-ERK signaling in gastric cancer cells[J].Oncotarget,2015,6(9):7244-61.
[23]Smyth D,Mckay CM,Gulbransen BD,et al.Interferon-gamma signals via an ERK1/2-ARF6 pathway to promote bacterial internalization by gut epithelia[J].Cell Microbio,2012,14(8):1257-70.
[24]Parri M,Chiarugi P.Rac and Rho GTPases in cancer cell motility control[J].Cell Commun Signal,2010,8:23.
[25]Kobayashi T,Inoue T,Shimizu Y,et al.Activation of Rac1 is closely related to androgen-independent cell proliferation of prostate cancer cells both in vitro and in vivo[J].Mol Endocrinol,2010,24(4):722-734.
[26]Palacios F,D'Souza-Schorey C.Modulation of Rac1 and ARF6activation during epithelial cell scattering[J].J Biol Chem,2003,278(19):17395-400.