shRNA敲减Toll样受体4对乳腺癌细胞MDA–MB–231侵袭性的影响
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摘要
目的:探讨沉默Toll样受体4(TLR4)基因后对乳腺癌细胞侵袭性的影响,为乳腺癌治疗提供新思路。方法:(1)用重组慢病毒感染乳腺癌MD–MBA–231细胞,干扰TLR4基因的表达,Western blot法检测MDA–MB–231/shTLR4(沉默)的TLR4蛋白表达水平。(2)加入外源性刺激因子高迁移率族蛋白1(HMGB1)并设置空白对照磷酸盐缓冲液(PBS),Western blot法检测在乳腺癌细胞MDA–MB–231/shTLR4(沉默)及MDA–MB–231/shControl(对照)细胞中下游信号因子相关蛋白核因子κB(NF–κB)、白细胞介素(IL)–6、血管内皮生长因子a(VEGFa)的表达水平。结果:(1)在沉默TLR4基因后,MDA–MB–231/shTLR4细胞的TLR4蛋白水平均明显降低,差异具有统计学意义(P<0.05)。(2)沉默TLR4基因后,HMGB1对于MDA–MB–231/shTLR4(沉默)细胞中下游信号因子相关蛋白NF–κB、IL–6、VEGFa的表达水平促进作用较MDA–MB–231/sh Control(对照)明显降低,差异具有统计学意义(P<0.01)。结论:沉默TLR4基因后,MDA–MB–231/sh TLR4(沉默)下游信号因子相关蛋白NF–κB、IL–6、VEGFa表达明显降低,从而降低了乳腺癌细胞的侵袭性。
Objective To investigate the effect of the toll-like receptor 4(TLR4) silencing on the invasion of breast cancer cells and therefore pave the way for developing a novel breast cancer treatment strategy. Method(1) Recombinant lentiviruses were used to infect MD-MBA-231 cell lines to interfere the expression of TLR4. The expression of TLR4 was examined in MDA–MB–231/sh TLR4 via Western Blot.(2) Exogenous high mobility group box-1(HMGB1) was used to stimulate the TLR4-silenced MDAMB-231 cells(MDA-MB-231/sh TLR4) and TLR-4 expressing MDA-MB-231 cells(MDA-MB-231/sh Control), where phosphate buffer saline(PBS) was used as negative control. The expressions of downstream signaling factor were examined, including nudear factor κB(NF-κB), IL-6 and VEGFa. Result(1) The expression of TLR4 significantly decreased in TLR4-silenced MDA-MB-231 cells(P < 0.05).(2) The enhancing effect of HMGB1 on the expression of NF-κB, IL-6 and VEGFa was significantly reduced in MDA-MB-231/sh TLR4, compared with MDA-MB-231/sh Control.(P < 0.01). Conclusion After silence TLR4 gene, the expression of NF-κB, IL-6 and VEGFa downstream of MDA-MB-231/sh TLR4(silenced) signalling was significantly reduced, which reduced the invasiveness of breast cancer cells.
Objective To investigate the effect of the toll-like receptor 4(TLR4) silencing on the invasion of breast cancer cells and therefore pave the way for developing a novel breast cancer treatment strategy. Method(1) Recombinant lentiviruses were used to infect MD-MBA-231 cell lines to interfere the expression of TLR4. The expression of TLR4 was examined in MDA–MB–231/sh TLR4 via Western Blot.(2) Exogenous high mobility group box-1(HMGB1) was used to stimulate the TLR4-silenced MDAMB-231 cells(MDA-MB-231/sh TLR4) and TLR-4 expressing MDA-MB-231 cells(MDA-MB-231/sh Control), where phosphate buffer saline(PBS) was used as negative control. The expressions of downstream signaling factor were examined, including nudear factor κB(NF-κB), IL-6 and VEGFa. Result(1) The expression of TLR4 significantly decreased in TLR4-silenced MDA-MB-231 cells(P < 0.05).(2) The enhancing effect of HMGB1 on the expression of NF-κB, IL-6 and VEGFa was significantly reduced in MDA-MB-231/sh TLR4, compared with MDA-MB-231/sh Control.(P < 0.01). Conclusion After silence TLR4 gene, the expression of NF-κB, IL-6 and VEGFa downstream of MDA-MB-231/sh TLR4(silenced) signalling was significantly reduced, which reduced the invasiveness of breast cancer cells.
引文
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[10]Maru Y.premetastatic milieu explained by tlr4 agonist-mediated homeostatic inflammation[J].Cell Mol Immunol.2010,7(2):94-99.
[11]Hollier BG,Tinnirello AA,Werden SJ,et al.FOXC2expression links epithelial-mesenchymal transition and stem cell properties in breast cancer.[J].Cancer Res.2013,73(6):1981-1992.
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[13]Li D,Xie K,Ding G,Li J,et al.Tumor resistance to anti-VEGF therapy through up-regulation of VEGF-C expression[J].Cancer Lett,2013,346(1):45-52.
[2]吴坤琳,陈祥锦,张惠灏,等.Toll样因子受体4/髓样分化因子88信号通路对乳腺癌细胞侵袭性的影响[J].中华实验外科杂志,2015,32(5):966-968.
[3]Mantovani A.Cancer:Infl aming metastasis[J].Nature,2009,457(7225):36-37.
[4]Zhang J,Kou YB,Zhu JS,et al.Knockdown of HMGB1inhibits growth and invasion of gastric cancer cells through the NF-κB pathway in vitro and in vivo[J].International Journal of Oncology,2014,44(4):1268-1276.
[5]Huang B,Zhao J,Li H,et al.Toll-like receptors ontumor cells facilitatevasion of immune surveillance[J].Cancer research,2005,65(12):5009-5014.
[6]Sas L,Lardon F,Vermeulen PB,et al.The interaction between ER and NF-κB in resistance to endocrine therapy[J].Breast Cancer Res,2012,14(4):1-14.
[7]Baldwin AS.Regulation of cell death and autophagy by IKK and NF-κB:critical mechanisms in immune function and cancer[J].Immunol Rev,2012,246(1):327–345.
[8]Hua D,Liu MY,Cheng ZD,et al.Small interfering RNA-directed targeting of Toll-like receptor 4 inhibits human prostate cancer cell invasion,survival,and tumorigenicity[J].Molecular Immunology,2009,46(15):2876-2884.
[9]Morrison WB.infl ammation and cancer:a comparative view[J].J Vet Intern Med,2012,26(1):18-31.
[10]Maru Y.premetastatic milieu explained by tlr4 agonist-mediated homeostatic inflammation[J].Cell Mol Immunol.2010,7(2):94-99.
[11]Hollier BG,Tinnirello AA,Werden SJ,et al.FOXC2expression links epithelial-mesenchymal transition and stem cell properties in breast cancer.[J].Cancer Res.2013,73(6):1981-1992.
[12]Xie G,Yao Q,Liu Y,et al.IL-6-induced epithelialmesenchymal transition promotes the generation of breast cancer stem-like cells analogous to mammosphere cultures[J].Int J Oncol,2012,40(4):1171-1179.
[13]Li D,Xie K,Ding G,Li J,et al.Tumor resistance to anti-VEGF therapy through up-regulation of VEGF-C expression[J].Cancer Lett,2013,346(1):45-52.
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