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目的:探讨LiCl对乳腺癌细胞系MDA-MB-231侵袭能力的影响,及其对侵袭关键因子NGAL表达的调控。方法:LiCI处理后,MTT测定细胞活力,光镜观察细胞伪足形成;Transwell观察细胞侵袭能力的变化;实时定量PCR及Western blotting观察NGAL在转录和翻译水平的表达;使用小分子药物Bay117082(NF-KB通路抑制剂)和FH535(Wnt/β-catenin通路抑制剂)处理细胞,检测关键通路对NGAL表达的调控。结果:在浓度低于10mmol/L时,LiCl对细胞活性的影响无显著性差异。5mmol/L或10mmol/LLiCl处理细胞24 h后,细胞形态发生明显改变,其伪足形成受到影响,但在NHEl抑制剂Cariporide作用下,细胞伪足破坏的情况有所减轻;Transwell结果显示5、10mmol/L LiCl分别处理细胞24 h后,细胞的侵袭能力受到明显抑制。实时定量PCR和Western blotting结果显示,LiCl处理后NGALmRNA和蛋白表达均明显下降,表明LiCl对NGAL的调控可能始于转录水平。小分子药物处理分别使Wnt/β-catenin和NF-kB信号通路抑制后,NGAL的表达均受到明显抑制,表明这两条通路都参与了NGAL表达的调控。结论:LiCl通过抑制NF-kB调控NGAL的表达,抑制伪足形成以及细胞侵袭。
Objective: To investigate the effect of LiCl on invasion of breast cancer cell line MDA-MB-231 and its regulation on the expression of NGAL. Methods: Cell viability was measured by MTT assay. Cell pseudopodia were observed by light microscopy. Cell invasion was observed by Transwell assay. Expression of NGAL was detected by real-time PCR and Western blotting. Small molecule drug Bay117082 (NF -KB pathway inhibitor) and FH535 (Wnt / β-catenin pathway inhibitor) to detect the regulation of NGAL expression by key pathways. Results: At concentrations below 10 mmol / L, the effect of LiCl on cell viability was not significantly different. After treated with 5 mmol / L or 10 mmol / L HCl for 24 h, the morphology of cells changed obviously and the formation of pseudopodia was affected. However, under the action of Cariporide, NHE1 inhibitor, the damage of pseudopodia was alleviated. After being treated with 10mmol / L LiCl for 24 h, the invasiveness of cells was significantly inhibited. Real-time PCR and Western blotting showed that the expression of NGAL mRNA and protein were significantly decreased after LiCl treatment, indicating that the regulation of NGAL by LiCl may start at the transcriptional level. Small molecule drug treatment inhibited Wnt / β-catenin and NF-κB signaling pathway, respectively, and NGAL expression was significantly inhibited, indicating that both pathways are involved in the regulation of NGAL expression. Conclusion: LiCl can inhibit the expression of NGAL through inhibition of NF-κB and inhibit the formation of pseudopodia and cell invasion.