FOXC2介导Hedgehog/Gli信号通路对乳腺癌侵袭及迁移的影响

doi:10.3877/cma.j.issn.1674-0793.2020.05.002

目的

探讨FOXC2介导Hedgehog/Gli信号通路通过调控上皮间质转化（EMT）途径参与乳腺癌侵袭及迁移的机制。

方法

应用不同浓度环靶明（Cyclopamine）处理乳腺癌MDA-MB-231细胞，MTT法检测细胞增殖抑制率及计算药物半数抑制浓度（IC₅₀）；采用Cyclopamine或沉默FOXC2基因表达以阻断Hedgehog/Gli信号通路活化；通过Transwell小室体外侵袭实验及划痕实验分别检测阻断Hedgehog/Gli信号通路对MDA-MB-231细胞侵袭及迁移影响；Western blotting检测阻断前后Hedgehog/Gli信号通路分子Smoothened（Smo）、Gli1和EMT相关标志物FOXC2，E-cadherin及Vimentin蛋白表达变化。

结果

与空白对照组比较，Cyclopamine可显著地抑制MDA-MB-231细胞增殖并呈现时效-量效关系，48 h的IC₅₀为25 μmol/L。Transwell小室体外侵袭实验及划痕实验均显示，与未转染组及Control-siRNA组相比，FOXC2-siRNA组和Cyclopamine组细胞穿膜数及迁移率均显著降低，差异有统计学意义（P<0.05）。Western blotting显示，与未转染组及Control-siRNA组相比较，FOXC2-siRNA组及Cyclopamine组Smo、Gli1及FOXC2蛋白表达显著降低，差异有统计学意义（P<0.05）。而沉默FOXC2表达可显著降低Vimentin蛋白表达及增加E-cadherin蛋白表达，差异有统计学意义（P<0.05）。

结论

FOXC2通过介导Hedgehog/Gli信号通路从而调控EMT促进乳腺癌侵袭及迁移，提示阻断该信号通路有望成为乳腺癌靶向治疗新线索。

关键词: FOXC2, Hedgehog/Gli信号通路, 上皮间质转化, 乳腺肿瘤, 侵袭迁移

Objective

To explore the mechanism of FOXC2 mediated Hedgehog/Gli signaling pathway involved in breast cancer invasion and migration by regulating epithelium mesenchymal transformation (EMT).

Methods

Breast cancer MDA-MB-231 cells were treated with different concentrations Cyclopamine-a Hedgehog/Gli signaling pathway inhibitor. MTT method was used to detect the cell proliferation inhibition rate and calculated the drug half inhibitory concentration (IC₅₀). Cyclopamine or silencing FOXC2 expression by RNA interference (siRNA) was used to block the Hedgehog/Gli signaling pathway activation. Transwell chamber invasion assay and scratch assay were used to detect invasion and migration ability of MDA-MB-231 cells after blocking Hedgehog/Gli signal pathway respectively. Western blotting was used to detect the expression of Hedgehog/Gli signaling pathway molecules Smoothened (Smo), Gli1 and EMT-related markers E-cadherin and Vimentin protein before and after blocking Hedgehog/Gli signaling pathway.

Results

Compared with the blank control group, Cyclopamine could significantly inhibit the MDA-MB-231 cells proliferation and showed a time-dose effect relationship. The half inhibition inhibitory concentration was 25 μmol/L after 48 hour. Transwell chamber invasion assay and wound healing assay showed that FOXC2-siRNA group and Cyclopamine group penetrating cells and migration rates were significantly lower, as compared with the untransfected group and Control-siRNA group. The difference was statistically significant (P<0.05). Western blotting results also showed that the expression of Smo, Gli1 and FOXC2 protein was significantly decreased in FOXC2-siRNA group and Cyclopamine group, as compared with the untransfected group and Control-siRNA group (P<0.05). The expression of Vimentin protein was significantly decreased and the expression of E-cadherin protein was increased when silencing FOXC2 expression, the differences were statistically significant (P<0.05).

Conclusions

FOXC2 mediates Hedgehog/Gli signaling pathway involved in breast cancer invasion and migration by regulating EMT. It is suggested that blocking the Hedgehog/Gli signal pathway may be a new targeted therapy of breast cancer.

Key words: FOXC2, Hedgehog/Gli signal pathway, Epithelial-mesenchymal transition, Breast tumor, Invasion and metastasis

表1 不同浓度Cyclopamine处理的MDA-MB-231细胞增殖抑制率（n=20, %,±s）

组别	24 h	48 h	72 h
空白对照组	10.32±1.13	10.47±1.25	11.62±1.04
低浓度组(10.0 μmol/L)	12.39±1.43	15.35±1.72	16.03±1.07
中浓度组(20.0 μmol/L)	15.24±1.26	45.08±2.17^a	71.25±2.21^a
高浓度组(40.0 μmol/L)	21.18±1.37^a	64.15±3.24^a	87.37±3.43^a

表1 不同浓度Cyclopamine处理的MDA-MB-231细胞增殖抑制率（n=20, %,±s）

组别	24 h	48 h	72 h
空白对照组	10.32±1.13	10.47±1.25	11.62±1.04
低浓度组(10.0 μmol/L)	12.39±1.43	15.35±1.72	16.03±1.07
中浓度组(20.0 μmol/L)	15.24±1.26	45.08±2.17^a	71.25±2.21^a
高浓度组(40.0 μmol/L)	21.18±1.37^a	64.15±3.24^a	87.37±3.43^a

图1 PCR鉴定重组慢病毒载体阳性克隆　1、2为阴性对照组（289 bp）；3~8为阳性对照组（343 bp）；9为Marker；10为空白对照组（289 bp）；11为阴性对照组（ddH₂O）

图2 阻断Hedgehog/Gli信号通路或沉默FOXC2表达对细胞侵袭能力的影响（×200） A为未转染组；B为Control-siRNA组；C为FOXC2-siRNA组；D为Cyclopamine组

图3 阻断Hedgehog/Gli信号通路或沉默FOXC2表达对细胞迁移能力的影响（×200） A、E为未转染组；B、F为Control-siRNA组；C、G为FOXC2-siRNA组；D、F为Cyclopamine组

图4 Western blotting检测细胞中Hedgehog/Gli信号通路分子及EMT相关标志物蛋白表达　1为空白对照组；2为Cyclopamine组；3为未转染组；4为Control-siRNA组；5为FOXC2-siRNA组

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FOXC2 affects the invasion and migration ability of breast cancer by Hedgehog/Gli signaling pathway

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FOXC2 affects the invasion and migration ability of breast cancer by Hedgehog/Gli signaling pathway