粒细胞集落刺激因子及其受体通过JAK2/STAT3信号通路促进食管鳞状细胞癌细胞增殖、迁移和上皮间质转化

G-CSF/G-CSFR Promotes Proliferation,Migration and Epithelial-Mesenchymal Transition of Esophageal Squamous Cell Carcinoma Cells through JAK2/STAT3 Signaling Pathway

[目的]探讨粒细胞集落刺激因子(granulocyte colony-stimulating factor,G-CSF)及G-CSF受体(G-CSF receptor,G-CSFR)在食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)细胞增殖、迁移和上皮间质转化中的作用。[方法]48例ESCC组织来自2019年1—9月在林州市肿瘤医院行食管癌根治术的患者。采用qRT-PCR检测ESCC组织及细胞系(KYSE70、KYSE150、KYSE450和Het-1A)中G-CSFR m RNA的表达,流式细胞术检测ESCC细胞系中G-CSFR阳性细胞百分率。CCK-8法、划痕实验、Transwell实验和流式细胞术分别检测rhG-CSF对KYSE450和KYSE150细胞增殖、迁移和JAK2/STAT3信号通路相关分子的影响。Western Blot检测E-cadherin、Ncadherin和Vimentin蛋白的表达。[结果]G-CSFR mRNA在ESCC组织和细胞系中的表达水平均显著高于正常食管上皮组织和正常食管上皮细胞系(P均<0.05)。G-CSFR阳性细胞率在ESCC细胞系中显著高于正常食管上皮细胞系(P均<0.01)。G-CSFR mRNA的高表达与ESCC患者的TNM分期相关(χ^(2)=5.421,P=0.020)。KYSE150和KYSE450细胞经不同浓度rhG-CSF处理后,与对照组相比,增殖活性显著增加(P均<0.01),而在加入JAK2/STAT3抑制剂AG490后,增殖活性显著减弱(P均<0.01)。与对照组相比,G-CSF(20 ng/mL)组KYSE150和KYSE450细胞划痕愈合率和迁移细胞数明显增加(P均<0.01),p-JAK2和p-STAT3水平显著增加(P均<0.01),N-cadherin和Vimentin蛋白表达显著增加,E-cadherin蛋白表达显著下降(P均<0.05)。而在加入AG490阻断JAK2/STAT3通路后,KYSE150和KYSE450细胞N-cadherin和Vimentin蛋白表达显著下降,E-cadherin蛋白表达显著增加(P均<0.05)。[结论]G-CSFR在ESCC中高表达,G-CSF/G-CSFR通过激活JAK2/STAT3信号通路促进ESCC细胞的增殖、迁移和上皮间质转化。

[Objective]To investigate the effects of granulocyte colony-stimulating factor/granulocyte colony-stimulating factor receptor(G-CSF/G-CSFR)on proliferation,migration and epithelial-mesenchymal transition(EMT)of esophageal squamous cell carcinoma(ESCC)cells.[Methods]ESCC tissue samples were collected from 48 ESCC patients who underwent surgical treatment in Linzhou Cancer Hospital from January to September 2019.The G-CSFR mRNA expression in ESCC tissues and ESCC cell lines KYSE450 and KYSE150 and normal esophageal epithelial cell line Het-1A were determined by qRT-PCR,and the percentage of G-CSFR positive cells was measured by flow cytometry.The effects of rhG-CSF on the proliferation,migration,and protein phosphorylation of the JAK2/STAT3 signaling pathway in KYSE450 and KYSE150 cells were investigated by CCK-8,scratch assay,Transwell assay,and flow cytometry,respectively.Western blot was used to detect E-cadherin,N-cadherin and Vimentin protein expression levels.[Results]G-CSFR mRNA expression level was upregulated in ESCC tissues and cell lines compared with normal esophageal epithelial tissues and cell line(all P<0.05).The percentage of G-CSFR positive cells was significantly increased in ESCC cell lines compared with normal esophageal cell line(all P<0.01).High expression level of G-CSFR mRNA was correlated with TNM stage in ESCC patients(χ^(2)=5.421,P=0.020).The proliferation of KYSE450 and KYSE150 cells treated with rhG-CSF was significantly increased(all P<0.01)compared with control.Whereas adding JAK2/STAT3 blocker AG490,the cell proliferation was significantly decreased(both P<0.01).After KYSE450 and KYSE150 cells being treated with rhG-CSF(20 ng/mL),the migration ability and p-JAK2 and p-STAT3 levels were significantly increased(all P<0.01),N-cadherin and Vimentin protein levels were significantly increased(all P<0.05),and E-cadherin protein level was decreased(P<0.05).In contrast,upon adding AG490 to block the JAK2/STAT3 signaling pathway,N-cadherin and Vimentin protein were downregulated and E-cadherin protein was upregulated(all P<0.05).[Conclusion]G-CSFR is highly expressed in ESCC.G-CSF/G-CSFR promotes proliferation,migration,and epithelial-mesenchymal transition of ESCC cells by activating the JAK2/STAT3 signaling pathway.