NGF/TrkA轴对宫颈癌SiHa细胞增殖、凋亡和侵袭的影响

doi:10.11958/20230318

摘要/Abstract

摘要：

目的探讨神经生长因子（NGF）/原肌球蛋白受体激酶A（TrkA）轴对宫颈癌SiHa细胞增殖、凋亡和侵袭的影响。方法体外培养正常宫颈上皮细胞HUCEC和宫颈癌细胞SiHa，将SiHa细胞分为对照组（正常培养）、L-NGF组（50 μg/L重组人NGF蛋白）、H-NGF组（100 μg/L重组人NGF蛋白）、H-NGF+L-K252a组（100 μg/L重组人NGF蛋白+50 μg/L K252a）、H-NGF+H-K252a组（100 μg/L重组人NGF蛋白+100 μg/L K252a）。Western blot检测NGF、TrkA、E-钙黏蛋白（E-cadherin）、N-钙黏蛋白（N-cadherin）、波形蛋白（Vimentin）表达；CCK-8法检测SiHa细胞增殖情况；流式细胞术检测SiHa细胞凋亡；划痕愈合实验测定细胞迁移；Transwell试验测定细胞侵袭。结果 SiHa细胞较HUCEC细胞NGF、TrkA水平升高（P<0.01）。与对照组相比，L-NGF组、H-NGF组NGF、TrkA水平，增殖活力，迁移率，侵袭细胞数量以及N-cadherin、Vimentin蛋白水平显著增加，凋亡率、E-cadherin蛋白水平显著下降（P<0.05），且H-NGF组较L-NGF组以上指标差异更显著（P<0.05）；与H-NGF组相比，H-NGF+L-K252a组、H-NGF+H-K252a组NGF、TrkA水平，增殖活力，迁移率，侵袭细胞数量以及N-cadherin、Vimentin蛋白水平显著降低，凋亡率、E-cadherin蛋白水平显著升高（P<0.05），且H-NGF+H-K252a组较H-NGF+L-K252a组以上指标差异更显著（P<0.05）。结论下调NGF/TrkA轴可以抑制宫颈癌SiHa细胞增殖、侵袭，促进其凋亡。

关键词: 神经生长因子, 受体, TrkA, 宫颈肿瘤, 细胞增殖, 细胞凋亡, 肿瘤浸润

Abstract:

Objective To investigate the impact of nerve growth factor (NGF)/tropomyosin receptor kinase A (TrkA) axis on the proliferation, apoptosis and invasion of cervical cancer SiHa cells. Methods Normal cervical epithelial cells HUCEC and cervical cancer cells SiHa were cultured in vitro. SiHa cells were grouped into the control group (normal culture), the L-NGF group (50 μg/L recombinant human NGF protein), the H-NGF group (100 μg/L recombinant human NGF protein), the H-NGF+L-K252a group (100 μg/L recombinant human NGF protein+50 μg/L K252a) and H-NGF+H-K252a group (100 μg/L recombinant human NGF protein+100 μg/L K252a). The protein expression levels of NGF, TrkA, E-cadherin, N-cadherin and Vimentin were detected by Western blot assay. The proliferation of SiHa cells was detected by CCK-8 method. Apoptosis of SiHa cells was detected by flow cytometry. Cell migration was determined by scratch healing test. Cell invasion was measured by Transwell assay. Results The levels of NGF and TrkA were obviously higher in SiHa cells than those in HUCEC cells (P<0.01). Compared with the control group, levels of NGF and TrkA, proliferative activity, migration rate, number of invasive cells and N-cadherin and Vimentin proteins were obviously higher in the L-NGF group and the H-NGF group (P<0.05), and the apoptosis rate and the level of E-cadherin protein were obviously lower (P<0.05). The above indexes were more obviously different in the H-NGF group than those of the L-NGF group (P<0.05). Compared with the H-NGF group, levels of NGF, TrkA, proliferative activity, migration rate, number of invasive cells and N-cadherin and Vimentin proteins were obviously lower in the H-NGF+L-K252a group and the H-NGF+H-K252a group (P<0.05), and the apoptosis rate and the level of E-cadherin protein were obviously higher (P<0.05). The above indexes in the H-NGF+H-K252a group were more obviously different than those in the H-NGF+L-K252a group (P<0.05). Conclusion Down-regulation of NGF/TrkA axis can inhibit the proliferation and invasion of cervical cancer SiHa cells and promote their apoptosis.

Key words: nerve growth factor, receptor, TrkA, uterine cervical neoplasms, cell proliferation, cell apoptosis, neoplasm invasiveness

中图分类号:

R737.3，R349.54

图/表 12

图1 HUCEC及SiHa细胞中NGF、TrkA蛋白表达免疫印迹图

Fig.1 Immunoblotting of NGF and TrkA protein expression in SiHa cells and HUCEC cells

表1 HUCEC及SiHa细胞中NGF、TrkA蛋白表达水平比较（n=6，$\bar{x}±s$）

Tab.1 Comparison of NGF and TrkA protein expression between HUCEC and SiHa cells

细胞	NGF	TrkA
HUCEC	0.70±0.06	0.38±0.04
SiHa	1.32±0.17	1.05±0.13
t	8.424^＊＊	12.066^＊＊

表2 各组SiHa细胞中NGF、TrkA蛋白表达水平比较（n=6，$\bar{x}±s$）

Tab.2 Comparison of NGF and TrkA protein expression levels between five groups of SiHa cells

组别	NGF	TrkA
对照组	1.20±0.14	1.02±0.09
L-NGF组	1.56±0.16^a	1.29±0.11^a
H-NGF组	1.89±0.24^ab	1.60±0.14^ab
H-NGF+L-K252a组	1.58±0.18^c	1.28±0.14^c
H-NGF+H-K252a组	1.25±0.16^cd	1.06±0.13^cd
F	14.670^＊＊	21.035^＊＊

图2 各组SiHa细胞中NGF、TrkA蛋白表达免疫印迹图 A：对照组；B：L-NGF组；C：H-NGF组；D：H-NGF+L-K252a组；E：H-NGF+H-K252a组。

Fig.2 Immunoblotting of NGF and TrkA protein expression in five groups of SiHa cells

表3 各组SiHa细胞在不同时间增殖活力比较（n=6，OD450值，$\bar{x}±s$）

Tab.3 Comparison of proliferation activity of SiHa cells at different time points between five groups

组别		4 h	24 h	48 h
对照组		0.15±0.01	0.20±0.03	0.31±0.05
L-NGF组		0.16±0.02	0.35±0.04^a	0.45±0.06^a
H-NGF组		0.15±0.01	0.49±0.06^ab	0.65±0.08^ab
H-NGF+L-K252a组		0.14±0.01	0.39±0.02^c	0.52±0.03^c
H-NGF+H-K252a组		0.15±0.02	0.28±0.01^cd	0.37±0.01^cd
F		1.364	54.864^＊＊	39.111^＊＊
组别	72 h		96 h	120 h
对照组	0.40±0.05		0.77±0.05	0.90±0.05
L-NGF组	0.60±0.07^a		1.01±0.12^a	1.18±0.12^a
H-NGF组	0.78±0.09^ab		1.28±0.09^ab	1.45±0.09^ab
H-NGF+L-K252a组	0.63±0.04^c		1.07±0.09^c	1.24±0.15^c
H-NGF+H-K252a组	0.44±0.02^cd		0.86±0.04^cd	0.99±0.10^cd
F	40.457^＊＊		33.844^＊＊	24.402^＊＊

图3 流式细胞术检测各组SiHa细胞凋亡水平

Fig.3 The apoptosis level of SiHa cells in each group detected by flow cytometry

图4 各组SiHa细胞凋亡率的比较 A：对照组；B：L-NGF组；C：H-NGF组；D：H-NGF+L-K252a组；E：H-NGF+H-K252a组。＊＊P<0.01：a与对照组比较，b与L-NGF组比较，c与H-NGF组比较，d与H-NGF+L-K252a组比较，P<0.05。

Fig.4 Comparison of SiHa cell apoptosis rate between five groups

图5 各组SiHa细胞迁移情况（划痕愈合实验，×100）

Fig.5 Migration of SiHa cells in five groups (scratch healing experiment, ×100）

图6 各组SiHa细胞侵袭情况（结晶紫染色，×200）

Fig.6 Invasion of SiHa cells in five groups (crystal violet staining, ×200)

图7 各组SiHa细胞中E-cadherin、N-cadherin、Vimentin蛋白表达免疫印迹图 A：对照组；B：L-NGF组；C：H-NGF组；D：H-NGF+L-K252a组；E：H-NGF+H-K252a组。

Fig.7 Western blot results of E-cadherin, N-cadherin and Vimentin expression in SiHa cells of each group

表4 各组SiHa细胞迁移率和侵袭细胞数量的比较（n=6，$\bar{x}±s$）

Tab.4 Comparison of invasive cell number and migration rate between five groups of SiHa cells

组别	迁移率/%	侵袭细胞数量/个
对照组	43.96±3.85	84.43±6.30
L-NGF组	65.48±5.21^a	105.35±8.48^a
H-NGF组	86.46±9.71^ab	124.69±10.37^ab
H-NGF+L-K252a组	67.73±7.13^c	108.65±9.59^c
H-NGF+H-K252a组	48.93±5.34^cd	93.80±7.38^cd
F	92.987^＊＊	19.216^＊＊

表5 各组SiHa细胞中EMT相关蛋白表达水平比较（n=6，$\bar{x}±s$）

Tab.5 Comparison of EMT related protein expression between five groups of SiHa cells

组别	E-cadherin	N-cadherin	Vimentin
对照组	0.73±0.06	0.43±0.04	0.53±0.10
L-NGF组	0.55±0.04^a	0.66±0.05^a	1.08±0.13^a
H-NGF组	0.32±0.03^ab	0.81±0.09^ab	1.32±0.15^ab
H-NGF+L-K252a组	0.56±0.05^c	0.65±0.07^c	0.95±0.08^c
H-NGF+H-K252a组	0.70±0.08^cd	0.47±0.05^cd	0.70±0.07^cd
F	52.740^＊＊	36.704^＊＊	47.807^＊＊

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