ARTN通过PI3K/Akt通路促进恶性外周神经鞘瘤细胞的增殖和侵袭
目的:探讨神经鞘胚素（ARTN）在恶性外周神经鞘瘤（MPNST）中的表达及其对MPNST细胞恶性行为的影响与作用通路。方法:收集1995年1月至2011年11月于天津医科大学肿瘤医院手术切除的51例MPNST石蜡包埋组织标本，采用免疫组织化学染色检测ARTN蛋白的表达，并分析ARTN蛋白表达与临床病理特征、预后的关系。利用人MPNST细胞系ST-8814（NF-1型）和STS26T（散发型），通过转染ARTN过表达质粒和ARTN小干扰RNA（siRNA）分别构建ARTN过表达和低表达细胞。采用实时定量聚合酶链反应检测ARTN mRNA的表达，Western bolt检测ARTN蛋白及磷脂酰肌醇...
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| Published in | 中华肿瘤杂志 Vol. 47; no. 2; pp. 149 - 159 |
|---|---|
| Main Authors | , , , , , , , , , , , |
| Format | Journal Article |
| Language | Chinese |
| Published |
天津市天津医院骨与软组织肿瘤科，天津300211%天津医科大学肿瘤医院骨与软组织肿瘤科，国家恶性肿瘤临床医学研究中心，天津市恶性肿瘤临床医学研究中心，天津市肿瘤分子流行病重点实验室，天津300202
23.02.2025
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| Subjects | |
| Online Access | Get full text |
| ISSN | 0253-3766 |
| DOI | 10.3760/cma.j.cn112152-20240531-00229 |
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| Abstract | 目的:探讨神经鞘胚素（ARTN）在恶性外周神经鞘瘤（MPNST）中的表达及其对MPNST细胞恶性行为的影响与作用通路。方法:收集1995年1月至2011年11月于天津医科大学肿瘤医院手术切除的51例MPNST石蜡包埋组织标本，采用免疫组织化学染色检测ARTN蛋白的表达，并分析ARTN蛋白表达与临床病理特征、预后的关系。利用人MPNST细胞系ST-8814（NF-1型）和STS26T（散发型），通过转染ARTN过表达质粒和ARTN小干扰RNA（siRNA）分别构建ARTN过表达和低表达细胞。采用实时定量聚合酶链反应检测ARTN mRNA的表达，Western bolt检测ARTN蛋白及磷脂酰肌醇3-激酶（PI3K）/Akt信号通路相关蛋白的表达，细胞计数盒8（CCK-8）法检测细胞增殖能力，细胞侵袭实验检测细胞的侵袭能力。利用STRING数据库检索与ARTN相互作用的通路蛋白，通过京都基因与基因组百科全书（KEGG）富集分析明确其作用通路。使用PI3K/Akt通路特异性抑制剂LY294002阻断ST-8814和STS26T细胞的PI3K/Akt通路，观察细胞增殖和侵袭能力的变化。结果:51例MPNST组织标本中，ARTN蛋白高表达22例，低表达29例。ARTN蛋白高表达与较大的肿瘤长径和疾病进展（复发或转移）有关（均
P＜0.05）。ARTN蛋白低表达组患者的中位无病生存时间（DFS）为26.2个月，中位总生存时间（OS）为66.9个月。ARTN蛋白高表达组患者的中位DFS为10.7个月，中位OS为53.8个月。Log rank检验结果显示，ARTN蛋白高表达组患者的无进展生存率比低表达组患者差（
P=0.027），但两组患者的总生存率差异无统计学意义（
P=0.790），Cox回归分析也证实了这一结果。CCK-8法检测结果显示，转染48 h，ARTN过表达组ST-8814和STS26T细胞的吸光度（
A）值分别为1.35±0.01和1.10±0.02，均高于空质粒对照组（1.05±0.01和0.78±0.01，均
P＜0.01），而ARTN siRNA组ST-8814和STS26T细胞的
A值分别为0.35±0.01和0.61±0.01，均低于对照siRNA组（0.74±0.01和1.10±0.04，均
P＜0.01）。转染72和96 h亦如此。细胞侵袭实验结果显示，ARTN过表达组ST-8814和STS26T细 |
|---|---|
| AbstractList | 目的:探讨神经鞘胚素（ARTN）在恶性外周神经鞘瘤（MPNST）中的表达及其对MPNST细胞恶性行为的影响与作用通路。方法:收集1995年1月至2011年11月于天津医科大学肿瘤医院手术切除的51例MPNST石蜡包埋组织标本，采用免疫组织化学染色检测ARTN蛋白的表达，并分析ARTN蛋白表达与临床病理特征、预后的关系。利用人MPNST细胞系ST-8814（NF-1型）和STS26T（散发型），通过转染ARTN过表达质粒和ARTN小干扰RNA（siRNA）分别构建ARTN过表达和低表达细胞。采用实时定量聚合酶链反应检测ARTN mRNA的表达，Western bolt检测ARTN蛋白及磷脂酰肌醇3-激酶（PI3K）/Akt信号通路相关蛋白的表达，细胞计数盒8（CCK-8）法检测细胞增殖能力，细胞侵袭实验检测细胞的侵袭能力。利用STRING数据库检索与ARTN相互作用的通路蛋白，通过京都基因与基因组百科全书（KEGG）富集分析明确其作用通路。使用PI3K/Akt通路特异性抑制剂LY294002阻断ST-8814和STS26T细胞的PI3K/Akt通路，观察细胞增殖和侵袭能力的变化。结果:51例MPNST组织标本中，ARTN蛋白高表达22例，低表达29例。ARTN蛋白高表达与较大的肿瘤长径和疾病进展（复发或转移）有关（均
P＜0.05）。ARTN蛋白低表达组患者的中位无病生存时间（DFS）为26.2个月，中位总生存时间（OS）为66.9个月。ARTN蛋白高表达组患者的中位DFS为10.7个月，中位OS为53.8个月。Log rank检验结果显示，ARTN蛋白高表达组患者的无进展生存率比低表达组患者差（
P=0.027），但两组患者的总生存率差异无统计学意义（
P=0.790），Cox回归分析也证实了这一结果。CCK-8法检测结果显示，转染48 h，ARTN过表达组ST-8814和STS26T细胞的吸光度（
A）值分别为1.35±0.01和1.10±0.02，均高于空质粒对照组（1.05±0.01和0.78±0.01，均
P＜0.01），而ARTN siRNA组ST-8814和STS26T细胞的
A值分别为0.35±0.01和0.61±0.01，均低于对照siRNA组（0.74±0.01和1.10±0.04，均
P＜0.01）。转染72和96 h亦如此。细胞侵袭实验结果显示，ARTN过表达组ST-8814和STS26T细 |
| Abstract_FL | Objective:To investigate the expression of Artemin (ARTN) in malignant peripheral nerve sheath tumor (MPNST), its effect on the malignant behavior of MPNST cells, and its signaling pathway.Methods:Fifty-one MPNST paraffin embedded tissues through surgical resection at Tianjin Medical University Cancer Hospital from January 1995 to November 2011 were collected, the expression of the ARTN protein was detected by immunohistochemistry, and the relationship between the ARTN protein expression and the clinical pathological characteristics and prognosis were analyzed. In human MPNST cell lines ST-8814 (NF-1) and STS26T(sporadic), ARTN overexpression and low expression cell lines were constructed by transfecting ARTN overexpression plasmids and ARTN small interfering RNA (siRNA), respectively. The expression of ARTN mRNA was detected by real time quantitative polymerase chain reaction (RT-qPCR), the expression of the ARTN protein and Phosphoinositide 3-kinase(PI3K)/Akt signaling pathway related proteins were detected by Western blot. CCK-8 assay was used to detect cell proliferation ability, and cell invasion assay was used to detect cell invasion ability. The pathway proteins that interacted with ARTN were searched in the STRING database, and the functional pathways were clarified by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The PI3K/Akt pathway specific inhibitor LY294002 was used to block the PI3K/Akt pathway of ST-8814 and STS26T cells to observe the changes in cell proliferation and invasion.Results:Among the 51 MPNST tissue specimens, 22 cases showed a high expression of the ARTN protein and 29 cases showed a low expression of the protein. Higher expressions of the ARTN protein was associated with larger tumor diameters and disease progression (recurrence or metastasis) (both
P＜0.05). The median disease-free survival (DFS) of patients with a low expression of the ARTN protein was 26.2 months, and the median overall survival (OS) was 66.9 months. The median DFS and median OS of patients with a high expression of the ARTN protein were 10.7 months and 53.8 months, respectively. The log rank test results showed that the progression free survival rate of patients with a high expression of the ARTN protein was worse than that of patients with a low expression (
P=0.027), but the difference in overall survival rate between the two groups was not statistically significant (
P=0.790), which was also confirmed by Cox regression analysis. The CCK-8 assay results showed that after 48 hours of transfection, the absorbance (
A) values of ST-8814 and STS26T cells in the ARTN overexpression group were 1.35±0.01 and 1.10±0.02, respectively, which were higher than those in the empty plasmid control group (1.05±0.01 and 0.78±0.01, both
P＜0.01), while the
A values of ST-8814 and STS26T cells in the ARTN siRNA group were 0.35±0.01 and 0.61±0.01, respectively, which were lower than those in the control siRNA group (0.74±0.01 and 1.10±0.04, both
P＜0.01). The results of cell invasion assay showed that the number of transmembrane cells in ST-8814 and STS26T cells overexpressing ARTN was (29.67±2.08) and (31.67±2.08), respectively, which were higher than those in the empty plasmid control group [(20.00±1.00) and (24.33±1.15), both
P＜0.01]. The number of transmembrane cells in ST-8814 and STS26T cells in the ARTN siRNA group were (14.00±2.00) and (19.33±1.53), respectively, which were lower than those in the control siRNA group [(19.33±2.52) and (23.33±0.58), both
P＜0.05].The KEGG results showed that ARTN is associated with multiple tumor signaling pathways, especially the PI3K/Akt signaling pathway. Western blot results showed that overexpression of ARTN upregulated the expression of p-PI3K and p-Akt proteins in ST-8814 and STS26T cells (both
P＜0.01).After knocking down ARTN expression, the expression of p-PI3K and p-Akt proteins was significantly down regulated (both
P＜0.01). LY294002 could significantly inhibit the effect of ARTN overexpression on ST-8814 and STS26T cells after blocking the PI3K/Akt pathway. The
A values of ST-8814 and STS26T cells in the ARTN overexpression+LY294002 group were 1.09±0.06 and 0.82±0.01, respectively, which were lower than those in the ARTN overexpression group (1.50±0.01 and 1.29±0.01, respectively, both
P＜0.01). The numbers of transmembrane cells in the cell invasion assay were 16.67±3.21 and 19.67±2.31, respectively, which were also lower than those in the ARTN overexpression group (29.67±2.08 and 31.67±2.08, respectively, both
P＜0.01).
Conclusions:In MPNST, a high expression of the ARTN protein was associated with larger tumor size, disease progression, and worse DFS. ARTN promotes the proliferation and invasion of MPNST cells through the PI3K/Akt signaling pathway. |
| Author | 刘金伟 廖智超 刘艳成 李爽 李婷 张洪亮 杨吉龙 刘昊天 张净宇 张超 朱锴 刘俊阳 |
| AuthorAffiliation | 天津市天津医院骨与软组织肿瘤科，天津300211%天津医科大学肿瘤医院骨与软组织肿瘤科，国家恶性肿瘤临床医学研究中心，天津市恶性肿瘤临床医学研究中心，天津市肿瘤分子流行病重点实验室，天津300202 |
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| Author_FL | Li Ting Zhang Jingyu Zhang Chao Zhu Kai Yang Jilong Zhang Hongliang Liao Zhichao Liu Jinwei Liu Junyang Liu Haotian Li Shuang Liu Yancheng |
| Author_FL_xml | – sequence: 1 fullname: Zhang Hongliang – sequence: 2 fullname: Liu Haotian – sequence: 3 fullname: Liu Junyang – sequence: 4 fullname: Zhang Chao – sequence: 5 fullname: Li Ting – sequence: 6 fullname: Liao Zhichao – sequence: 7 fullname: Liu Yancheng – sequence: 8 fullname: Zhang Jingyu – sequence: 9 fullname: Zhu Kai – sequence: 10 fullname: Li Shuang – sequence: 11 fullname: Liu Jinwei – sequence: 12 fullname: Yang Jilong |
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| Keywords | 侵袭 磷脂酰肌醇3-激酶/Akt通路 增殖 Phosphoinositide 3-kinase/Akt pathway Malignant peripheral nerve sheath tumor 神经鞘胚素 Proliferation 恶性外周神经鞘瘤 Invasion Artemin |
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| Snippet | 目的:探讨神经鞘胚素（ARTN）在恶性外周神经鞘瘤（MPNST）中的表达及其对MPNST细胞恶性行为的影响与作用通路。方法:收集1995年1月至2011年11月于天津医科大学肿瘤医院手术... |
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| Title | ARTN通过PI3K/Akt通路促进恶性外周神经鞘瘤细胞的增殖和侵袭 |
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