[1]李青山,郭红生,贾天阳.ISLR 通过活化PI3K-AKT 通路促进上皮- 间质转化影响骨肉瘤细胞恶性进展研究[J].现代检验医学杂志,2024,39(05):17-21+29.[doi:10.3969/j.issn.1671-7414.2024.05.004]
　LI Qingshan,GUO Hongsheng,JIA Tianyang.ISLR Promotes Epithelial-mesenchymal Transition Through Activating PI3KAKT Pathway and Influences the Malignant Progression of Osteosarcoma Cells[J].Journal of Modern Laboratory Medicine,2024,39(05):17-21+29.[doi:10.3969/j.issn.1671-7414.2024.05.004]
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ISLR 通过活化PI3K-AKT 通路促进上皮- 间质转化影响骨肉瘤细胞恶性进展研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第39卷
期数:
2024年05期
页码:
17-21+29
栏目:
论著
出版日期:
2024-09-15

文章信息/Info

Title:
ISLR Promotes Epithelial-mesenchymal Transition Through Activating PI3KAKT Pathway and Influences the Malignant Progression of Osteosarcoma Cells
文章编号:
1671-7414（2024）05-017-06
作者:
李青山; 郭红生; 贾天阳
（邯郸市中心医院骨科，河北邯郸 056001）
Author(s):
LI Qingshan; GUO Hongsheng; JIA Tianyang
（Department of Orthopedics, Handan Central Hospital, Hebei Handan 056001, China）
关键词:
骨肉瘤; 细胞增殖; 上皮- 间质转化; 含免疫球蛋白超家族亮氨酸丰富重复蛋白; PI3K/AKT 通路
分类号:
R738.1；R730.43
DOI:
10.3969/j.issn.1671-7414.2024.05.004
文献标志码:
A
摘要:
目的 研究含免疫球蛋白超家族亮氨酸丰富重复蛋白（immunoglobulin superfamily containing leucine-rich repeatprotein，ISLR）参与骨肉瘤细胞恶性进展的作用及其潜在调节机制。方法 通过实时定量聚合酶链反应（qRT-PCR）检测骨肉瘤组织和细胞中ISLR mRNA 水平。通过转染ISLR 短发夹RNA（short hairpin RNA，shRNA）序列或阴性对照shRNA（negative-control shRNA，NC shRNA）序列至U2OS 细胞，后用磷脂酰肌醇3 激酶（phosphatidylinositol3 kinase，PI3K）激活剂740 Y-P 处理细胞。通过CCK-8 法、Transwell 实验和流式细胞术分别检测细胞活力、侵袭能力和细胞凋亡率。蛋白印迹实验（Western blot）检测ISLR 蛋白、上皮- 间质转化（epithelial-mesenchymal transition，EMT）相关蛋白[ 上皮钙黏蛋白（epitheia-cadherin，E-cadherin）、神经钙黏蛋白（nerve-cadherin，N-cadherin）、波形蛋白（Vimentin），Snail]、PI3K/ 蛋白激酶B（protein kinase B，AKT）通路相关蛋白、细胞凋亡相关蛋白[ 半胱天冬氨酸蛋白酶3（cysteinyl aspartate-specific proteinase -3，Caspase-3），B 淋巴细胞瘤-2（B cell lymphoma/leukemia-2，Bcl-2），Bcl-2 相关X 蛋白（Bcl-2 associated X ，Bax）] 和肿瘤增殖标志物Ki67 蛋白表达。采用慢病毒转染的U2OS细胞注射裸鼠构建异种移植瘤模型，监测肿瘤生长情况。结果 与癌旁组织（1.01±0.02）相比，骨肉瘤组织（5.14±1.63）中ISLR mRNA 水平显著上调，差异具有统计学意义（t=-14.332，P<0.001）。与正常人成骨细胞hFOB1.19（1.01±0.01）相比，骨肉瘤细胞MG63（3.05±0.57），U2OS（4.55±0.79），HOS（2.46±0.41），Saos-2（2.62±0.44）和143B（3.62±0.51）中ISLR mRNA 相对表达均显著升高，差异具有统计学意义（t=4.883，8.473，3.471，3.854，6.247，均P<0.05）。沉默ISLR 明显抑制了U2OS 细胞增殖（t=6.593，6.835）及侵袭（t=8.621，8.448），促进细胞凋亡（t=25.505，25.574），差异具有统计学意义（均P<0.05）。沉默ISLR 明显促进U2OS 细胞中Caspase-3 活性（t=13.489，13.366）及Bax 蛋白（t=8.628，8.524）表达，抑制Bcl-2 蛋白（t=10.948，10.775）表达，差异具有统计学意义（均P<0.05）。沉默ISLR显著促进EMT相关蛋白E-cadherin（t=15.168，15.087）表达，抑制N-cadherin（t=10.220，10.058），Vimentin（t=8.303，8.164）和Snail（t=9.211，9.384）蛋白表达，降低PI3K/AKT 通路关键蛋白PI3K 和AKT 磷酸化水平（t=17.441，14.452），差异具有统计学意义（均P<0.05）。740 Y-P 处理可逆转ISLR 沉默对U2OS 细胞的影响。裸鼠体内实验显示敲低ISLR显著抑制了肿瘤生长。结论 ISLR 可能通过激活PI3K/AKT 通路促进骨肉瘤EMT 及细胞增殖、侵袭，抑制细胞凋亡，从而促进骨肉瘤进展。
Abstract:
Objective To investigate the role of immunoglobulin superfamily containing leucine-rich repeat protein (ISLR) in the malignant progression of osteosarcoma cells and its potential regulatory mechanism. Methods ISLR mRNA levels in osteosarcoma tissues and cells were detected by quantitative real time polymerase chain reaction (qRT-PCR). U2OS cells were transfected with ISLR short hairpin RNA (shRNA) sequence or negative-control shRNA (NC shRNA) sequence, thus the cells were treated with phosphatidylinositol 3 kinase (PI3K) activator 740 Y-P. The cell viability, invasion ability and apoptosis rate were detected by CCK-8 assay, Transwell assay and flow cytometry, respectively. Western blot was used to detect the expressions of ISLR protein, epithelial-mesenchymal transition (EMT)-related proteins [Epitheia-cadherin (E-cadherin), Nerve cadherin (N-cadherin), Vimentin, Snail], PI3K/protein kinase B (AKT) pathline-related proteins, apoptotic proteins [Cysteinyl aspartatespecific proteinase-3 (Caspase-3), B cell lymphoma/leukemia-2 (Bcl-2), Bcl-2 associated X protein (Bax)] and proliferation marker Ki67 protein. Lentivirus was used to transfect U2OS cells, and the cells were injected into nude mice to construct a xenograft tumor model, and tumor growth was monitored. Results ISLR mRNA level in osteosarcoma tissue (5.14±1.63) was up-regulated compared with para-cancerous tissue (1.01±0.02), and the difference was significant (t=-14.332, P<0.001). Compared with normal osteoblasts hFOB1.19 (1.01±0.01), osteosarcoma cells MG63 (3.05±0.57), U2OS (4.55±0.79), HOS (2.46±0.41), the relative expression of ISLR mRNA in Saos-2 (2.62±0.44) and 143B (3.62±0.51) were increased, and differences were significant (t=4.883, 8.473, 3.471, 3.854, 6.247, all P<0.05). Silencing ISLR inhibited the proliferation of U2OS cells (t=6.593, 6.835) and invasion (t=8.621, 8.448), but promoted cell apoptosis (t=25.505, 25.574), and the differences were significant (all P<0.05). Silencing ISLR promoted Caspase-3 activity in U2OS cells (t=13.489, 13.366) and Bax protein (t=8.628, 8.524), but inhibited Bcl-2 protein expression (t=10.948, 10.775), with significant differences (all P<0.05). Silencing ISLR promoted EMT-related protein E-cadherin (t=15.168, 15.087), inhibited N-cadherin (t=10.220, 10.058), Vimentin (t=8.303, 8.164) and Snail (t=9.211, 9.384), but reduced the phosphorylation levels of PI3K and AKT (t=17.441, 14.452), with significant differences (all P<0.05). Additionally, 740 Y-P treatment reversed the effect of silencing ISLR on U2OS cells. Experimental results in vivo showed that knockdown of ISLR significantly inhibited tumor growth. Conclusion ISLR could promote EMT, proliferation and invasion, but inhibit apoptosis of osteosarcoma cells by activating the PI3K/AKT pathway, there by promoting osteosarcoma progression.

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备注/Memo

备注/Memo:
基金项目：河北省2022 年度医学科学研究课题计划项目（20220582）。
作者简介：李青山（1982-），男，硕士，副主任医师，研究方向：骨关节，E-mail：LQs15630003868@126.com。
更新日期/Last Update: 2024-09-15