正五聚蛋白3调控TLR4/NF-κB信号通路促进小儿神经母细胞瘤细胞的增殖侵袭和耐药

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目的:探讨正五聚蛋白3(PTX3)对小儿神经母细胞瘤细胞增殖、侵袭和耐药的影响及其作用机制。方法:将si-RNA(si-RNA组)、si-PTX3(si-PTX3组)、siRNA+pcDNA3.1(siRNA+pcDNA3.1组)、si-PTX3+pcDNA3.1(si-PTX3+pcDNA3.1组)、siRNA+pcDNA3.1-TLR4(siRNA+pcDNA3.1-TLR4组)和si-PTX3+pcDNA3.1-TLR4(si-PTX3+pcDNA3.1-TLR4组)转染至SH-SY5Y细胞中。收集2016年7月至2019年8月就诊于驻马店市中心医院的小儿神经母细胞瘤患儿的肿瘤组织和癌旁正常组织32例。采用实时荧光定量聚合酶链反应(qRT-PCR)和免疫组化检测小儿神经母细胞瘤组织和癌旁正常组织中PTX3 mRNA和蛋白的表达水平,EdU增殖实验检测各组SH-SY5Y细胞增殖能力,Western blot检测血管内皮生长因子(VEGF)、耐药相关蛋白P-糖蛋白(P-gp)和多药耐药相关蛋白1(MRP-1)以及基质金属蛋白酶1(MMP-1)的表达水平。结果:小儿神经母细胞瘤组织中PTX3 mRNA的表达水平为0.87±0.07,高于癌旁正常组织(0.13±0.06, n P<0.05);免疫组化检测PTX3蛋白的表达情况与qRT-PCR结果一致。si-PTX3组细胞中PTX3 mRNA和蛋白的表达水平分别为0.25±0.05和0.45±0.66,低于si-RNA组(分别为0.95±0.08和1.02±0.10;均n P<0.05)。si-PTX3组细胞EdU阳性率、侵袭率、VEGF、MMP-1、P-gp和MRP-1蛋白的表达水平分别为(19.73±1.22)%、(8.45±1.06)%、0.25±0.05、0.19±0.03、0.19±0.06和0.16±0.07,均低于si-RNA组[分别为(31.86±1.86)%、(28.12±2.96)%、0.58±0.07、0.44±0.06、0.46±0.08和0.51±0.05;均n P<0.05]。si-PTX3+pcDNA3.1组细胞EdU阳性率、侵袭率、VEGF、P-gp蛋白、TLR4、p-p65蛋白的表达水平分别为(19.49±1.68)%、(8.48±1.36)%、0.10±0.15、0.18±0.07、0.45±0.06、0.25±0.05,均低于siRNA+pcDNA3.1组[分别为(38.21±2.67)%、(26.39±2.14)%、0.49±0.05、0.52±0.06、0.93±0.14和0.82±0.06;均n P<0.05]。siRNA+pcDNA3.1-TLR4组细胞EdU阳性率、侵袭率、VEGF、P-gp、TLR4和p-p65蛋白的表达水平分别为(62.73±5.18)%、(50.45±3.25)%、2.17±0.17、2.15±0.16、2.68±0.16、2.48±0.13,均高于siRNA+pcDNA3.1组(均n P<0.05)。n 结论:降低PTX3的表达能抑制小儿神经母细胞瘤细胞SH-SY5Y的增殖和侵袭能力,并降低其耐药性,其作用机制可能是通过调控TLR4/NF-κB信号通路来实现,为小儿神经母细胞瘤的诊断和临床治疗提供新视角。“,”Objective:To investigate the effect of pentraxin 3 (PTX3) on the proliferation, invasion and drug resistance of pediatric neuroblastoma cells and its mechanism.Methods:si-RNA (si-RNA group), si-PTX3 (si-PTX3 group), siRNA+ pcDNA3.1 (siRNA+ pcDNA3.1 group), si-PTX3+ pcDNA3.1 (si-PTX3+ pcDNA3.1 group), siRNA+ pcDNA3.1-Toll-like receptor 4 (siRNA+ pcDNA3.1-TLR4 group) and si-PTX3+ pcDNA3.1-TLR4 (si-PTX3+ pcDNA3.1-TLR4 group) were transfected into SH-SY5Y cells. Collected 32 cases of tumor tissue and cancerous tissue in children with childhood neuromaternal cells who were treated at Zhumadian center hospital from July 2016 to August 2019. Real-time fluorescent quantitative polymerase chain (RT-qPCR) reaction and immunohistochemistry experiments were used to detect the protein expressions of PTX3 in neuroblastoma tissues and normal tissues. 5-Ethynyl-2′-deoxyuridine (EdU) was used to detect the proliferation effect of PTX3 on neuroblastoma cell SH-SY5Y. Western blot experiment was used to detect the protein expression levels of vascular endothelial growth factor (VEGF), resistance-related proteins including P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP-1), and invasion-related protein matrix metalloproteinase-1 (MMP-1).Results:PTX3 mRNA expressions in neuroblastoma tissues were 0.87±0.07, higher than 0.13±0.06 of normal tissues, and the differences were statistically significant (n P<0.05), The expression of the immunohistochemistry test PTX3 protein was consistent with the qRT-PCR results. Compared with the si-RNA group (0.95±0.08; 1.02±0.10), the mRNA and protein expressions of PTX3 in the si-PTX3 group (0.25±0.05; 0.45±0.66) decreased, the differences were statistically significant (alln P<0.05). The number of EdU positive cells, invasion rate, VEGF, MMP-1, P-gp and MRP-1 protein expressions in si-RNA group were (31.86±1.86)%, (28.12±2.96)%, (0.58±0.07), (0.44±0.06), (0.46±0.08) and (0.51±0.05), respectively, higher than (19.73±1.22)%, (8.45±1.06)%, (0.25±0.05), (0.19±0.03), (0.19±0.06) and (0.16±0.07) in si-PTX3 group, and the differences were statistically significant (alln P<0.05). The Number of EdU positive cells [(19.49±1.68)%], invasion rate [(8.48±1.36)%], VEGF protein expression (0.10±0.15), P-gp (0.18±0.07) , TLR4 (0.45±0.06), p-p65 (0.25±0.05) protein expressions in si-PTX3+ pcDNA3.1 group were relatively lower compared with siRNA+ pcDNA3.1 group [(38.21±2.67)%, (26.39±2.14)%, 0.49±0.05, 0.52±0.06, 0.93±0.14 and 0.82±0.06] (alln P<0.05). The number of EdU-positive cells [(62.73±5.18)%], invasion rate [(50.45±3.25)%], VEGF protein expression (2.17±0.17), P-gp (2.15±0.16), TLR4 (2.68±0.16), p-p65 (2.48±0.13) protein expressions in the siRNA+ pcDNA3.1-TLR4 group increased compared with siRNA+ pcDNA3.1 group (alln P<0.05).n Conclusions:Inhibition of PTX3 can inhibit the proliferation and invasion of neuroblastoma cells SH-SY5Y, and reduce drug resistance. Its mechanism may be achieved by regulating the TLR4/NF-κB signaling pathway. This result can provide a new perspective for pediatric neuroblasts tumor diagnosis and clinical treatment.
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