神经母细胞瘤SPNS2表达及相关机制探讨

Expression of SPNS2 in children with neuroblastoma and its influences on cell proliferation,invasion and migration

目的探讨鞘氨醇-1-磷酸转运体2(SPNS2)在神经母细胞瘤(NB)患儿中的表达以及利用小分子干扰RNA技术沉默SPNS2,观察沉默SPNS2对NB细胞增殖、侵袭、迁移的影响。方法收集2016-12-16-2021-12-16郑州大学附属儿童医院接受手术治疗的30例NB患儿的瘤组织和瘤旁组织,实时定量PCR(qRT-PCR)、蛋白质印迹法和免疫组织化学法分别检测组织中SPNS2 mRNA、SPNS2蛋白表达,并分析SPNS2蛋白表达与NB患儿临床病理特征的关系。NB细胞SH-SY5Y分为CK组、si-SPNS2组、si-NC组、pcDNA-SPNS2组和pcDNA组,qRT-PCR、CCK-8、平板克隆实验、Transwell、划痕实验分别检测细胞中SPNS2 mRNA表达、细胞增殖、克隆形成、侵袭和迁移能力;蛋白质印迹法检测细胞中SPNS2、增殖细胞核抗原(PCNA)、基质金属蛋白酶9(MMP-9)、MMP-2蛋白表达;体内移植瘤实验检测肿瘤生长情况。结果瘤旁组织和NB组织中SPNS2 mRNA表达量分别为1.00±0.03和2.99±0.16,t=66.956,P<0.001,SPNS2蛋白表达量分别为0.24±0.02和1.35±0.14,t=42.990,P<0.001;SPNS2蛋白表达与INSS分期(χ^(2)=13.097,P<0.001)、淋巴结转移(χ^(2)=5.748,P=0.017)、病理亚分型(χ^(2)=3.967,P=0.046)有关,与性别(χ^(2)=0.144,P=0.705)、年龄(χ^(2)=0.215,P=0.643)、肿瘤大小(χ^(2)=0.433,P=0.510)无关;CK组、si-NC组、si-SPNS2组、pcDNA组、pcDNA-SPNS2组D(450 nm)值分别为1.14±0.13、1.16±0.14、0.51±0.04、1.15±0.12和1.56±0.13,差异有统计学意义,F=61.310,P<0.001;CK组、si-NC组、si-SPNS2组、pcDNA组、pcDNA-SPNS2组克隆形成率分别为(78.87±2.08)%、(78.76±2.16)%、(34.54±3.89)%、(77.43±2.36)%和(93.32±2.11)%,差异有统计学意义,F=434.563,P<0.001;CK组、si-NC组、si-SPNS2组、pcDNA组、pcDNA-SPNS2组侵袭细胞数目分别为(86.67±7.12)、(88.34±7.25)、(29.67±3.17)、(87.76±7.13)和(115.52±8.96)个,差异有统计学意义,F=121.399,P<0.001;CK组、si-NC组、si-SPNS2组、pcDNA组、pcDNA-SPNS2组划痕愈合率分别为(56.78±4.31)%、(57.36±4.29)%、(21.13±2.02)%、(56.92±4.36)%和(74.42±6.14)%,差异有统计学意义,F=116.804,P<0.001;CK组、si-NC组、si-SPNS2组、pcDNA组、pcDNA-SPNS2组裸鼠体内移植瘤质量分别为(0.52±0.04)、(0.54±0.06)、(0.19±0.01)、(0.53±0.04)和(0.72±0.06)g,差异有统计学意义,F=87.738,P<0.001;CK组、si-NC组、si-SPNS2组、pcDNA组、pcDNA-SPNS2组SH-SY5Y细胞中SPNS2 mRNA、SPNS2、PCNA、MMP-9、MMP-2蛋白表达差异有统计学意义,均P<0.001。结论SPNS2在NB患儿瘤组织中高表达,沉默SPNS2对SH-SY5Y细胞增殖、侵袭和迁移起到抑制作用,SPNS2可能是NB的促癌因子之一。

Objective To explore the expression of sphingosine-1-phosphate transporter 2(SPNS2)in children with neuroblastoma(NB)and silenced SPNS2by using small interfering RNA technology to observe the effect of silencing SPNS2on NB cell proliferation,invasion,migration.Methods The tumor tissues and paratumor tissues of 30children with NB who underwent surgical treatment in the Children’s Hospital of Zhengzhou University from December 16,2016to December 16,2021were collected.qRT-PCR,western blot and immunohistochemistry were used to detect the expression of SPNS2mRNA and SPNS2protein in tissues,and the relationship between the expression of SPNS2protein and the clinicopathological characteristics of children with NB was analyzed.NB cells SH-SY5Ywere divided into CK group,si-SPNS2group,si-NC group,pcDNA-SPNS2group and pcDNA group.qRT-PCR,CCK-8,plate cloning experiment,Transwell and scratch assay were used to detect SPNS2mRNA expression,cell proliferation,clone formation,invasion,migration ability in cells,respectively;Western blot was used to detect the expression of SPNS2,proliferating cell nuclear antigen(PCNA),matrix metalloproteinase(MMP)-9,and MMP-2proteins in cells;in vivo xenograft experiment was performed to detect tumor growth.Results The expression levels of SPNS2mRNA in paratumor tissue and NB tissue were 1.00±0.03and 2.99±0.16,t=66.956,P<0.001.SPNS2protein expressions were 0.24±0.02and 1.35±0.14,t=42.990,P<0.001,respectively.SPNS2protein expression was related to INSS stage(χ^(2)=13.097,P<0.001),lymph node metastasis(χ^(2)=5.748,P=0.017)and pathological subtype(χ^(2)=3.967,P=0.046),it was not related to gender(χ^(2)=0.144,P=0.705),age(χ^(2)=0.215,P=0.643)and tumor size(χ^(2)=0.433,P=0.510).The D(450nm)values of CK group,si-NC group,si-SPNS2group,pcDNA group and pcDNA-SPNS2group were 1.14±0.13,1.16±0.14,0.51±0.04,1.15±0.12and 1.56±0.13,respectively,the difference was statistically significant,F=61.310,P<0.001.The clone formation rates of CK group,si-NC group,si-SPNS2group,pcDNA group and pcDNA-SPNS2group were(78.87±2.08)%,(78.76±2.16)%,(34.54±3.89)%,(77.43±2.36)%and(93.32±2.11)%,the difference was statistically significant,F=434.563,P<0.001.The number of invasive cells in CK group,si-NC group,si-SPNS2group,pcDNA group and pcDNA-SPNS2group were 86.67±7.12,88.34±7.25,29.67±3.17,87.76±7.13and115.52±8.96,the difference was statistically significant,F=121.399,P<0.001.The wound healing rates of CK group,si-NC group,si-SPNS2group,pcDNA group and pcDNA-SPNS2group were(56.78±4.31)%,(57.36±4.29)%,(21.13±2.02)%,(56.92±4.36)%and(74.42±6.14)%,the difference was statistically significant,F=116.804,P<0.001.The mass of transplanted tumor in nude mice in CK group,si-NC group,si-SPNS2group,pcDNA group and pcDNA-SPNS2group were(0.52±0.04),(0.54±0.06),(0.19±0.01),(0.53±0.04)and(0.72±0.06)g,the difference was statistically significant,F=87.738,P<0.001.There were significant differences in SPNS2mRNA,SPNS2,PCNA,MMP-9and MMP-2protein expressions in SH-SY5Y cells of CK group,si-NC group,si-SPNS2group,pcDNA group and pcDNA-SPNS2group,all P<0.001.Conclusions SPNS2is highly expressed in tumor tissues of children with NB,and silencing SPNS2can inhibit the proliferation,invasion and migration of SH-SY5Y cells.SPNS2may be one of the tumor-promoting factors of NB.