SNHG7-miR-653-5p-STAT2反馈通路在神经母细胞瘤进展中的调节作用研究

Role of SNHG7-miR-653-5p-STAT2 feedback loop in regulating neuroblastoma progression

目的研究lncRNA SNHG7在神经母细胞瘤(neuroblastoma,NB)进展中的作用,以及SNHG7-miR-653-5p-STAT2反馈通路在神经母细胞瘤进展中的调节作用。方法从青岛大学附属医院收治的NB患儿体内获得92对NB组织及相邻非肿瘤组织。采用qRT-PCR检测SNHG7在神经母细胞瘤肿瘤组织及细胞中的表达情况。采用Kaplan-Meier分析神经母细胞瘤患儿的总体存活率。采用比色法(MTT法)和集落形成法检测SNHG7对SK-N-SH和SH-SY5Y细胞的作用。采用Transwell侵袭及迁移试验检测SK-N-SH和SH-SY5Y细胞的侵袭和迁移能力。采用荧光素酶检测miR-653-5p和SNHG7、STAT2之间的作用。采用RIP测定及RNA下拉测定检验SNHG7和miR-653-5p在NB中的相对表达情况。采用Spearman相关分析探究SNHG7与miR-653-5p、STAT2的相关性。结果qRT-PCR显示,92对NB组织及相邻非肿瘤组织中,肿瘤组织(n=53)中SNHG7的表达量高于非肿瘤组织(n=39)。SNHG7高表达的NB患儿(n=53)总生存期随月份的增加而逐渐降低,SNHG7低表达的NB患儿(n=39)总生存期随月份的增加也逐渐降低,两组差异有统计学意义(P=0.004)。细胞功能试验:①MTT法和集落形成法检测结果显示,SNHG7的下调对SK-N-SH和SH-SY5Y细胞的存活和增殖有明显抑制作用;②Transwell试验检测结果显示,敲低SNHG7可明显抑制SK-N-SH和SH-SY5Y细胞的细胞迁移和侵袭能力(P<0.05);③荧光素酶检测显示,miR-653-5p能降低SNHG7-WT(野生型)的荧光素酶活性,且miR-653-5p与STAT2-WT(野生型)之间存在特异性的相互作用。Spearman相关分析显示:①NB组织中SNHG7与miR-653-5p表达水平呈负相关(r=-0.281,P=0.007);②STAT2的表达量与NB组织中miR-653-5p表达水平呈负相关(r=-0.295,P=0.004),STAT2的表达量与NB组织中SNHG7表达水平呈正相关(r=0.296,P=0.004)。结论SNHG7通过miR-653-5p/STAT2通路促使NB进展,这为NB提供了一个新的治疗靶点和预测预后的生物标志物。

Objective To explore the role of lncRNASNHG7 in the progression of neuroblastoma(NB)and examine the regulatory role of snhg7-mir-653-5p-stat2 feedback pathway in NB.Long noncoding RNAs(lncRNAs)have been implicated in the pathogenesis of various cancers,including neuroblastoma(NB).However,the role of lncRNASNHG7 in NB progression remains elusive.Methods The expression of SNHG7 in NB tissues and cells was detected by quantitative realtime-polymerase chain reaction(qRT-PCR).Cellular invasion and migration were detected by Transwell invasion and migration assay.Luciferase assay was utilized for detecting the interaction between mir-653-5p,SNHG7 and STAT2.The relative expressions of SNHG7 and mir-653-5p in NB were determined by RNA-binding protein immunoprecipitation(RIP)assay and RNA pull-down assay.Spearman's correlation curve was employed for examining the relationship between SNHG7 and mir-653-5p and STAT2.Overall survival was measured by Kaplan-Meier analysis.The effects of SNHG7 on SK-N-SH/SH-SY5Y cells were determined by methylthiazolyldiphenyl-tetrazolium bromide(MTT)and colony formation.Results qRT-PCR demonstrated that,among 92 pairs of neuroblastoma(NB)and adjacent non-tumor tissues,expression quantity of SNHG7 was higher in tumor tissues(n=53)than that in non-tumor tissues(n=39).Kaplan Meier analysis revealed that overall survival decreased with advancing months in high SNHG7 patients(n=53)and similarly in low SNHG7 counterparts(n=39)with a lower extent(P=0.004).An up-regulation of SNHG7 was correlated with a poor overall survival rate of NB patients.Cell function test indicated that a down-regulation of SNHG7 by MTT and colony formation significantly suppressed the survival and proliferation of SK-N-SH/SH-SY5Y cells.Transwell test indicated that knocking down SNHG7 significantly suppressed the migration and invasion of SK-N-SH/SH-SY5Y cells(P<0.01).Luciferase assay revealed that miR-653-5p lowered the luciferase activity of SNHG7-wt(wildtype).And a specific interaction existed between miR-653-5p and STAT2-wt(wildtype).Spearman's correlation curve analysis indicated that SNHG7 in NB tissues was negatively correlated with miR-653-5p(r=-0.281,P=0.007).The expression of STAT2 was negatively correlated with miR-653-5p in NB tissues(r=-0.295,P=0.004)and positively correlated with SNHG7 in NB tissues(r=0.296,P=0.004).These data suggested that SNHG7 played a role in NB progression by regulating the pathway of miR-653-5p/STAT2.Conclusion SNHG7 promotes NB progression through the pathway of miR-653-5p/STAT2.Thus it may provide a novel therapeutic target and prognostic biomarker for NB.