中华小儿外科杂志
中華小兒外科雜誌
중화소인외과잡지
Chinese Journal of Pediatric Surgery
2015年
10期
775-779
,共5页
朱永洁%陈鑫%韩芦芦%段于河%董蒨
硃永潔%陳鑫%韓蘆蘆%段于河%董蒨
주영길%진흠%한호호%단우하%동천
神经母细胞瘤%侵袭%转染
神經母細胞瘤%侵襲%轉染
신경모세포류%침습%전염
Neuroblastoma%Invasion%Transfection
目的 神经母细胞瘤是儿童常见的恶性疾病,转移是致死的主要因素,其中微小RNA(microRNA,miRNA)在该疾病的转移中具有很重要的作用.本研究旨在探索miR-195是否通过RET调节神经母细胞瘤细胞的侵袭.方法 在神经母细胞瘤细胞中转染mimics control和miR-195mimics,建立过表达miR-195的细胞模型.通过Transwell侵袭实验研究过表达miR-195对侵袭的影响.利用生物信息学检测miR-195的靶基因,并应用荧光素酶报告基因鉴定其对靶基因3'UTR的结合序列.实时定量PCR及western检测过表达miR-195对靶基因mRNA及蛋白的改变.应用siRNA干扰建立抑制靶基因的细胞模型,通过Transwell侵袭实验检测敲减靶基因对侵袭的影响.最后建立恢复靶基因表达的细胞模型,通过Transwell侵袭实验验证能力的恢复.结果 在SH-SY5Y和SK-N-SH内,均成功过表达miR-195的相对水平分别为34±1.19和43±1.71,与对照组相比具有显著差异(P＜0.001).SH-SY5Y过表达miR-195后发生侵袭的细胞数由93±2降至62±1,SK-N-SH过表达miR-195后发生侵袭的细胞数由82±2降至56±5,过表达miR-195后显著抑制细胞侵袭(P＜0.05);生物信息学检测miR-195能够结合RET mRNA的3 'UTR.过表达miR-195显著抑制RET的mRNA和蛋白质水平(P＜0.05).过表达miR-195显著抑制报告质粒RET 3' UTRWT的荧光素酶的相对活性(P＜0.05).RNA干扰均抑制两种细胞内RET(一个在转化中发生重排的原癌基因,RET原癌基因)的mRNA和蛋白的水平(P＜0.05).在SH-SY5Y内,敲减RET后发生侵袭的细胞数目由89±2降至62±5,在SK-N-SH中由78±2降至63±1,均具有显著差异(P＜0.05).在恢复实验中,对照组和实验组发生侵袭的数目分别为84±3、64±9、65±5和82±1,恢复RET表达恢复细胞的侵袭能力.结论 miR-195与神经母细胞瘤侵袭能力相关,并且能够通过靶向RET调节神经母细胞瘤细胞的侵袭.
目的 神經母細胞瘤是兒童常見的噁性疾病,轉移是緻死的主要因素,其中微小RNA(microRNA,miRNA)在該疾病的轉移中具有很重要的作用.本研究旨在探索miR-195是否通過RET調節神經母細胞瘤細胞的侵襲.方法 在神經母細胞瘤細胞中轉染mimics control和miR-195mimics,建立過錶達miR-195的細胞模型.通過Transwell侵襲實驗研究過錶達miR-195對侵襲的影響.利用生物信息學檢測miR-195的靶基因,併應用熒光素酶報告基因鑒定其對靶基因3'UTR的結閤序列.實時定量PCR及western檢測過錶達miR-195對靶基因mRNA及蛋白的改變.應用siRNA榦擾建立抑製靶基因的細胞模型,通過Transwell侵襲實驗檢測敲減靶基因對侵襲的影響.最後建立恢複靶基因錶達的細胞模型,通過Transwell侵襲實驗驗證能力的恢複.結果 在SH-SY5Y和SK-N-SH內,均成功過錶達miR-195的相對水平分彆為34±1.19和43±1.71,與對照組相比具有顯著差異(P＜0.001).SH-SY5Y過錶達miR-195後髮生侵襲的細胞數由93±2降至62±1,SK-N-SH過錶達miR-195後髮生侵襲的細胞數由82±2降至56±5,過錶達miR-195後顯著抑製細胞侵襲(P＜0.05);生物信息學檢測miR-195能夠結閤RET mRNA的3 'UTR.過錶達miR-195顯著抑製RET的mRNA和蛋白質水平(P＜0.05).過錶達miR-195顯著抑製報告質粒RET 3' UTRWT的熒光素酶的相對活性(P＜0.05).RNA榦擾均抑製兩種細胞內RET(一箇在轉化中髮生重排的原癌基因,RET原癌基因)的mRNA和蛋白的水平(P＜0.05).在SH-SY5Y內,敲減RET後髮生侵襲的細胞數目由89±2降至62±5,在SK-N-SH中由78±2降至63±1,均具有顯著差異(P＜0.05).在恢複實驗中,對照組和實驗組髮生侵襲的數目分彆為84±3、64±9、65±5和82±1,恢複RET錶達恢複細胞的侵襲能力.結論 miR-195與神經母細胞瘤侵襲能力相關,併且能夠通過靶嚮RET調節神經母細胞瘤細胞的侵襲.
목적 신경모세포류시인동상견적악성질병,전이시치사적주요인소,기중미소RNA(microRNA,miRNA)재해질병적전이중구유흔중요적작용.본연구지재탐색miR-195시부통과RET조절신경모세포류세포적침습.방법 재신경모세포류세포중전염mimics control화miR-195mimics,건립과표체miR-195적세포모형.통과Transwell침습실험연구과표체miR-195대침습적영향.이용생물신식학검측miR-195적파기인,병응용형광소매보고기인감정기대파기인3'UTR적결합서렬.실시정량PCR급western검측과표체miR-195대파기인mRNA급단백적개변.응용siRNA간우건립억제파기인적세포모형,통과Transwell침습실험검측고감파기인대침습적영향.최후건립회복파기인표체적세포모형,통과Transwell침습실험험증능력적회복.결과 재SH-SY5Y화SK-N-SH내,균성공과표체miR-195적상대수평분별위34±1.19화43±1.71,여대조조상비구유현저차이(P＜0.001).SH-SY5Y과표체miR-195후발생침습적세포수유93±2강지62±1,SK-N-SH과표체miR-195후발생침습적세포수유82±2강지56±5,과표체miR-195후현저억제세포침습(P＜0.05);생물신식학검측miR-195능구결합RET mRNA적3 'UTR.과표체miR-195현저억제RET적mRNA화단백질수평(P＜0.05).과표체miR-195현저억제보고질립RET 3' UTRWT적형광소매적상대활성(P＜0.05).RNA간우균억제량충세포내RET(일개재전화중발생중배적원암기인,RET원암기인)적mRNA화단백적수평(P＜0.05).재SH-SY5Y내,고감RET후발생침습적세포수목유89±2강지62±5,재SK-N-SH중유78±2강지63±1,균구유현저차이(P＜0.05).재회복실험중,대조조화실험조발생침습적수목분별위84±3、64±9、65±5화82±1,회복RET표체회복세포적침습능력.결론 miR-195여신경모세포류침습능력상관,병차능구통과파향RET조절신경모세포류세포적침습.
Objective To detect whether miR-195 regulates neuroblastoma cell invasion by targeting RET.Methods A miR-195-overexpressing cell model was established by transfecting cells with mimics control and miR-195 mimics.Transwell invasion assay was performed for detecting cell invasion activity.Bioinformatics was used to predict the target of miR-195,together with luciferase reporter assay.Real-time quantitative polymerase chain reaction (PCR) and Western blot were performed for detecting the target gene expression.Then RET-inhibiting cell model was established by RNAi interference and used for examining cell invasion activity.And the expression of RET was restored and the invasion ability detected.Results The expression of miR-195 was enhanced by 34 ± 1.19 and 43 ± 1.71 folds in SH-SY5Y and SK-N-SH respectively.They showed significant difference compared with control group (P＜0.05).An overexpresion of miR-195 decreased cell invasion count from 93 ± 2 to 62 ± 1 in SH-SY5Y and from 82 ± 2 to 56 ± 5 in SK-N-SH.An overexpression of miR-195 inhibited cell invasion significantly (P＜0.05).Bioinformatics indicated that miR-195 could bind to 3'UTR of RET mRNA.An over-expression of miR-195 could significantly inhibit the mRNA and protein expressions of RET (P ＜0.05).And an overexpression of miR-195 could also inhibit significantly the relative luciferase activity of RET 3'UTR WT (P＜0.05).A knockdown of RET decreased cell invasion count from 89 ± 2 to 62 ± 5 in SH-SY5Y and from 78 ± 2 to 63 ± 1 in SK-N-SH.A knockdown of RET inhibited significantly cell invasion (P＜0.05).In rescue assay,invasion cell counts of control and experimental groups were 84 ± 3,64 ± 9,65± 5 and 82 ± 1 respectively.Restored expression of RET rescued cell invasion activity.Conclusions Correlated with neuroblastoma cell invasion,MiR-195 regulates neuroblastoma cell invasion by targeting RET.
