中华小儿外科杂志
中華小兒外科雜誌
중화소인외과잡지
CHINESE JOURNAL OF PEDIATRIC SURGERY
2013年
7期
524-528
,共5页
鹿洪亭%董蒨%陈鑫%张虹%郝希伟%韩芦芦
鹿洪亭%董蒨%陳鑫%張虹%郝希偉%韓蘆蘆
록홍정%동천%진흠%장홍%학희위%한호호
神经母细胞瘤%细胞增殖%微RNAs
神經母細胞瘤%細胞增殖%微RNAs
신경모세포류%세포증식%미RNAs
Neuroblastoma%Cell proliferation%MicroRNAs
目的 探讨microRNA-338-3p(miR-338-3p)对神经母细胞瘤细胞系SK-N-SH和SK-N-MC增殖、侵袭和迁移能力的影响,miR靶基因的确定及靶基因功能的研究.方法 miRNA芯片结果发现miR在神经母细胞瘤中低表达,利用实时定量PCR验证其结果.利用脂质体LipofectamineTM2000将miR-338-3p mimics和miR-338-3p的反义寡核苷酸链(ASO)以及对照质粒分别瞬时转染SK-N-SH和SK-N-MC两种细胞系,MTT法和克隆形成实验检测细胞的增殖能力,Transwell实验检测细胞的体外侵袭和迁移能力.利用生物信息学技术预测miR-338-3p的靶基因,并应用EGFP荧光报告载体实验,实时荧光定量PCR和Western blot实验对靶基因进行验证.并利用实时定量PCR检测神经母细胞瘤中靶基因的表达.最后,将靶基因的敲降质粒和过表达质粒以及对照空质粒分别瞬时转染细胞,MTT法和克隆形成实验检测细胞的增殖能力,Transwell实验来检测细胞的体外侵袭和迁移能力.结果 实时定量PCR显示与癌旁正常组织相比,miR-338-3p在神经母细胞瘤细胞中低表达(P＜0.01)；癌细胞转染miR-338-3p mimics后,细胞活性、克隆形成能力、侵袭和迁移能力与对照组相比是减弱的(P＜0.01),而在转染了miR-338-3p的ASO封闭miR-338-3p的表达后,细胞活性、克隆形成能力、侵袭和迁移能力与对照组相比是增强的(P＜0.01).利用生物信息学方法将RAB14基因作为miR-338-3p的候选靶基因,EGFP荧光报告载体实验显示过表达miR-338-3p可以抑制含有miR-338-3p结合位点的报告载体的荧光量(P＜0.01),而将结合位点的碱基进行突变后miR-338-3p对靶基因3'UTR的抑制作用则消失.实时荧光定量PCR和Western blot结果表明过表达miR-338-3p可以同时在mRNA和蛋白水平上抑制RAB14的表达(P＜0.01),而用ASO抑制miR-338-3p的表达后RAB14的水平升高(P＜0.01).实时定量PCR结果显示靶基因RAB14在神经母细胞瘤中呈现高表达(P＜0.01).最后,癌细胞转染敲降RAB14的质粒pSilencer/shR-RAB14后,细胞活性、克隆形成能力、侵袭和迁移能力降低(P＜0.01),而过表达靶基因RAB14后,细胞活性、克隆形成能力、侵袭和迁移能力增强(P＜0.01).结论 RAB14是miR-338-3p的直接靶基因,miR-338-3p通过抑制RAB14的表达而抑制神经母细胞瘤细胞的增殖及侵袭和迁移能力.
目的 探討microRNA-338-3p(miR-338-3p)對神經母細胞瘤細胞繫SK-N-SH和SK-N-MC增殖、侵襲和遷移能力的影響,miR靶基因的確定及靶基因功能的研究.方法 miRNA芯片結果髮現miR在神經母細胞瘤中低錶達,利用實時定量PCR驗證其結果.利用脂質體LipofectamineTM2000將miR-338-3p mimics和miR-338-3p的反義寡覈苷痠鏈(ASO)以及對照質粒分彆瞬時轉染SK-N-SH和SK-N-MC兩種細胞繫,MTT法和剋隆形成實驗檢測細胞的增殖能力,Transwell實驗檢測細胞的體外侵襲和遷移能力.利用生物信息學技術預測miR-338-3p的靶基因,併應用EGFP熒光報告載體實驗,實時熒光定量PCR和Western blot實驗對靶基因進行驗證.併利用實時定量PCR檢測神經母細胞瘤中靶基因的錶達.最後,將靶基因的敲降質粒和過錶達質粒以及對照空質粒分彆瞬時轉染細胞,MTT法和剋隆形成實驗檢測細胞的增殖能力,Transwell實驗來檢測細胞的體外侵襲和遷移能力.結果 實時定量PCR顯示與癌徬正常組織相比,miR-338-3p在神經母細胞瘤細胞中低錶達(P＜0.01)；癌細胞轉染miR-338-3p mimics後,細胞活性、剋隆形成能力、侵襲和遷移能力與對照組相比是減弱的(P＜0.01),而在轉染瞭miR-338-3p的ASO封閉miR-338-3p的錶達後,細胞活性、剋隆形成能力、侵襲和遷移能力與對照組相比是增彊的(P＜0.01).利用生物信息學方法將RAB14基因作為miR-338-3p的候選靶基因,EGFP熒光報告載體實驗顯示過錶達miR-338-3p可以抑製含有miR-338-3p結閤位點的報告載體的熒光量(P＜0.01),而將結閤位點的堿基進行突變後miR-338-3p對靶基因3'UTR的抑製作用則消失.實時熒光定量PCR和Western blot結果錶明過錶達miR-338-3p可以同時在mRNA和蛋白水平上抑製RAB14的錶達(P＜0.01),而用ASO抑製miR-338-3p的錶達後RAB14的水平升高(P＜0.01).實時定量PCR結果顯示靶基因RAB14在神經母細胞瘤中呈現高錶達(P＜0.01).最後,癌細胞轉染敲降RAB14的質粒pSilencer/shR-RAB14後,細胞活性、剋隆形成能力、侵襲和遷移能力降低(P＜0.01),而過錶達靶基因RAB14後,細胞活性、剋隆形成能力、侵襲和遷移能力增彊(P＜0.01).結論 RAB14是miR-338-3p的直接靶基因,miR-338-3p通過抑製RAB14的錶達而抑製神經母細胞瘤細胞的增殖及侵襲和遷移能力.
목적 탐토microRNA-338-3p(miR-338-3p)대신경모세포류세포계SK-N-SH화SK-N-MC증식、침습화천이능력적영향,miR파기인적학정급파기인공능적연구.방법 miRNA심편결과발현miR재신경모세포류중저표체,이용실시정량PCR험증기결과.이용지질체LipofectamineTM2000장miR-338-3p mimics화miR-338-3p적반의과핵감산련(ASO)이급대조질립분별순시전염SK-N-SH화SK-N-MC량충세포계,MTT법화극륭형성실험검측세포적증식능력,Transwell실험검측세포적체외침습화천이능력.이용생물신식학기술예측miR-338-3p적파기인,병응용EGFP형광보고재체실험,실시형광정량PCR화Western blot실험대파기인진행험증.병이용실시정량PCR검측신경모세포류중파기인적표체.최후,장파기인적고강질립화과표체질립이급대조공질립분별순시전염세포,MTT법화극륭형성실험검측세포적증식능력,Transwell실험래검측세포적체외침습화천이능력.결과 실시정량PCR현시여암방정상조직상비,miR-338-3p재신경모세포류세포중저표체(P＜0.01)；암세포전염miR-338-3p mimics후,세포활성、극륭형성능력、침습화천이능력여대조조상비시감약적(P＜0.01),이재전염료miR-338-3p적ASO봉폐miR-338-3p적표체후,세포활성、극륭형성능력、침습화천이능력여대조조상비시증강적(P＜0.01).이용생물신식학방법장RAB14기인작위miR-338-3p적후선파기인,EGFP형광보고재체실험현시과표체miR-338-3p가이억제함유miR-338-3p결합위점적보고재체적형광량(P＜0.01),이장결합위점적감기진행돌변후miR-338-3p대파기인3'UTR적억제작용칙소실.실시형광정량PCR화Western blot결과표명과표체miR-338-3p가이동시재mRNA화단백수평상억제RAB14적표체(P＜0.01),이용ASO억제miR-338-3p적표체후RAB14적수평승고(P＜0.01).실시정량PCR결과현시파기인RAB14재신경모세포류중정현고표체(P＜0.01).최후,암세포전염고강RAB14적질립pSilencer/shR-RAB14후,세포활성、극륭형성능력、침습화천이능력강저(P＜0.01),이과표체파기인RAB14후,세포활성、극륭형성능력、침습화천이능력증강(P＜0.01).결론 RAB14시miR-338-3p적직접파기인,miR-338-3p통과억제RAB14적표체이억제신경모세포류세포적증식급침습화천이능력.
Objective To explore the potential roles of miR-338-3p on the cell proliferation,migration and invasion abilities and identify the target gene for miR-338-3p in human neuroblastoma cell lines.Methods Our previous results from miRNA array showed that miR-338-3p was downregulated in neuroblastoma.In the current study,quantitative Real-time PCR was performed to detect the expression of miR-338-3p in the neuroblastoma tissues and the adjacent non-tumor tissues.Then we transfected the neuroblastoma cells with miR-338-3p mimics or ASO-miR-338-3p,or the controls using LipofectamineTM 2000 reagent.The cell proliferation was measured by MTT and colony formation assay,and the migration and invasion abilities were detected by the Transwell chamber kit.The bioinformatics was used to predict the target gene for miR-338-3p,and EGFP reporter assay,quantitative Real-time PCR and Western blot were performed to validate the target gene.Finally,we transfected the cells with pSilencer/shR-RAB14 and pcDNA3/RAB14 respectively,and then detected the cell proliferation,migration and invasion abilities.Results The miR-338-3p was downregulated in neuroblastoma tissues,compared to the adjacent non-tumor tissues (P＜0.01).The miR-338-3p overexpression inhibited the cell viability,colony formation rate,migration and invasion abilities in the cells transfected with miR-338-3p mimics (P＜0.01).In contrast,the inhibition of miR-338-3p had the opposite roles (P＜0.01).RAB14 was predicted as a candidate target gene for miR-338-3p.EGFP reporter assay showed that miR-338-3p reduced the GFP intensity controlled by the RAB14 3'UTR (P＜0.01),while neither miR-338-3p nor inhibition of miR-338-3p had effect on the GFP intensity controlled by the mutated 3'UTR of RAB14.miR-338-3p reduced the RAB14 expression on both mRNA and protein levels (P＜0.01).In contrast,the inhibition of miR-338-3p increased the RAB14 levels.In addition,RAB14 was upregulated in neuroblastoma tissues (P＜ 0.01).Finally,silencing RAB14 inhibited the cell viability,colony formation,migration and invasion abilities (P＜0.01),but overexpression of RAB14 had the opposite roles (P＜0.01).Conclusions RAB14 is a direct target gene for miR-338-3p,and miR-338-3p inhibits the cell proliferation,migration and invasion abilities by the suppression of RAB14.
