中华小儿外科杂志
中華小兒外科雜誌
중화소인외과잡지
CHINESE JOURNAL OF PEDIATRIC SURGERY
2012年
5期
382-385
,共4页
蒋志慧%董蒨%高会江%鹿洪亭%刘世海%赵静
蔣誌慧%董蒨%高會江%鹿洪亭%劉世海%趙靜
장지혜%동천%고회강%록홍정%류세해%조정
微小RNA%受体,趋化因子%神经母细胞瘤
微小RNA%受體,趨化因子%神經母細胞瘤
미소RNA%수체,추화인자%신경모세포류
MicroRNA%Receptors,chemokine%Neuroblastoma
目的 微小RNA( microRNA,miRNA)是一类非编码小分子RNA组成的家族,能够通过降解靶mRNA或抑制其翻译过程参与调节基因表达.本研究旨在探索上调miR338水平后人神经母细胞瘤细胞系SH-SY5Y中CXCR4表达变化,进而了解miR338调节神经母细胞瘤侵袭与转移的分子机制.方法 将人工合成的成熟miR338前体(pre-mir-338)转染入SH-SY5Y细胞,而后利用实时荧光定量RT-PCR检测转染前后各组细胞miR338的表达水平.应用半定量RT-PCR及Western蛋白印迹法分别从mRNA水平、蛋白水平检测各实验组CXCR4的表达变化.结果 实时荧光定量PCR法检测对照组及各实验组细胞miR338基因含量,发现转染了pre-mir-338的10 nM,30 nM,50 nM,100 nM组miR338含量分别为空白对照组的1.32倍,1.62倍,1.90倍及1.86倍,差异具有统计学意义(P＜0.05),成功将pre-mir-338转染入细胞达到miR338过表达的目的；转染组(10 nM组、30 nM组、50nM组)CXCR4基因mRNA相对光密度值分别为0.75±0.06,0.58±0.08,0.24±0).05,依次降低,均明显低于空白对照组1.11±0.08,空载对照组1.04±0.08(P＜0.05).转染组CX-CR4蛋白0).24±0.06较未转染组0.56±0.08降低(P＜0.05).结论 miR338能够通过下调神经母细胞瘤细胞CXCR4 miRNA及蛋白的表达参与调节肿瘤的侵袭与转移,人工合成的pre-mir-338有望通过替代方式为神经母细胞瘤的治疗提供新的途径.
目的 微小RNA( microRNA,miRNA)是一類非編碼小分子RNA組成的傢族,能夠通過降解靶mRNA或抑製其翻譯過程參與調節基因錶達.本研究旨在探索上調miR338水平後人神經母細胞瘤細胞繫SH-SY5Y中CXCR4錶達變化,進而瞭解miR338調節神經母細胞瘤侵襲與轉移的分子機製.方法 將人工閤成的成熟miR338前體(pre-mir-338)轉染入SH-SY5Y細胞,而後利用實時熒光定量RT-PCR檢測轉染前後各組細胞miR338的錶達水平.應用半定量RT-PCR及Western蛋白印跡法分彆從mRNA水平、蛋白水平檢測各實驗組CXCR4的錶達變化.結果 實時熒光定量PCR法檢測對照組及各實驗組細胞miR338基因含量,髮現轉染瞭pre-mir-338的10 nM,30 nM,50 nM,100 nM組miR338含量分彆為空白對照組的1.32倍,1.62倍,1.90倍及1.86倍,差異具有統計學意義(P＜0.05),成功將pre-mir-338轉染入細胞達到miR338過錶達的目的；轉染組(10 nM組、30 nM組、50nM組)CXCR4基因mRNA相對光密度值分彆為0.75±0.06,0.58±0.08,0.24±0).05,依次降低,均明顯低于空白對照組1.11±0.08,空載對照組1.04±0.08(P＜0.05).轉染組CX-CR4蛋白0).24±0.06較未轉染組0.56±0.08降低(P＜0.05).結論 miR338能夠通過下調神經母細胞瘤細胞CXCR4 miRNA及蛋白的錶達參與調節腫瘤的侵襲與轉移,人工閤成的pre-mir-338有望通過替代方式為神經母細胞瘤的治療提供新的途徑.
목적 미소RNA( microRNA,miRNA)시일류비편마소분자RNA조성적가족,능구통과강해파mRNA혹억제기번역과정삼여조절기인표체.본연구지재탐색상조miR338수평후인신경모세포류세포계SH-SY5Y중CXCR4표체변화,진이료해miR338조절신경모세포류침습여전이적분자궤제.방법 장인공합성적성숙miR338전체(pre-mir-338)전염입SH-SY5Y세포,이후이용실시형광정량RT-PCR검측전염전후각조세포miR338적표체수평.응용반정량RT-PCR급Western단백인적법분별종mRNA수평、단백수평검측각실험조CXCR4적표체변화.결과 실시형광정량PCR법검측대조조급각실험조세포miR338기인함량,발현전염료pre-mir-338적10 nM,30 nM,50 nM,100 nM조miR338함량분별위공백대조조적1.32배,1.62배,1.90배급1.86배,차이구유통계학의의(P＜0.05),성공장pre-mir-338전염입세포체도miR338과표체적목적；전염조(10 nM조、30 nM조、50nM조)CXCR4기인mRNA상대광밀도치분별위0.75±0.06,0.58±0.08,0.24±0).05,의차강저,균명현저우공백대조조1.11±0.08,공재대조조1.04±0.08(P＜0.05).전염조CX-CR4단백0).24±0.06교미전염조0.56±0.08강저(P＜0.05).결론 miR338능구통과하조신경모세포류세포CXCR4 miRNA급단백적표체삼여조절종류적침습여전이,인공합성적pre-mir-338유망통과체대방식위신경모세포류적치료제공신적도경.
Objective MicroRNAs (miRNAs) are a class of small non-coding RNAs that can regulate gene expression by mRNA degradation or translation repression.In this study,our goal was to investigate the variation of CXCR4 in human neuroblastoma cell line SH-SY5Y over-expressed with miR338 and explored the mechanisms by which it influenced invasion,migration of neuroblastoma.Methods Pre-miRNA-338 was transfected into SH-SY5Y cells.Then we used real-time RT PCR to quantify the expression level of miR338 in these cells.CXCR4 expression at mRNA and protein levels were detected by semi-quantitive RT-PCR and Western blotting.Results Compared with control groups,real-time RT-PCR identified an increase in miRNA338 expression in premiRNA-338 transfected cells.CXCR4 mRNA in transfected cells significantly decreased (0.75 ± 0.06,0.58 ± 0.08,0.24± 0.05 VS 1.11± 0.08,1.04 ± 0.08,P＜0.05 ),CXCR4 protein was also decreased (0.24 ± 0.06 VS0.56± 0.08,P＜0.05).Conclusions Our results suggested that miR338 had potential tumor migration suppressor activity,acting through inhibition of CXCR4.miRNA338 replacement therapy through delivery of pre-miRNA 338 may be a novel therapeutic strategy.
