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34 "

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2012 $

12 % &'()*++, Journal of Ningxia Medical University -.

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1674 - 6309( 2012)

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1249 -

04 ・ Yt・ PPARα ?§¨ miRNA N?~??`

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1 ( 1. %&6?"*n8c', +, 750001;

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3. %&6?"*OO', +, 750001) 9:: pN ??*>l PPARα g?H6 miRNA.qr bc????$8??? PPARα g?H miRNA. PCR 7? PPARα H 3'UTR, *??・P4QQ^), ;

?0 miRNA ?r? 293T °±, lR3???è8?c l?0 miRNA ;

PPARα H]V??.i 293T °±)Szr??0 miRNA T^, gj? RNA ???), e? real time PCR D Western blot ?? PPARα mRNA D?)H??.st ( 1)TargetScan ??U??| miR -

9 D miR -

124 U;

PPARα H 3'UTR ??B]V.( 2)miR -

9 b miR -

124 ???? pmirGLO - PPARα - 3'UTR R3???è( P < 0. 01) .( 3) i miR -

9 b miR -

124 c??H 293T °±), PPARα mRNA D?)?????( P < 0. 01) .s8 PPARα

3 miR -

9 D miR -

124 ?>Hg4Q, miR -

9 b miR -

124 i r???Vè~? PPARα H??. ;

?@A0: R373.

9 -BCDE: A FGH#:

2012 -08 -31 IJKL: W;

@, |, T"?, ??, ??? microRNA {U??. E -mail: guodonggeng@126. com xyIJ: >X(

1958 - ) , ?;

6, ??/, ¨????k?, ?? ?=#W$??. c?peroxisome proliferator - activated receptor,PPAR)?r6^? =r?Q?, ~Y?4Q??, ?????Z、 ? ??、 ?>??D??? [ 1].????, PPARα [ ??C??m ? [

2 - 3].8 RNA(microRNA, miRNA)_`?i5 C?、 ??4Q7) [ 4], ~?6? 30% H4Q? ? [ 5]H?W??W c ? [ 6].fa miRNA >b????aHb?÷?, H miRNAs Vcs?_, g4 QH??D>l?r?? miRNA {UHG"d, ?.ò miRNA gXH>l@??, ?m6$"> b???"Hd#8?, ???? PPARα g?H6 miRNA, *bc]@$ D{U$?N??GA?0??]W, r PPARα i?^?)H{U??el4?.

1 z{|qr 1.

1 ?HEK293T °±??? ê)'*;

pmirGLO lR3??・P4QQ^?? ?è8?yF?? Promega;

DNA 4=、 !P?R ??g?????? Omega ??;

Taq PCR Master Mix、 T4 u??、 ??ènd? XbaⅠ、 EcoR V、 ° ±????、 fg°±8A# ??X¨???;

Trizol、 Lipofectamine2000 r?? ??? invitrogen ??;

Realtime SYBR Green PCR ??(uh????;

miR -

9 D miR -

124 mimics ??è@:( Negtive control, NC) ?? Qiagen ??;

Western Blot sigma ??. 1.

2 IJ???U MiRBase: http:/ / www. miR Base. org (April 2011) ;

TargetScan Human:http:/ / www. TargetScan. org / archives. html (Release 6. 0: November2011) ;

Premier v5;

primer blast (http:/ / www. ncbi. nlm. nih. gov / tools/ primer - blast/ ) ;

NC BI - Pubmed (http:/ / www. ncbi. nlm. nih. gov / gene) . 1.

3 ・P4QQ^@u PCR 7?VH89:

8 A¤?li?p?? PBMC, Trizol Ogj? RNA, i? Oligo( dT)6?、 ?r??e^y 25μL, ?r ?.J: 37℃, 60min;

72℃, 10min.PCR ?e^y 25μL, PCR ?e.J: 94℃, 3min.i? Primerv5 IJ??KL6?à?, *6, XbaⅠ4?d?? ( PCR ???/ EcoR V ?d??) : PPARα(NM_ 005037)5 ' - CTAGTCTAGAGATATCACTGATGCCA CCA CCTGA - 3', PCR 7? PPARα 3'j??kl ?( 3'untranslational region,

3 'UTR) , 3391bp,

30 cy cles: 94℃,30 s;

55℃,30 s;

72℃,1 min;

72℃,5 ・

9 4

2 1 ・

34 ? min.?e]??, ?? 2% m??????, i2 pQn??ld?!P、 4=. ?VH89*?? pmirGlo Q^: Q^? PCR ?????è??nd? XbaⅠ?=;

T4 u?? 16℃u?c&;

r=;

W?è LB ??#o?, 37℃, 200rpm, 40min;

pq¨???è LB m???4 37℃r??? 14h;

gjR?, EcoR V ?d>l? 7R? pmirGLO - PPARα - 3'UTR. 1.

4 °±???Szr? HEK293T °±?? .J [

7 - 8]: >? DMEM, 10% ????, 37℃, 5% CO2 ?sD3°.tb?#,

2 ~ 3d ??.r?ü 24h, 0. 25% ???= 293T °±*??

24 ±^, M r? b ° ± ? 90% Q V.S z r ? ? Lipo fectamine 2000( Cat. No. 11668)??;

??.c? ?Z?z, 4r miRNA: W????4u? Lipo fectamine

2000 r???、 miR - 9,-

124 H mimic ? NC[Lipofectamine

2000 (μL)∶ siRNA(pmoL)= 5∶ 100] ;

R

3 ? ? ? è

8 ? z,? r miRNA D pmirGLO - 3'UTR[Lipofectamine 2000(μL)∶ siRNA ( pmoL) ∶ DNA( μg)= 5∶ 100∶ 2] .??÷^?vw -,°±??±;

37℃, 5% CO2 ???q?z?, P=°±, gj? RNA b??lR3???è8

3 ], ?"??

3 "?±. 1.

5 ? RNA gj?>l ?: Trizol Om?(ht tp:/ / www. invitrogen. com/ )gj? RNA, W RNase ??M,- 80℃ ??.1% m??????>l? RNA ü?è;

OD

260 / OD 280, ???°D4°. 1.

6 ZzR3l? PCR(Real time SYBR Green PCR) §r?: Oligo dT ?r§r?6?,

20 μL ?e^y?: "PrimeScrip RT reagent Kit Perfect Real Time "(TAKARA,http:/ / www. takara. com. cn / ) ??;

6?, ?¨???