Upload
others
View
5
Download
0
Embed Size (px)
Citation preview
DOI:10.13350/j.cjpb.190602 ·!"
·
miR133a3p456789:;<=>?@
LX2!AαSMABCDE
op
1,2qr
1,stu
1,,vv
1,2,wx
1,yz{|}
·~�
1,���
1,3
(1.¯ü:B#�Bü&°e³(C$ùúÑÅ}[
,DEF{78ö¿GuF=HIF8û3=
,DEJKLM830054;
2.DE78<N{8³ÈÉ8=
,DE<�ËFde"ÞúÑÅ}[
;3.DEF{78dOF8=<��8³A*<�8Pû[
)
【!"
】 #$ QBmiR133a3púfR¶S&T
(recombinantadenoassociatedvirus,rAAV)vÍUVazW-XY
Zop
,¬T W-JÝ'~l
。 %& a[��XYZopiLX2op
,s}AfDMEMQ�f
,AAV8Scram
bledQ�f
,AAV8miR133a3pf
,�lqRTPCRPVW miR133a3pW-ÍUU
,VW\H;<
(COL1A1)、�]
^^_;<
(alphasmoothmuscleactin,αSMA)@TGFβ/SmadÁÂà`deZÍUU
。 '( úfR¶S&T°
aQBzW- LX2op
,AAV8miR133a3pW- LX2opfZ miR133a3p¶QÍUUf11000±343.10,³
DMEMQ�f����ËÇý8bc
(狋=32.07,犘<0.01)。AAV8miR133a3pW-f³DMEM fαSMAmRNA
Z¶QÍUUAjf0.42±0.06vs1.00±0.09(狋=5.72,犘<0.05),COL1A1mRNAZ¶QÍUUf0.34±0.03vs
1.00±0.04(狋=12.66,犘<0.01)。 ') úfR¶S&T°aW-LX2op
,ï@ø*XYZop����$�α
SMAZÍU
,f�dmiR133a3p�e)XYZop.��#Z~lfT8g!dO
。
【*+,
】 R¶S&T
;XYZop
;miR133a3p
【-./01
】 R373.9 【23456
】 A 【2781
】 16735234(2019)06062504
[犑狅狌狉狀犪犾狅犳犘犪狋犺狅犵犲狀犅犻狅犾狅犵狔.2019Jun;14(6):625-628.]
犆狅狀狊狋狉狌犮狋犻狅狀狅犳犃犃犞8狋犺犪狋狅狏犲狉犲狓狆狉犲狊狊犲狊犿犻犚133犪3狆犪狀犱犻狋狊犻狀犺犻犫犻狋狅狉狔犲犳犳犲犮狋狅狀α犛犕犃犲狓狆狉犲狊狊犻狅狀犻狀
犔犡2犮犲犾犾狊
LILiang1,ZHANGChuanshan1,BIXiaojuan
1,MAYangyang1,2,WANGHui1,TUERGANAILI
Aji1,LINRenyong
1,3 (1.犛狋犪狋犲犓犲狔犔犪犫狅狉犪狋狅狉狔狅犳犘犪狋犺狅犵犲狀犲狊犻狊,犘狉犲狏犲狀狋犻狅狀犪狀犱犜狉犲犪狋犿犲狀狋狅犳犎犻犵犺犾狔犘狉犲狏犪
犾犲狀狋犇犻狊犲犪狊犲狊犻狀犆犲狀狋狉犪犾犃狊犻犪,犆犾犻狀犻犮犪犾犕犲犱犻犮犪犾犚犲狊犲犪狉犮犺犐狀狊狋犻狋狌狋犲,犜犺犲犉犻狉狊狋犎狅狊狆犻狋犪犾犃犳犳犻犾犻犪狋犲犱狑犻狋犺犡犻狀犼犻犪狀犵犕犲犱犻
犮犪犾犝狀犻狏犲狉狊犻狋狔,犝狉狌犿狇犻,犆犺犻狀犪830054;2.犡犻狀犼犻犪狀犵犓犲狔犔犪犫狅狉犪狋狅狉狔狅犳犅犻狅犾狅犵犻犮犪犾犚犲狊狅狌狉犮犲狊犪狀犱犌犲狀犲狋犻犮犈狀犵犻狀犲犲狉犻狀犵,
犆狅犾犾犲犵犲狅犳犔犻犳犲犛犮犻犲狀犮犲犪狀犱犜犲犮犺狀狅犾狅犵狔,犡犻狀犼犻犪狀犵犝狀犻狏犲狉狊犻狋狔;3.犇犲狆犪狉狋犿犲狀狋狅犳犅犻狅犮犺犲犿犻狊狋狉狔,犆狅犾犾犲犵犲狅犳犅犪狊犻犮犕犲犱
犻犮犻狀犲,犡犻狀犼犻犪狀犵犕犲犱犻犮犪犾犝狀犻狏犲狉狊犻狋狔)
【犃犫狊狋狉犪犮狋】 犗犫犼犲犮狋犻狏犲狊 ToconstructanAAVvectorthatoverexpressesmiR133a3pandtoexamineitsinhibitory
effectonαSMAmRNAexpressionintheLX2hepaticstellatecellline. 犕犲狋犺狅犱狊 LX2cellswereculturedinvitro,di
videdintothreegroups,andtransfectedwiththreeagents(DMEMcontrol,AAV8ScrambledgroupandAAV8miR
133a3pgroup).TherelativeexpressionofmiR133a3pandtheexpressionofαSMA,COL1A1,caspase9,andTGFβ/
SmadmRNAweredetectedusingqRTPCR. 犚犲狊狌犾狋狊 AAV8miR133a3pwassuccessfullyconstructedandtransfected
intoLX2cells.miR133a3pwasoverexpressedintheAAV8miR133a3pgroup.Therelativelevelofexpressionofα
SMAmRNAinLX2cellstransfectedwithAAV8miR133a3pwas0.42±0.06.Theleveldifferedsignificantlyfrom
thatintheDMEMcontrolgroup(狋=5.72,犘<0.05).TherelativelevelofexpressionofCOL1A1mRNAwas0.34±
0.03.TheleveldifferedsignificantlyfromthatintheDMEMcontrolgroup(狋=12.66,犘<0.01).Therewerenosignifi
cantdifferencesinexpressionofTGFβ/SmadmRNA (犘>0.05). 犆狅狀犮犾狌狊犻狅狀 AAV8miR133a3pwassuccessfully
constructedandtransfectedintotheLX2cellline.TheresultsofinhibitionofoverexpressionofmiR133a3pindicated
thatAAV8miR133a3pisanoveltoolforfurtherresearchonliverfibrosisregulatedbyHSCandmiR133a3p.
【犓犲狔狑狅狉犱狊】 Adenoassociatedviruses(AAVs);hepaticstellatecell(HSC);microRNA133a3p
·526·
5 6 7 8 9 : ; < =
犑狅狌狉狀犪犾狅犳犘犪狋犺狅犵犲狀犅犻狅犾狅犵狔 2019ô6õ ö14÷ö6ø
Jun.2019, Vol.14,No.6
【9:;#
】 DE�hi`C6`j{8dpÿÒ
(No.2016D01C329);$ù`j{8dpÿÒ
(No.81371838,81660108);DE�
hi`C6kD��
(0l
、dm
)ÿÒ
(No.201705120)。
【<=>?
】 nop
,Email:renyong_lin@sina.com
【>?@A
】 ý q
(1983-),r,5s7�0
,12
,Å}t
。òóû3O4
:XuÜv³w÷ZA*�*
。
Email:lilisx83@163.com
X���
(Liverfibrosis)¾À�xGXÜv
(&
TGXy
、z{GX&
、|z{G>öX
、#TGX&
、
`}~�GX&ß
)�Z¿�`}w÷Ç�
, Å:¾
�íⅠh
、Ⅲh\HZop[d:
(extracellularma
trixc,ECM)Ұ7��r
,çèECM7U¹Ã
,èX
YZop
(hepaticstellatecell,HSC)¾ECM Ò°Z
òóopÚF
,�Z�½
、º�¾���ZS��®@
#+à`
,eU�X���ZC���òó��ø*
HSCZ½�
、�{ ��
、��ECMZ¹Ãß
[1]。Õ
�RNA(MicroRNA,miRNA)¾:�18~25ntZÉ
�¼�½
,³�deZ3'|��6
(3'UntranslatedRe
gions,3'UTR)XÒ
,��de�r²ø* ��v
Þ
[2]。Fengß
[3]��û3ü¦ miRNA �X���
ü�vÞ#ÊËúó~l
,miR133a3pu�^op
��G miR206ù�
,ø*^opZº�@A�v
Þ
。�Å}ÀlmiR133a3pR¶S&TvÍUVa
W-0FXYZopLX2,VWECM òó°�\H
(collagen)、HSC½��$�α�]^^_;<
(al
phasmoothmuscleactin,αSMA)、e)���ü<
ZTGFβ/SmadÁÂà`¶Sdeß mRNA ZÍ
U
,fû3miR133a3pe)XYZop.èX��
�vÞ#Z~l�*M�g!dO
。
noD%&
1 BC
0XYZopLX2opig`#${8=op
�
;AAV8CBAGFPU6miR133a3pvÍUR¶S
&TJ AAV8CBAGFPU6ScrambledQ�f&T
X40���9m< @��
;B=DMEM ��d
,
�¡¢£bOFBS,L¤¥¦§@0.05%¨;<«
EDTA,© mRNA �Mqrb Trizol,ö¿�cDNA
Ò°stu
,miRNAÇWªstu�g`}$Ther
moFisherScientific9m
;MicroRNAAssayTUs
tug`}$ThermoFisherScientific9m
;mRNA
RSTUstug`n$QIAGEN9m Ð
;��X
L«<"9mÒ°��
。
2 %&
2.1 ^��LX2LM�RNA_` LX2op]1
×105 ��6¬¨
,10%bO
、37℃��24h,á&
T1×106 Vg/cell,Ðo��d�é&T
,Aj��
,
DMEM f
、AAV8Scrambled(1×106 Vg/cell)f、
AAV8miR133a3p(1×106 Vg/cell)f。W-24h
>?op<:®¯
。48h§��b®¯op
,PBS¶
op3¸
,�¬á¨1mlTrizol,°±
,~opqrb
²�ÕRNA«1.5ml¤+µ#
,[t³�5min;á
200μl�´�°±15s,³�10min。4℃,12000r/
min(¤+¥¦10.2cm)¤+15min;µNL¶Õ·
m¶�DZ1.5ml¤+µ#
,á¨500μl�¸¹
,º
»°±
,[t¬10min。4℃,12000r/min(¤+¥
¦10.2cm)¤+10min,§LO
;á1ml75%¼¹
,
°±
,4℃,7500r/min(¤+¥¦10.2cm)¤+5
min;§LO
,[t³�10min,½ÝRNA¹º
;á¨
30μlRNAarb
,�l Nanodrop1000ÕU¾[±
DASS�ýWT©RNA��
。
2.2 ���@efgh PCR �� N1μg©
RNA,¿À±²³HIÁWªÒ°ö¿�cDNA。N
200ng©RNA,¿À MicroRNAÁWªstu±²
³HIÒ°Â�XQ¼½ �
。-20℃\]Øl
。
RS��PVWmiR133a3pÍUU
,Ç�a¾
:20×
TaqMan?SmallRNAAssay,1.00μl;ÁWª �
,1.
33μl;2×PCR MasterMix;Nucleasefreewater,7.
67μl;PCRÇ���
:95℃'ÁG10min;95℃ÁG
10s,60 ℃ Â Ã Ä Å 30s,: 40 Ï Æ �
。SYBR
GREEN-ÌPVWTGFβ/SmadÁÂà`¶Sde
mRNAÍU
。Ç�a¾
:2×PCRMasterMix,10μl;
ForwardPrimer,0.40μl;Rreverseprimer,0.40μl;
cDNA,2.00μl;RNaseFreeH2O,7.20μl;PCRÇ�
��
:95℃'ÁG5min;95℃ÁG10s,60℃ÂÃÄ
Å30s,:40ÏÆ�
;65~95℃Ãar_ÄAB
。
2.3 kl;�� ÅÀRSTUÓÀkl2△△Ct
PýÆdeÍUU
,�lSPSS22.0ÇýÈ�{|Ó
ÀAB
?VWXY]�ӱ��
(x±s)ÍÎ
,�f
f¤��kl狋V}
,α=0.05。
' (
1 pq*rstSuvwxy犔犡2K\z{
RS»�ÇÕÈ�
,>? AAV8miR133a3p、
AAV8miRScrambledW-LX2op24h�opÉ
Ê
,¥¦Ë·RS
(Ì1),�� miRNAvÍUR¶S
&T°aW-LX2op
。
2 狇犚犜犘犆犚|}pq*rsxy 犔犡2K\ 犿犻犚
133犪3狆$q~SuP
AjMNW- AAV8miR133a3p、AAV8miR
Scrambled�ZLX2op©RNA,{|ÁWª
。]
DMEM、AAV8miRScrambled f f Q � f
,� l
TaqMan��PVWLX2op# miR133a3pZ¶
QÍUm�
,XYDÌ2。Å}f miR133a3p¶Q
ÍUUf11000±343.10,³Q�f����ËÇý
8bc
(狋=32.07,犘<0.01),Ͳ miR133a3púf
R¶S&T@B·vÍUmiR133a3p。
·626·
5 6 7 8 9 : ; < =
犑狅狌狉狀犪犾狅犳犘犪狋犺狅犵犲狀犅犻狅犾狅犵狔 2019ô6õ ö14÷ö6ø
Jun.2019, Vol.14,No.6
.1 MN���������pq*rsxy犔犡2K\
犉犻犵.1 犚犲犮狅犿犫犻狀犪狀狋犪犱犲狀狅狉犲犾犪狋犲犱狏犻狉狌狊犲狊狑犲狉犲狋狉犪狀狊犳犲犮狋犲犱犔犡2犮犲犾犾狊狅狀犿犻犮狉狅狊犮狅狆犲
.2 pq*rsxy犔犡2K\犿犻犚133犪3狆q~SuP
犉犻犵.2 犚犲犾犪狋犻狏犲犲狓狆狉犲狊狊犻狅狀狅犳犿犻犚133犪3狆狅犳犔犡2狋狉犪狀狊犳犲犮狋犲犱狉犃犃犞8
3 狇犚犜犘犆犚|}pq*rsxy犔犡2K\��
、α
犛犕犃、犜犌犉β/犛犿犪犱�1<�9�Su��
klqRTPCRPVW AAV8miR133a3pW-LX
2op�TGFβ/SmadÁÂà`J���¶SdeÍ
UU
,XYDÌ3。TGFβ/SmadÁÂà`¶Sde
mRNAÍUUÕ²ÇÁ�
(犘>0.05);HSC½��$
� αSMA mRNA � AAV8miR133a3p f ³
DMEMfZ¶QÍUUAjf0.42±0.06vs1.00
±0.09,���ËÇý8bc
(狋=5.72,犘<0.05;);
ECMòó°�\H;<COL1A1mRNA� AAV8
miR133a3pf³ DMEM fZ¶QÍUUAjf
0.34±0.03vs1.00±0.04,��ËÇý8bc
(狋=
12.66,犘<0.01)。
A TGFβreceptorⅠ(TGFβRⅠ) B TGFβreceptorⅡ(TGFβRⅡ) C TGFβⅠ D Smad2 E Smad3 F Smad4 G αSMA H
COL1A1ICaspase9.3 ��MNOP犘犆犚|}犃犃犞8犿犻犚133xy犔犡2,犿犚犖犃q~SuP
犉犻犵.3 犚犲犾犪狋犻狏犲犲狓狆狉犲狊狊犻狅狀狅犳犿犚犖犃狅犳犔犡2狋狉犪狀狊犳犲犮狋犲犱犃犃犞8犿犻犚133犱犲狋犲犮狋犲犱犫狔狇犚犜犘犆犚
e )
û3Ͳ
,XYZop�X���{Þ#ÍS�
~l
,XYZop�ÎCXu#¥³ÏÊ
,ÊËÐ]�
<ÊAze®�<ÊAÑ&Za@
,ÒXuü<Üv
,XYZopWÁ°�½Ê
,Ê˺�G
、ÅÓG
,A
�°^°��op
,¾ ECM Ò°ZòóÚF
[4]。
ECM��\H;<
(Ⅰ、Ⅲ、Ⅳ、V、Ⅵß
)、ÔG;<
、
·726·
5 6 7 8 9 : ; < =
犑狅狌狉狀犪犾狅犳犘犪狋犺狅犵犲狀犅犻狅犾狅犵狔 2019ô6õ ö14÷ö6ø
Jun.2019, Vol.14,No.6
¶Õ;<
、ÖÕ;<
、ÔG;<
、;<×=ß°�
[5]。
XYZopZ�½àvØAÙope* TGFβ1,
TNFα、EGF、PDGFß
,²`AÙope* TGFβ1,
�TNFαZ{¿H½�
,TGFβ1ÚÛf¾¿�úó
Z�{ HSC½�@ECM <Z§a@e®;<
[6]。
d�TGFβ/SmadÁÂà`�e)XYZop�½
JECMÒ°AÙOÜS�G~l
,°f����Z
úó�Ñ
,�l �a
、�A*Ý�t²ø*tÞZÁ
ÂA*,ç
,�±ÍÙø*²ÞZ HSC½�Z~
l
[7-8]。
miRNA¾¿?�{�LB�\ß
、:�à22nt
Z|��RNA,°ámiRNAàvÐoâã²�Ðo
âã³mRNA3'|��6XÒ
,��Ge)�deZ
��½G
,�Wª�e)�deZÍU
[9]。miRNA@
àv§�ÁÂà`[\ HSCZ½�
、º�@��
,
miR133u�^op��GmiRNA206ù�
,�e)
äå^
、+^���OÜÊËø*~l
[10-11]。miR
133Ë miR133a@ miR133b,àv TGFβ/SmadÁ
Âà`ø* HSC\HÒ°
,¾æ�ZC��ѲY
Z�$�
[12]。úfR¶S&TÊËB·
、çTW-é
¼opß�G
,¾èé�deC�ZDhVa
,8hR
¶S&TQXuopBê-G
[13]。
�û3ëìXfíîïÜëëZ^op��G
miR133a3p,Q B AAV8miR133a3p V a
,W -
LX2op�WÙB·ÍU
,ÍUUàfQ�fZ104
�
,RSTUPCRVWÙ HSCop½���G�$
�αSMA@ECMòó°�COL1A1mRNAÍU�
�
,³Roderburgß
[12]Àl miR133amimicsÝ'X
YZop\HÒ°ZXY¿è
,CvÍU miR133ú
fR¶S&T�Ý' TGFβ/SmadÁÂà`LúÍ
¦ëø*~l
。
û3kl AAV8miR133a3p±B·W- HSC
zø*αSMA、COL1A1Z¶QÍUU
,miR133a3p
vÍUa¾QB°Y
,f�d miR133e)ÁÂà`
�X��#Z~lfT8Å}@g!dO
。
【fg23
】
[1] PinzaniM.Pathophysiologyofliverfibrosis[J].DigDis,2015
(33):492-7.
[2] KitanoM,BloomstonPM.Hepaticstellatecellsandmicrornasin
pathogenesisofliverfibrosis[J].JClinMed,2016,5(3):E38.
[3] FengY,NiuLL,WeiW,etal.AfeedbackcircuitbetweenmiR
133andtheERK1/2pathwayinvolvinganexquisitemechanism
forregulatingmyoblastproliferationanddifferentiation[J].Cell
DeathDis,2013,4(11):e934.
[4] PucheJE,SaimanY,FriedmanSL,etal.Hepaticstellatecells
andliverfibrosis[J].ComprPhysiol,2013,3(4):1473-92.
[5] ArriazuE,RuizdeGalarretaM,CuberoFJ,etal.Extracellular
matrixandliverdisease[J].AntioxidRedoxSignal.2014,21
(7):1078-97.
[6] YoshidaK,MurataM,YamaguchiT,etal.TGFβ/Smadsigna
lingduringhepaticfibrocarcinogenesis[J].IntJOncol,2014,45
(4):1363-71.
[7] GordonKJ,BlobeGC.Roleoftransforminggrowthfactorbeta
superfamilysignalingpathwaysinhumandisease[J].Biochim
BiophysActa,2008,1782(4):197-228.
[8] ð;)
,ñòó
,óÎ.W�<:e*βQXYZop½�Ze
)~l
[J].HIXò&9$
,2016,32(11):2195-98.
[9] SanjayS,GirishC.RoleofmiRNAanditspotentialasanoveldi
agnosticbiomarkerindruginducedliverinjury[J].EurJClin
Pharmacol,2017,73(4):399-407.
[10] DuanLJ,QiJ,KongXJ,etal.MiR133modulatesTGFβ1in
ducedbladdersmoothmusclecellhypertrophicandfibroticre
sponse:implicationforaroleofmicroRNAinbladderwallremod
elingcausedbybladderoutletobstruction[J].CellSignal,2015,
27(2):215-27.
[11] WangL,LiX,ZhouY,etal.DownregulationofmiR133via
MAPK/ERKsignalingpathwayinvolvedinnicotineinducedcar
diomyocyteapoptosis[J].NaunynSchmiedebergsArchPharma
col,2014,387(2):197-206.
[12] RoderburgC,LueddeM,VargasCardenasD,etal.miR133a
mediatesTGFβdependentderepressionofcollagensynthesisin
hepaticstellatecellsduringliverfibrosis[J].JHepatol,2013,58
(4):736-42.
[13] OhnoK,ItoM,KawakamiY,etal.CollagenQisakeyplayer
fordevelopingrationaltherapyforcongenitalmyastheniaandfor
dissectingthemechanismsofantiMuSKmyastheniagravis[J].J
MolNeurosci,2014,53(3):359-61.
【hijk
】 20190305 【lmjk
】 20190510
·826·
5 6 7 8 9 : ; < =
犑狅狌狉狀犪犾狅犳犘犪狋犺狅犵犲狀犅犻狅犾狅犵狔 2019ô6õ ö14÷ö6ø
Jun.2019, Vol.14,No.6