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Explore the influence of CYP21A2 silencing on H295R human adrenal cortical carcinoma cell gene expression function

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Explore the influence of CYP21A2 silencing on H295R human adrenal cortical carcinoma cell gene expression function

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Content C o p y ri g h t 2 0 2 0 Y i m a n H u
Ex p l o r e t h e i n f l u e n c e o f C Y P2 1 A2 s i l e n c i n g o n H2 9 5 R h u m a n a d r e n a l
c o r t i c a l c a r c i n o m a c e l l g e n e e x p r e s s i o n f u n c t i o n
b y
Yi m a n Hu
A Th e s i s P r e s e n t e d t o t h e
F AC UL TY OF THE US C S c h o o l o f P h a r m a c y
UNI V ER S I TY OF S OUTH ER N C ALI F OR NI A
I n P a r t i a l F u l f i l l m e n t o f t h e
R e q u i r e m e n t s f o r t h e De g r e e
MASTER OF SCIENCE
Pharmaceutical Sciences
M a y 2 0 2 0
i i
T A B L E O F C O N T E N T S
L i s t of T a bl e s i i i
L i s t of F i gur e s i v
A bs t r a c t v
C ha pt e r 1: I n t r oduc t i on 1
1.1 C ong e ni t a l A dr e na l H y p e r pl a s i a ( C A H ) 1
1.2 P a t hw a y s of s t e r oi d hor m one s y n t he s i s 4
1.3 21- hy d r ox y l a s e ( 21O H ) de f i c i e nc y 8
1.4 11β - hy d r ox y l a s e ( 11 β O H ) de f i c i e nc y 10
1.5 3β – hy d r ox y s t e r oi d de hy d r oge na s e t y p e 2 ( 3 β H S D 2) de f i c i e nc y 11
1.6 17- hy d r ox y l a s e de f i c i e nc y 12
1.7 P 450 oxi dor e duc t a s e ( P O R ) de f i c i e nc y 12
1.8 L i poi d c onge ni t a l a dr e na l hy p e r pl a s i a 13
1.9 P 450 c hol e s t e r ol s i de - c ha i n c l e a va ge ( S C C ) de f i c i e nc y 14
1.10 H 295R C e l l l i ne 14
C ha pt e r 2 M a t e r i a l a nd M e t hod 16
2.1 C e l l c ul t ur e 16
2.2 R N A i nt e r f e r e nc e a s s a y 17
2.3 R N A e xt r a c t i on 17
2.4 Q ua nt i t a t i ve R e a l - T i m e P C R 18
2.5 B C A pr ot e i n a s s a y 20
2.6 E L I S A A s s a y f or C or t i s ol 20
2.7 E L I S A A s s a y f or A l dos t e r one 21
C ha pt e r 3 R e s ul t s 23
3.1 T he g e ne e x pr e s s i on c ha nge s i n H 295R c e l l s a f t e r dow n- r e gul a t i on of C Y P 21A 2
g e n e 23
3.2 S t e r oi d pr oduc t i on c ha nge s i n H 295R a f t e r knoc k dow n C Y P 21A 2 g e ne 27
C ha pt e r 4 D i s c us s i on 29
R e f e r e nc e s 31
i i i
L i s t o f T a b l e s
T a bl e 1. qP C R pr i m e r s 19
i v
L i s t o f F i g u r e s
F i gur e 1 A dr e na l s t e r oi doge ne s i s pa t hw a y s 7
F i gur e 2 S c he m a t i c r e pr e s e nt a t i on of a g e ne c onve r s i on m e c ha ni s m 9
F i gur e 3 G e ne e x pr e s s i on c ha nge a f t e r R N A i nt e r f e r e nc e A s s a y 26
F i gur e 4 E f f e c t of dow n- r e gul a t e d C Y P 21A 2 on S t e r oi d P r oduc t i on i n H 295R C e l l 28
v
A b s t r a c t
C ong e ni t a l a dr e na l hy p e r pl a s i a i s one of t he m os t c om m on g e ne t i c di s or de r s . T he c l i ni c a l
m a ni f e s t a t i ons of C A H i nc l ude a dr e na l i ns uf f i c i e nc y , g e ni t a l a m bi g ui t y , s hor t s t a t ur e , a nd a n
i nc r e a s e d r i s k of m e t a bol i c s y n dr om e a nd i nf e r t i l i t y dur i ng a dol e s c e nc e a nd a dul t hood. C A H i s a
f a m i l y of a ut os om a l r e c e s s i ve he r e di t a r y di s e a s e s c ha r a c t e r i z e d by de c r e a s e d a dr e na l s t e r oi d
pr oduc t i on r e s ul t i ng f r om m ut a t i ons of g e ne s e nc odi ng e nz y m e s i nvol ve d i n s t e r oi dog e ne s i s . T he
g e ne a f f e c t e d by m ut a t i ons a r e : C Y P 21A , C Y P 11B , C Y P 17A 1, H SD 3B 2, C Y P 11A 1, a nd St A R .
D i ve r s e g e ne m ut a t i ons i nt e r r upt s y n t he s i s pa t hw a y i n di f f e r e nt poi nt s , l e a di ng t o s e ve r a l t y p e s
of c onge ni t a l a dr e na l hy p e r pl a s i a . T he m os t c om m on C A H s ubt y p e i s 21- hy d r ox y l a s e de f i c i e nc y
( 21O H D ) . 21O H D i s di vi de d i nt o c l a s s i c a nd non- c l a s s i c a l f or m s . T he c l a s s i c a l f or m of 21O H D
i s f ur t he r di vi de d i nt o " s a l t w a s t i ng " a nd " s i m pl e vi r i l i z i ng " s ubt y p e s . A s 21- hy d r ox y l a s e
dy s f unc t i on c a us e d by m ut a t i on of C Y P 21A 2 g e ne c oul d l e a d t o s e ve r e C A H , w e a r e i nt e r e s t e d
i n t he c ha nge s of H 295R c e l l a c t i vi t i e s a nd s e c r e t i on t ha t oc c ur w he n t he C Y P 21A 2 g e ne i s
knoc ke d dow n. T o obt a i n f ur t he r e vi de nc e r e ga r di ng t he r ol e of C Y P 21A 2 i n t he hum a n H 295R
a dr e noc or t i c a l c a nc e r c e l l l i ne , t he e x pr e s s i on of C Y P 21A 2 w a s s i l e nc e d by s i R N A t r a ns f e c t i on.
W he n C Y P 21A 2 ge n e i s s i l e nc e d, t he e x pr e s s i on s of dow n s t r e a m ge n e s , C Y P 11B 1 a nd C Y P 11B 2 ,
w e r e i nhi bi t e d. A nd C Y P 17A 1 a nd H SD 3B 2 g e ne , e nc ode d e nz y m e s w or ki ng on ot he r pa t hw a y s ,
w e r e up- r e gul a t e d w he n t he c or t i s ol , a l dos t e r one a nd c or t i c os t e r one s y n t he s i s pa t hw a y s w e r e
bl oc ke d. T hi s pa t t e r n of g e ne e x pr e s s i on c ha nge s i s c ons i s t e nt w i t h t he phe nom e non i n C A H .
1
C h a p t e r 1 I n t r o d u c t i o n
1 . 1 C o n g e n i t a l A d r e n a l H y p e r p l a s i a ( C A H )
C ong e ni t a l a dr e na l hy p e r pl a s i a r e f e r s t o a f a m i l y of a ut os om a l r e c e s s i ve he r e di t a r y di s e a s e s
c ha r a c t e r i z e d by de c r e a s e d a dr e na l s t e r oi d pr oduc t i on r e s ul t i ng f r om m ut a t i ons of g e ne s
e nc odi ng e nz y m e s i nvol ve d i n s t e r oi doge ne s i s . W he n t he r e duc t i on of t he a c t i vi t y of one of t he
e nz y m e s i nvol ve d i n t he s t e r oi dog e ni c pa t hw a y i n t he a dr e na l c or t e x ha ppe ns , i t l e a ds t o r e duc e d
c or t i s ol pr oduc t i on by t he a dr e na l g l a nd. T he ne ga t i ve f e e dba c k t o t he hy p ot ha l a m us a nd
pi t ui t a r y i s r e duc e d by i m pa i r e d s y n t he s i s of c or t i s ol . T he w e a ke n f e e dba c k s t i m ul a t e s t he
s e c r e t i on of c or t i c ot r opi n- r e l e a s i ng hor m one ( C R H ) a nd A C T H , l e a di ng t o a dr e na l hy p e r pl a s i a .
E nz y m e s ha ve a w i de r a nge of f unc t i ona l de f e c t s , f r om s l i g ht t o c om pl e t e , f r om t he i ne f f i c i e nc y
of a s i ng l e e nz y m e t o t he i ne f f i c i e nc y of m ul t i pl e e nz y m e s ( T ur c u a nd A uc hus , 2015) .
T he e nz y m e s a f f e c t e d by m ut a t i ons a r e : 21- hy d r ox y l a s e ( 21O H ; C Y P 21A ) , 11β - H y d r ox y l a s e
( 11 β O H ; C Y P 11B ) , 17α - hy d r ox y l a s e ( 17O H ; a l s o know n a s 17, 20- l y a s e ; C Y P 17A 1) , t y p e 2 3β -
hy d r ox y s t e r oi d de hy d r oge na s e ( 3 β H S D 2; H S D 3B 2P 450 c hol e s t e r ol s i de - c ha i n c l e a va ge
( P 450s c c ; C Y P 11A 1) a nd P 450 oxi dor e duc t a s e ( P O R ) .) . A nd t he r e i s a pr ot e i n pa r t i c i pa t e d i n
s t e r oi doge ne s i s a f f e c t e d by m ut a t i ons a s w e l l : a c ut e r e gul a t or of s t e r oi d pr oduc t i on ( S t A R ) ,
( C ha t z i a g ge l ou e t a l ., 2019) . M or e t ha n 90% of C A H c a s e s ha ve r e s ul t e d f r om 21- hy d r ox y l a s e
de f i c i e nc y ( 21O H D ) , w hi c h m a ke s i t t he m os t c om m on t y p e . B a s e d on t he pr e s e nc e or a bs e nc e
of c or t i s ol de f i c i e nc y , 21O H D i s di vi de d i nt o c l a s s i c a nd non- c l a s s i c a l f or m s . A c c or di ng t o
w he t he r m i ne r a l oc or t i c oi d s y n t he s i s ha s be e n i m pa i r e d e nough t o c a us e l i f e - t hr e a t e ni ng s odi um -
de f i c i e nc y s y m pt om s i n i nf a nt s , t he c l a s s i c a l f or m of 21O H D i s f ur t he r di vi de d i nt o " s a l t w a s t i ng
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" a nd " s i m pl e vi r i l i z i ng " s ubt y p e s ( T ur c u a nd A uc hus , 2015) . I n “ s a l t - w a s t i ng ” C A H , w hi c h
a c c ount s f or 75% of c l a s s i c C A H , c om pl e t e l os s of t he e nz y m e a c t i vi t y r e s ul t s i n c or t i s ol a nd
a l dos t e r one de f i c i e nc y ( C ha t z i a g ge l ou e t a l ., 2019) . I n “ s i m pl e vi r i l i z i ng ” C A H , 1–2% e nz y m e
a c t i vi t y r e m a i ns w i t h nor m a l m i ne r a l oc or t i c oi d c onc e nt r a t i ons ( S ha r m a a nd S e t h, 2014) . N on-
c l a s s i c a l 21O H D i s c ha r a c t e r i z e d by pa r t i a l e nz y m e de f i c i e nc y l e a di ng t o s uf f i c i e nt c or t i s ol
bi os y n t he s i s , a s e nz y m e a c t i vi t y i s s a t i s f a c t or y ha vi ng 20–50% f unc t i on. T hus , pa t i e nt s a r e
a s y m pt om a t i c , or s y m pt om s a ppe a r m uc h l a t e r due t o e l e va t e d a c c um ul a t i on of pr e c ur s or s
bl oc ke d by t he e nz y m e ( S ha r m a a nd S e t h, 2014) . T hi s l e a ds t o i nc r e a s e d 17-
hy d r ox y p r oge s t e r one a nd 21- de ox y c o r t i s ol c onc e nt r a t i ons be i ng c onve r t e d t o a dr e na l a ndr oge ns
a nd a ndr oge n pr e c ur s or s , f ol l ow e d by a m i l d a ndr oge n ove r dos e . S i nc e c ha nge s i n e nz y m e
a c t i vi t y a r e c ont i nuous , i t i s e s s e nt i a l t o not e t ha t t he s e c l a s s i f i c a t i ons a r e a r t i f i c i a l .
C A H i s one of t he m os t c om m on g e ne t i c di s or de r s , oc c ur r i ng i n ne w bor ns a t a r a t e of
a ppr ox i m a t e l y 1: 10,000 t o 1: 20,000 ( K r one a nd A r l t , 2009) . T he c l a s s i c f or m of t he m os t
c om m on C A H s ubt y p e , 21- hy d r ox y l a s e de f i c i e nc y ( 21O H D ) , ha s a n i nc i de nc e of a ppr ox i m a t e l y
1: 10,000 t o 1: 15,000 ne w bor ns , w hi l e m i l de r non- c l a s s i c f or m s a r e m or e c om m on ( 1: 500-
1000 i n t he C a uc a s us popul a t i on) . T he f i r s t pa t i e nt w i t h a 21- hy d r ox y l a s e de f i c i e nc y w a s
r e por t e d by N a pl e s a na t om y L ui gi de C r e c c hi o i n 1865 ( W i t c he l , 2017) . A t ne c r ops y , t he
c a da ve r w a s de s c r i be d a s ha vi ng a s c r ot a l f us i on, a 10 c m c ur ve d pe ni s , hy p os pa di a s ,
unde s c e nde d bi l a t e r a l t e s t e s , va gi na , ut e r us , f a l l opi a n t ube s , ova r i e s , a nd s i g ni f i c a nt l y e nl a r ge d
a dr e na l g l a nds . T hi s m a n w a s bor n w i t h a m bi g uous g e ni t a l s a nd w a s r e por t e d t o be m a l e
t hr oughout a dul t hood, dy i ng of a dr e na l i ns uf f i c i e nc y due t o vom i t i ng , di a r r he a , a nd c ol l a ps e i n
hi s 40s ( W i t c he l , 2017) . T he c l i ni c a l m a ni f e s t a t i ons of C A H i nc l ude a dr e na l i ns uf f i c i e nc y ,
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g e ni t a l a m bi g ui t y , s hor t s t a t ur e , a nd a n i nc r e a s e d r i s k of m e t a bol i c s y n dr om e a nd i nf e r t i l i t y
dur i ng a dol e s c e nc e a nd a dul t hood.
F or t he c l a s s i c 21O H t r e a t m e nt , i t i s ne c e s s a r y t o r e s t or e t he de f i c i e nc y of g l uc oc or t i c oi ds a nd
m i ne r a l oc or t i c oi ds a nd t o r e gul a t e t he a dr e na l a ndr oge n e x c e s s . I d e a l l y , g l uc oc or t i c oi d a nd
m i ne r a l oc or t i c oi d r e pl a c e m e nt s houl d a voi d di s e a s e - r e l a t e d s i de e f f e c t s a nd pr ovi de a g ood
qua l i t y of l i f e a nd nor m a l l i f e e x pe c t a nc y . G l uc oc or t i c oi d r e pl a c e m e nt t he r a py s t a r t e d i n t he
1950s i s now a c l a s s i c t r e a t m e nt f or C A H . ( W e i t z m a n e t a l ., 1971) . G l uc oc or t i c oi d t r e a t m e nt
m a i nl y s uppl e m e nt s phy s i ol ogi c a l l y ne e de d g l uc oc or t i c oi ds t o i nhi bi t e xc e s s i ve s e c r e t i on of
C R H a nd A C T H by t he hy p ot ha l a m us a nd pi t ui t a r y g l a nd, s o t he s e c r e t i on of a dr e na l hor m one s
i s r e duc e d. G l uc oc or t i c oi d t he r a py i s c ur r e nt l y m a i nl y us e d i n pa t i e nt s w i t h s e ve r e C A H . I n or de r
t o s uppl e m e nt g l uc oc or t i c oi ds a dm i ni s t r a t i on, m i ne r a l oc or t i c oi d a nd f l udr oc or t i s one a r e of t e n
us e d i n c l i ni c a l pr a c t i c e t o s t r e ngt he n t he i nhi bi t i on of A C T H . S t udi e s ha ve s how n t ha t t r e a t m e nt
ove r s i x m ont hs r e duc e s t he g r ow t h r a t e , s o f l udr oc or t i s one s houl d a l s o be a ppl i e d i ndi vi dua l l y
( N guy e n e t a l ., 2006) . S i nc e c l a s s i c 21O H de f i c i e nc y pa t i e nt s s how i nc r e a s e d c om or bi di t i e s ( A r l t
e t a l ., 2010) ( C a br e r a e t a l ., 2001) a nd i nc r e a s e d m or t a l i t y ( F a l ha m m a r e t a l ., 2014) , t hi s g oa l ha s
not be e n a c hi e ve d. I n t he non- c l a s s i c a l t r e a t m e nt of 21O H D , i t i s us ua l l y no hor m one
r e pl a c e m e nt r e qui r e d s i nc e t he a i m i s s i m pl y t o r e s ol ve t he s y m pt om s c a us e d by a dr e na l
a ndr oge n e x c e s s . G l uc oc or t i c oi ds c a n a l s o be us e d t o t r e a t non- c l a s s i c a l 21O H D , but t he
t r e a t m e nt g oa l s c a n a l s o be r e a c he d t hr ough c os m e t i c m e a s ur e s , a nt i - a ndr oge ns , a nd a nt i -
a ndr oge n or a l c ont r a c e pt i ve s ( M a t t he w s a nd C he e t ha m , 2013) ( va n de r K a a y a nd va n de n A kke r ,
2014) .
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1 . 2 P a t h w a y s o f s t e r o i d h o r m o n e s y n t h e s i s
A dr e na l s t e r oi d pr oduc t i on i s a dy n a m i c pr oc e s s t ha t r e l i e s on de novo s y n t he s i s a nd doe s not
s t or e pr e - s y n t he s i z e d hor m one s f or i m m e di a t e s e c r e t i on. C hol e s t e r ol i s a c om m on pr e c ur s or of
a l l s t e r oi ds a nd c a n be e f f i c i e nt l y m e t a bol i z e d i nt o t he f i na l pr oduc t t hr ough a s e r i e s of
e nz y m a t i c r e a c t i ons . T o s t a r t s t e r oi d pr oduc t i on , une s t e r i f i e d " f r e e " c hol e s t e r ol m a y be
t r a ns por t e d f r om e ndos om e s t o m i t oc hondr i a vi a ve s i c l e t r a ns por t , i nvol vi ng m e m br a ne f us i on,
or t hr ough non- ve s i c ul a r t r a ns por t . S t e r oi dog e ni c a c ut e r e gul a t or y ( S t A R ) pr ot e i n a c t s on t he
out e r m i t oc hondr i a l m e m be r s t o a c c e l e r a t e c hol e s t e r ol i m por t i nt o t he i nne r m i t oc hondr i a l
m e m br a ne . C Y P 11A 1 c onve r t s c hol e s t e r ol t o pr e gne nol one on t he i nne r m i t oc hondr i a l
m e m br a ne a s t he f i r s t a nd r a t e - l i m i t i ng s t e ps of s t e r oi d pr oduc t i on ( M i l l e r a nd B os e , 2011) .
A dr e na l s t e r oi d s y n t he s i s oc c ur s t hr oug h a s e r i e s of s t e ps t ha t a r e pr om ot e d by a dr e na l r e gi on-
s pe c i f i c e nz y m e e x pr e s s i on. A nd di ve r s e g e ne m ut a t i ons i nt e r r upt s y n t he s i s pa t hw a y i n di f f e r e nt
poi nt s , l e a di ng t o s e ve r a l t y p e s of c onge ni t a l a dr e na l hy p e r pl a s i a . ( F i gur e 1 A )
F i gu r e 1
A A dr e na l hor m one s y n t he s i s
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B 21- hy d r ox y l a s e ( 21O H ) de f i c i e nc y
C 11β - hy d r ox y l a s e ( 11 β O H ) de f i c i e nc y
D 17- hy d r ox y l a s e ( 17O H ) de f i c i e nc y
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E 3β – hy d r ox y s t e r oi d de hy d r oge na s e t y p e 2 ( 3 β H S D 2) de f i c i e nc y
F P 450 oxi dor e duc t a s e ( P O R ) de f i c i e nc y
7
G L i poi d c onge ni t a l a dr e na l hy p e r pl a s i a a nd P 450 c hol e s t e r ol s i de c ha i n c l e a va ge ( S C C )
de f i c i e nc y
F i gu r e 1 A d r e n al s t e r oi d oge n e s i s p at h w ays
( A ) N or m a l s t e r oi doge ne s i s pa t hw a y , ( B ) 21- hy d r ox y l a s e de f i c i e nc y , ( C ) 11β - hy d r ox y l a s e
de f i c i e nc y , ( D ) 17- hy d r ox y l a s e de f i c i e nc y , ( E ) 3β - hy d r ox y s t e r oi d de hy d r oge na s e t y p e 2
de f i c i e nc y , ( F ) P 450 oxi dor e duc t a s e de f i c i e nc y , a nd ( G ) l i poi d c ong e ni t a l a dr e na l hy p e r pl a s i a
a nd P 450 c hol e s t e r ol s i de c ha i n c l e a va ge de f i c i e nc y .
G e ne s w hos e m ut a t i ons c a us e c onge ni t a l a dr e na l hy p e r pl a s i a a r e s how n i n r e d. L i ght g r a y
i ndi c a t e s hor m one s a t l ow e r c onc e nt r a t i ons due t o s uppr e s s i on of pr e vi ous s t e r oi d pr oduc t i on.
D a s he d a r r ow s i ndi c a t e i ndi r e c t i nhi bi t i on of s ubs e que nt hor m one s .
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D H E A = de hy d r oe pi a ndr os t e r one . P O R = P 450 oxi dor e duc t a s e .
1 . 3 2 1 - h y d r o x y l a s e ( 2 1 O H ) d e f i c i e n c y
21- hy d r ox y l a s e ( 21O H ) de f i c i e nc y a s s oc i a t e d w i t h pa t hoge ni c m ut a t i ons i n t he 21- hy d r ox y l a s e
( C Y P 21A 2 ) g e ne r e pr e s e nt s t he va s t m a j or i t y ( 95% ) of C A H c a s e s ( P i g na t e l l i e t a l ., 2019) . T he
g e ne f or 21- hy d r ox y l a s e , C Y P 21A 2 , i s l oc a t e d on t he l ong a r m of c hr om os om e 6, w i t hi n t he
hum a n l e ukoc y t e a nt i g e n c l a s s I I I r e gi on ( C a r r ol l e t a l ., 1985) ( P . C . W hi t e a nd S pe i s e r , 2000) . A
ps e udoge ne C Y P 21A 1P , w hi c h e nc ode s unf unc t i ona l e nz y m e s , l i e s a t a bout 30 kb f r om t he
C Y P 21A 2 g e ne . B ot h t he f unc t i ona l g e ne a nd t he ps e udoge ne c ont a i n t e n e xons a nd ha ve 98%
hom ol og y of t he nuc l e ot i de s e que nc e i n t he i r e x ons a nd 96% of t he s e que nc e i de nt i t y i n t he
i nt r on ( H i ga s hi e t a l ., 1986) ( P . C . W hi t e a nd S pe i s e r , 2000) . D ue t o l a r ge g e ne c onve r s i ons ,
c om pl e t e de l e t i ons , a nd nons e ns e or f r a m e - s hi f t m ut a t i ons , t he s e ps e udoge ne s C Y P 21A 1P
i na c t i va t e d a nd pr e ve nt i ng s y n t he s i s of f unc t i ona l pr ot e i n. T he pos i t i on a nd hi g h hom ol og y
be t w e e n t he t w o g e ne s pr om ot e m i s m a t c he s , l e a di ng t o t he r e c om bi na t i on be t w e e n t he g e ne a nd
t he ps e udoge ne . ( F i gur e 2) M os t 21O H D m ut a nt a l l e l e s a r e g e ne r a t e d by i nt e r ge ni c
r e c om bi na t i on a nd g e ne c onve r s i on e ve nt s be t w e e n t w o C Y P 21A g e ne s ( P i g na t e l l i e t a l ., 2019) .
T he s a l t w a s t i ng f or m s of C A H i s us ua l l y c a us e d by c om pl e t e de l e t i on, l a r ge g e ne
t r a ns f or m a t i on, a nd c om pl e t e l os s of 21- hy d r ox y l a s e a c t i vi t y c a us e d by nons e ns e or f r a m e s hi f t
m ut a t i ons . T he m ut a t i on r e s ul t s i n onl y 1- 2% e nz y m e a c t i vi t y r e m a i ni ng, w hi c h c a n pr oduc e
e nough a l dos t e r one i n s i m pl e vi r i l i z i ng f or m s of C A H . N on- c l a s s i c a l 21O H de f i c i e nc y pa t i e nt s
ha ve 20- 60% of e nz y m e a c t i vi t y w i t h no a dr e na l i ns uf f i c i e nc y . A non- c l a s s i c a l 21O H de f i c i e nc y
pa t i e nt c a n be a c om pound he t e r oz y go t e , w i t h one c l a s s i c a l a l l e l e a nd one non- c l a s s i c a l a l l e l e , or
a he t e r oz y go t e w i t h t w o non- c l a s s i c a l a l l e l e s . T he r e a r e c e r t a i n c or r e l a t i ons be t w e e n g e not y p e
9
a nd phe not y p e t e nd t o be m a i nt a i ne d i n t he m os t s e ve r e a nd m i l de s t c ondi t i on of t he . H ow e ve r ,
t he i nt e r m e di a t e f or m s a r e of t e n not c l os e l y a s s oc i a t e d w i t h s pe c i f i c g e ne t i c de f e c t s , i ndi c a t i ng
ot he r c a us e s of phe not y p i c e x pr e s s i on, s uc h a s g e ne t i c or e nvi r onm e nt a l ( T ur c u a nd A uc hus ,
2015) .
F i gu r e 2
F i gu r e 2 S c h e m at i c r e p r e s e n t at i on of a ge n e c on ve r s i on m e c h an i s m
T he m i s m a t c h be t w e e n t w o D N A s e que nc e s l e a di ng t o r e c om bi na t i on be t w e e n t he C Y P 21A 2
g e ne a nd t he C Y P 21A 1P ps e udoge ne .
D ue t o t he 21- hy d r ox y l a s e de f i c i e nc y , ups t r e a m s t e r oi ds e x c e s s a nd t ur n t o vi a bl e pa t hw a y s t o
pr oduc e e f f e c t i ve a ndr oge ns . 17- hy d r ox y p r oge s t e r one ( 17O H P ) i s t he m a i n s ubs t r a t e of
C Y P 21A 2 e nz y m e . T he a c c um ul a t i on of 17O H P i s a s i g n of 21O H de f i c i e nc y , s o 17O H P i s
us ua l l y us e d f or t he di a g nos i s a nd m oni t or i ng of t hi s di s e a s e . I n a ddi t i on, i na c t i va t i on of
C Y P 21A 2 e nz y m e e nc our a ge s t he a c c um ul a t i on of ot he r C 21 s t e r oi ds . C Y P 17A 1 e nz y m e
pe r f or m s s a m e e f f i c i e nc y i n hy d r ox y l a t i on of pr e gne nol one a nd pr og e s t e r one a t pos i t i on 17, but
t he r e i s s t i l l up t o 30% of pr oge s t e r one be i ng 16α - hy d r ox y l a t i on ( S w a r t e t a l ., 2010) . I n t he
10
r e gul a r pa t hw a y t o pr oduc e a l dos t e r one a nd c or t i s ol , pr oge s t e r one a nd 17O H P f i r s t l y ha ve
hy d r ox y l a t i on a t pos i t i on 21 t hr ough C Y P 21A 2 e nz y m e , a nd t he n g e t hy d r ox y l a t i on a t ot he r
pos i t i ons . T he c onc e nt r a t i on of pr oge s t e r one a nd 17O H P i nc r e a s e a s a r e s ul t of 21- hy d r ox y l a s e
dy s f unc t i on. B e c a us e t he y a r e s ubs t r a t e s of C Y P 11B 1e nz y m e , t he y a r e c onve r t e d t o 11β -
hy d r ox y p r oge s t e r one a nd 21- de ox y c or t i s ol pr oduc t i on, r e s pe c t i ve l y .
T he e x c e s s 17O H P c a us e d by C Y P 21A 2 de f i c i e nc y i s t r a ns f e r r e d t hr ough ot he r a va i l a bl e
pa t hw a y s t o f or m pos s i bl e a ndr oge ns , s uc h a s t e s t os t e r one a nd 5α - di hy d r ot e s t os t e r one ( D H T ) .
C Y P 17A 1 m e di a t e s t he c onve r s i on of 17- hy d r ox y p r e gne nol one t o D H E A ( Δ 5 pa t hw a y ) a nd
17O H P t o a ndr os t e ne di one ( Δ 4 pa t hw a y ) . H ow e ve r , c om pa r e d t o t he Δ 4 r e a c t i on, t he c a t a l y t i c
e f f i c i e nc y of hum a n 17,20- l y a s e t o t he Δ 5 r e a c t i on i s a bout 100 t i m e s hi g he r , w hi c h e x pl a i ns t he
hug e a c c um ul a t i on of 17O H P i n 21O H D . I n 21O H D pa t i e nt s , due t o t he hi g h 17O H P i n t he
a dr e na l g l a nd, a l a r ge a m ount of a ndr os t e ne di one s y n t he s i s m a y s t i l l oc c ur t hr ough t he Δ 4
pa t hw a y . ( F i gur e 1 B )
1 . 4 1 1 β - h y d r o x y l a s e ( 1 1 β O H ) d e f i c i e n c y
11β O H de f i c i e nc y i s c a us e d by a m ut a t i on i n C Y P 11B 1 . T he e nz y m e e nc ode d by C Y P 11B 1
w or ks i n t he a dr e na l g l a ndul a r t i s s ue , a nd unde r t he r e gul a t i on of A C T H , 11 de ox y c or t i s ol i s
c onve r t e d t o c or t i s ol a nd de ox y c or t i c os t e r one i s c onve r t e d c or t i c os t e r one . M os t C Y P 11B 1
m ut a t i ons a r e a s s oc i a t e d w i t h l i t t l e or l os t e nz y m e a c t i vi t y , a nd t hi s i s c ons i s t e nt w i t h t he c l a s s i c
C A H phe not y p e ( E l - M a ouc he e t a l ., 2017) .
11
D a m a ge d 11- hy d r ox y l a t i on c a us e s r e duc e d c or t i c os t e r oi d a nd c or t i s ol s y n t he s i s , a nd t he n A C T H
i nc r e a s e s due t o t he w e a ke n ne ga t i ve f e e dba c k. A s t he r e s ul t of bl oc ka ge of pa t hw a y s , a ndr oge n
pr oduc t i on i s pr om ot e d, a nd a ndr oge n a c c um ul a t e s . U nde r nor m a l c i r c um s t a nc e s , i t i s ve r y
unl i ke l y t ha t c or t i c os t e r one a nd de ox y c or t i c os t e r one a r e pr oduc e d by C Y P 11B 1 i n a dr e na l
he r pe s z os t e r , but unde r t he i nf l ue nc e of A C T H t he c onc e nt r a t i on of de ox y c or t i c os t e r one w i l l
i nc r e a s e s i g ni f i c a nt l y . D e ox y c or t i c os t e r one i s a w e a k m i ne r a l oc or t i c oi d, but i nc r e a s e d
de ox y c or t i c os t e r one c a n i nhi bi t t he r e ni n- a ngi ot e ns i n s y s t e m , l e a di ng t o vol um e e x pa ns i on,
hy p e r t e ns i on ( P e r r i n C . W hi t e , 2011) .
I n t he c l i ni c pr a c t i c e , pa t i e nt s w i t h 11β O H de f i c i e nc y a nd pa t i e nt s w i t h 21O H de f i c i e nc y ha ve
s i m i l a r s y m pt om s l i ke a ndr oge n a c c um ul a t i on. H ow e ve r , di f f e r e nt f r om pa t i e nt s w i t h 21O H
de f i c i e nc y , pa t i e nt s w i t h 11β O H de f i c i e nc y ha ve hi g h bl ood pr e s s ur e r a t he r t ha n s a l t l os s ( P . C .
W hi t e e t a l ., 1994) .
1 . 5 3 β – h y d r o x y s t e r o i d d e h y d r o g e n a s e t y p e 2 ( 3 β H S D 2 ) d e f i c i e n c y
D a m a ge d 3 β H S D 2 f unc t i on c a us e s l ow e r c onc e nt r a t i ons of a l dos t e r one , a ndr os t e ne di one a nd
c or t i s ol , a nd t he n f ol l ow s by e l e va t e d c onc e nt r a t i ons of r e ni n, A C T H a nd
de hy d r oe pi a ndr os t e r one .
T he r e a r e t he H SD 3B 1 a nd H SD 3B 2 g e ne s e nc ode t w o i s of or m s of 3β - hy d r ox y s t e r oi d
de hy d r oge na s e , w hi c h a r e t y p e 1 ( 3 β H S D 1) a nd t y p e 2 ( 3 β H S D 2) . H SD 3B 1 i s s t r ong l y
e x pr e s s e d i n t he pl a c e nt a a nd s ur r oundi ng t i s s ue s , a s H SD 3B 2 g e ne i s hi g hl y e x pr e s s e d i n t he
a dr e na l a nd g ona ds ( E l - M a ouc he e t a l ., 2017) .
12
B e c a us e i ni t i a l s t udi e s di d not ba s e on ge ne t i c f i ndi ngs by i de nt i f y i ng a pos s i bl e non- c l a s s i c f or m ,
t he f ol l ow i ng g e ne t i c s t udi e s f a i l e d t o c onf i r m t he di a gnos i s . O ve r t he pa s t s e ve r a l de c a de s ,
hor m ona l s t a nda r d f or t he di a gnos i s of 3β H S D 2 de f i c i e nc y ha s c ha nge d ( M e r m e j o e t a l ., 2005) .
T he r e i s non- c l a s s i c a l 3β H S D 2 de f i c i e nc y , but e x t r e m e l y r a r e .
1 . 6 1 7 - h y d r o x y l a s e d e f i c i e n c y
T he e nz y m e e nc ode d by t he C Y P 17A 1 g e ne e x pr e s s e s bot h 17α - hy d r ox y l a s e a nd 17, 20- l y a s e
a c t i vi t y . D ue t o t he e nz y m e ' s pos i t i on i n t he s t e r oi d pr oduc t i on pa t hw a y , s e ve r e m ut a t i ons i n t hi s
g e ne c a n da m a ge t he f unc t i on of pr oduc t i on of g ona da l a nd a dr e na l s t e r oi ds , l e a di ng t o s e x ua l
i m m a t ur e a nd a dol e s c e nt f a i l ur e ( A uc hus , 2017, p. 20) . C Y P 17A 1 i s e x pr e s s e d i n t he r e t i c ul a r
ba nds a nd a dr e na l g l a ndul a r , not i n t he g l om e r ul i . T hus , s t e r oi doge ne s i s , r e gul a t e d by A C T H ,
r e s ul t s i n e l e va t e d c onc e nt r a t i ons of c or t i c os t e r one a nd de ox y c or t i c os t e r one . H i g h l e ve l s of
de ox y c or t i c os t e r one i nhi bi t a l dos t e r one pr oduc t i on, l e a d t o s odi um r e t e nt i on, a nd hy p e r t e ns i on
a nd hy p oka l e m i a . D e s pi t e phe not y p i c va r i a t i ons , non- c l a s s i c f or m s w i t h s l i g ht c l i ni c a l
m a ni f e s t a t i ons ha ve not be e n i de nt i f i e d ( E l - M a ouc he e t a l ., 2017) .
1 . 7 P 4 5 0 o x i d o r e d u c t a s e ( P O R ) d e f i c i e n c y
P O R i s a n i m por t a nt pa r t i nvol vi ng i n t he e l e c t r oni c t r a ns por t of t he e ndopl a s m i c r e t i c ul um . T he
m e t a b ol i c c a pa c i t y of s e ve r a l e n z y m e s , i nc l ud i ng 17O H , 21O H , a n d a r om a t a s e , r e l i e s on of P O R .
P O R de f i c i e nc y w a s di s c ove r e d i n 2004 a nd i t pr ovi de s a n e x pl a na t i on f or m ul t i pl e hor m ona l
de f i c i e nc i e s , or i gi na l l y know n a s obvi ous c om bi na t i on of 17O H a nd 21O H de f i c i e nc y . ( A r l t e t
a l ., 2004) M os t P O R m ut a t i ons r e t a i n c e r t a i n e nz y m e f unc t i ons . A s s e e n i n r ode nt m ode l s , t he
13
e nz y m e s c om pl e t e l y l os s of f unc t i on i n hom oz y go us m ut a t i ons , w hi c h m a y not be vi a bl e ( O t t o e t
a l ., 2003) .
I n g e ne r a l , da m a ge d 17α - hy d r ox y l a s e i nc r e a s e s s y n t he s i s of m i ne r a l oc or t i c oi d i nt e r m e di a t e s ,
m i ne r a l oc or t i c oi d de f i c i e nc y i s not s how n i n pa t i e nt s w i t h P O R de f i c i e nc y . P a t i e nt s r e s pond
di f f e r e nt l y t o c or t i c os t e r oi ds , a nd m os t pa t i e nt s r e qui r e pe r m a ne nt or s t r e s s g l uc oc or t i c oi d
t he r a py ( K r one e t a l ., 2012) .
1 . 8 L i p o i d c o n g e n i t a l a d r e n a l h y p e r p l a s i a
C l a s s i c l i pi d c onge ni t a l a dr e na l hy p e r pl a s i a i s t he m os t s e ve r e f or m of C A H . I t i s c ha r a c t e r i z e d
by i m pa i r m e nt of a l l s t e r oi d hor m one s s y n t he s i s due t o S t A R m ut a t i ons . S t A R r e gul a t e s
c hol e s t e r ol t r a ns f e r w i t hi n t he m i t oc hondr i a , w hi c h i s a r a t e - l i m i t i ng s t e p i n t he i ni t i a t i on of
s t e r oi d pr oduc t i on. W he n c hol e s t e r ol i s f a i l e d t o be m obi l i z e d, l i pi d dr opl e t s a c c um ul a t e a nd c a n
be obs e r ve d a t a ut ops y , s o i t i s na m e d by l i pi d c onge ni t a l a dr e na l hy p e r pl a s i a . T he l i pi d f or m
c a us e s l i f e - t hr e a t e ni ng a dr e na l i ns uf f i c i e nc y , m a ki ng i t be one of t he r a r e s t f or m s of c onge ni t a l
a dr e na l hy p e r pl a s i a . I n 46, X X , a nd 46, X Y i nf a nt s , ne ona t a l c r i s i s a nd f e m a l e e x t e r na l g e ni t a l i a
m a y be pr e s e nt e d ( M i l l e r , 2017) .
T he r e i s a t w o- hi t m ode l t o e x pl a i n t he pa t hoge ne s i s of l i pi d c onge ni t a l a dr e na l hy p e r pl a s i a . T he
f i r s t hi t i s due t o t he i na c t i va t i on of S t A R , l e a di ng t o t he a c c um ul a t i on of i nt r a c e l l ul a r c hol e s t e r ol
a nd c hol e s t e r ol e s t e r s . A nd t he s e c ond hi t i s c a us e d by c e l l f unc t i on da m a ge r e s ul t i ng i n t he
i nc r e a s e d c onc e nt r a t i on of c hol e s t e r ol a nd c hol e s t e r ol e s t e r i n c e l l . T hi s t w o- hi t m e c ha ni s m
14
pr ovi de s e x pl a na t i ons f or s om e unus ua l c l i ni c a l phe not y p e s . I t r e por t e d t ha t 46, X X pa t i e nt s
s pont a ne ous l y e nt e r e d pube r t y due t o t he l ow e s t e x pr e s s i on of ova r i a n S t A R ( M i l l e r , 2017) .
T he f i r s t non- c l a s s i c f or m of c onge ni t a l l i pi d a dr e noc or t i c a l hy p e r pl a s i a w a s r e por t e d i n 2006
a nd a ppr ox i m a t e l y 20% t o 30% of S t A R a c t i vi t y w a s r e t a i ne d. P a t i e nt s m a y pr e s e nt non- c l a s s i c
l i poi d c onge ni t a l a dr e na l hy p e r pl a s i a a s e a r l y a s ne w bor ns or a s l a t e a s a dul t hood, w i t h
hy p e r pi gm e nt a t i on, g l uc oc or t i c oi d e f f i c i e nc y , a nd hi g h A C T H c onc e nt r a t i ons ( M e t he r e l l e t a l .,
2009) .
1 . 9 P 4 5 0 c h o l e s t e r o l s i d e - c h a i n c l e a v a g e ( S C C ) d e f i c i e n c y
P 450 c hol e s t e r ol s i de - c ha i n c l e a va ge de f i c i e nc y ( S C C ) , e nc ode d by C Y P 11A 1 , pa r t i c i pa t e s i n t he
c onve r s i on of c hol e s t e r ol t o pr e gne nol one , w hi c h i s t he f i r s t s t e p of s t e r oi doge ne s i s . M or e ove r , i t
ha s s i m i l a r i t i e s w i t h l i pi d c onge ni t a l a dr e na l hy p e r pl a s i a i n c l i ni c s a nd bi oc he m i s t r y .
N e ve r t he l e s s , pa t i e nt s us ua l l y ha ve g ona ds a nd a t r ophi c a dr e na l . T he r e a r e 40 c a s e s of S C C
de f i c i e nc y be i ng r e por t e d ( M i l l e r , 2017) .
S a m e a s non- c l a s s i c a l l i pi d c onge ni t a l a dr e na l hy p e r pl a s i a , non- c l a s s i c S C C de f i c i e nc y w i t h 7-
30% of e nz y m e a c t i vi t y r e t a i ne d a l s o pr e s e nt s a l t e r e d g ona da l f unc t i on a nd de l a y e d a dr e na l
i ns uf f i c i e nc y ( T e e e t a l ., 2013) .
1 . 1 0 H 2 9 5 R c e l l l i n e
I n 1994, a c ont i nuous c e l l l i ne , N C I - H 295, w a s f i r s t l y e s t a bl i s he d f r om a n i nva s i ve pr i m a r y
a dr e noc or t i c a l c a r c i nom a ( G a z da r e t a l ., 1990) . T he N C I - H 295 c e l l s e x pr e s s m ul t i pl e pa t hw a y s
15
of s t e r oi doge ne s i s a nd e na bl e t o pr oduc e s t e r oi ds . A s N C I - H 295 c e l l s a r e l oos e l y a t t a c he d a nd
ha ve l ong c e l l c y c l e t i m e s , t hr e e s ubs t r a i ns ha ve be e n e s t a bl i s he d by us i ng a l t e r na t i ve g r ow t h
c ondi t i ons on t he or i gi na l c e l l l i ne . C om pa r e d w i t h t he pa r e nt H 295 c e l l l i ne , H 295R c e l l s g r e w
i n a f i r m l y a dhe r e nt m onol a y e r , a nd t he t i m e c e l l s r e qui r e d t o doubl e t he i r popul a t i on w a s
r e duc e d f r om f i ve da y s t o t w o da y s ( R a i ne y e t a l ., 2004) .
T he hum a n H 295R a dr e noc or t i c a l c a nc e r c e l l l i ne ha s pr ove n t o be us e f ul t o be a vi t r o m ode l t o
s t udy s t e r oi d g e ne r a t i on pa t hw a y s a nd pr oc e s s e s ( H e c ke r e t a l ., 2006) ( H i l s c he r ova e t a l ., 2004)
( S a nde r s on e t a l ., 2002) . T he H 295R c e l l l i ne e x hi bi t s t he phy s i ol ogi c a l f e a t ur e s of
undi f f e r e nt i a t e d hum a n f e t a l a dr e na l c e l l s ( G a z da r e t a l ., 1990) . T he s e c e l l s pe r f or m a s pe c i a l i n
vi t r o s y s t e m be c a us e t he y ha ve t o c a pa bi l i t y t o pr oduc e s t e r oi d hor m one s t ha t f ound i n t he a dul t
a dr e na l c or t e x a nd g ona ds . I t i s us e f ul t o s t udy t he s y n t he s i s of c or t i c os t e r oi ds ,
A s 21- hy d r ox y l a s e dy s f unc t i on c a us e d by m ut a t i on of C Y P 21A 2 g e ne c oul d l e a d t o s e ve r e
c onge ni t a l a dr e na l hy p e r pl a s i a , a nd t hi s i s t he ove r w he l m i ng m a j or i t y , w e a r e i nt e r e s t e d i n t he
c ha nge s i n c e l l a c t i vi t i e s a nd s e c r e t i on t ha t oc c ur w he n t he C Y P 21A 2 g e ne i s knoc ke d dow n i n
H 295R c e l l l i ne . T o obt a i n f ur t he r e vi de nc e r e ga r di ng t he r ol e of C Y P 21A 2 i n t he hum a n H 295R
a dr e noc or t i c a l c a nc e r c e l l l i ne , t he e x pr e s s i on of C Y P 21A 2 w a s s i l e nc e d by s i R N A t r a ns f e c t i on.
16
C h a p t e r 2 M a t e r i a l a n d M e t h o d
2 . 1 C e l l c u l t u r e
2.1.1 H 295R c e l l s
H 295R c e l l s c ul t ur e m e di um w a s c om pos e d of D M E M - F 12 G l ut a m a x , 2.5% N u- S e r um G r ow t h
M e di um S uppl e m e nt ( C or ni ng I n c ., #355100) , 0.1% I T S P r e m i x U ni ve r s a l C ul t ur e S uppl e m e nt
( C or ni ng I n c ., #354351) , a nd 1% pe ni c i l l i n/ s t r e pt om y c i n. H 295R c e l l s m us t be c ul t ur e d i n
s pe c i f i c 75 c m
2
c ul t ur e f l a s k a nd m a i nt a i ne d a t s pe c i f i c c ondi t i on, w hi c h w a s 37℃ a nd 5% C O 2 .
H 295R c e l l s w e r e pa s s a ge d a t a r a t i o of 1: 5 e ve r y w e e k. T o pa s s H 295R c e l l s , c ul t ur e m e di um
w a s r e m ove d f r om t he f l a s k. A f t e r w a s hi ng t he c e l l s by P B S f or one t i m e , t he c e l l s w e r e t r e a t e d
by 2 m L t r y p s i n a nd i nc uba t e d a t 37℃ f or 2 m i nut e s ( m i n) . W he n t he c e l l s w e r e de t a c he d f r om
t he f l a s k, t o ne ut r a l i z e t he t r y p s i n a nd t o s t op t he r e a c t i on, 10 m L c ul t ur e m e di um w a s a dde d t o
t he f l a s k. T he n t he c e l l s w e r e c ol l e c t e d i n a 50 m L f a l c on t ube ( C or ni ng I n c ., #352098) a nd
c e nt r i f uge d a t 800 r pm f or t hr e e m i n a t r oom t e m pe r a t ur e . T he s upe r na t a nt w a s r e m ove d f r om
t he t ube a nd t he c e l l pe l l e t w a s r e s us pe nde d i n 10 m L f r e s h c ul t ur e m e di um . N e x t , 2 m L m e di um
c ont a i ni ng H 295R c e l l s w a s m i xe d w i t h 8 m L f r e s h c ul t ur e m e di um a nd a dde d t o a ne w 75 c m
2
c ul t ur e f l a s k.
H 295R c e l l s w e r e f r oz e n w he n t he de ns i t y of H 295R c e l l s r e a c he d 80- 90% . S i m i l a r t o w ha t w e
di d w he n pa s s i ng t he c e l l s , H 295R c e l l s w e r e di s s oc i a t e d by t r y p s i n c e nt r i f uge d a t 800 r pm f or
t hr e e m i n a t r oom t e m pe r a t ur e . A f t e r t ha t , e a c h of 1 × 10
6
c e l l s w e r e r e s us pe nde d i n 1 m L
f r e e z i ng m e di um t ha t w a s c om pos e d of 87.5% of c ul t ur e m e di um , 7.5% N u- S e r um G r ow t h
17
M e di um S uppl e m e nt , a nd 5% D M S O . T he n t he c e l l s w e r e put i nt o t he C ool C e l l L X c ont r ol l e d-
r a t e a l c ohol - f r e e c e l l f r e e z i ng c ont a i ne r ( B i oc i s i on, #B C S - 405) f or 24 hour s ( h) a nd pl a c e d i n t he
l i qui d ni t r og e n f or l ong - t e r m s t or a ge .
T o t ha w H 295R c e l l s , a vi a l of f r oz e n c e l l s w a s put i nt o 37℃ w a t e r ba t h unt i l t he f r oz e n m e di um
w a s c om pl e t e l y t ha w e d. T he m a xi m um w a i t i ng t i m e w a s 2 m i n. A f t e r t ha t , t he f r e e z i ng m e di um
c ont a i ni ng H 295R c e l l s w a s a dde d t o a ne w 75 c m
2
c ul t ur e f l a s k t ha t 15 m L f r e s h c ul t ur e
m e di um w a s pr e - a dde d. B e f or e t he f l a s k w a s i nt o t he i nc uba t or , t he f l a s k w a s s ha ke n c om pl e t e l y
t o m a ke c e l l s di s t r i but e e ve nl y i n t he f l a s k. T he m e di um w a s c ha nge d ne x t da y .
2 . 2 R N A i n t e r f e r e n c e a s s a y
T he H 295R c e l l s w e r e s e e de d 2 × 10
5
c e l l s / w e l l i n 24- w e l l pl a t e a nd c ul t ur e d w i t h t ot a l m e di um
f or 48 h. T he H 295R c e l l s w e r e t r a ns f e c t e d w i t h e i t he r of 20 nM ne ga t i ve c ont r ol s i R N A
( O r i ge ne , # S R 30004) , 20 nM H P R T pos i t i ve c ont r ol ( O r i ge ne , # S R 30003) or 6.6 nM
C Y P 21A 2( H um a n) s i R N A ( L o c us I D 1589, O r i ge ne , # S R 301121) us i ng s i T r a n 2.0 s i R N A
t r a ns f e c t i on r e a ge nt ( O r i ge ne , # T T 320001) a c c or di ng t o t he m a nuf a c t ur e r s ’ i ns t r uc t i ons . A f t e r
t r e a t m e nt w i t h s i R N A , H 295R c e l l s w e r e c ul t ur e d i n t ot a l m e di um . C ol l e c t m e di um a nd ha r ve s t
c e l l s 48 h a f t e r t r a ns f e c t i on.
2 . 3 R N A e x t r a c t i o n
R N A w a s i s ol a t e d f r om H 295R us i ng A m bi on ® R i boP ur e T M K i t ( T he r m oF i s he r S c i e nt i f i c ,
#A M 1924) a c c or di ng t o t he m a nuf a c t ur e r ’ s i ns t r uc t i ons . T he c e l l s w e r e di s r upt e d a nd
18
hom og e ni z e d i n T R I R e a ge nt . T he n R N A w a s e x t r a c t e d f r om hom og e na t e by us i ng c hl or of or m .
A que ous pha s e , w he r e R N A r e m a i ns , a nd or ga ni c pha s e w a s s e pa r a t e by c e nt r i f ugi ng a t 12000 x
g f or 10 m i n a t 4° C . F i l t e r C a r t r i dge - C ol l e c t i on T ube w a s us e d t o pur i f y R N A by bi ndi ng R N A
t o t he f i l t e r i n t he F i l t e r C a r t r i dge . D N A a nd pr ot e i ns w e r e w a s he d by W a s h S ol ut i on. A nd R N A
w a s c ol l e c t e d by E l ut i on B uf f e r . T he c onc e nt r a t i on of R N A s a m pl e w a s m e a s ur e d by t he
N a noD r op 1000A S pe c t r ophot om e t e r . R N A s a m pl e s a r e s t or e d a t - 20 ° C .
F i r s t - s t r a nd c D N A w a s s y n t he s i z e d f r om R N A us i ng t he A m bi on ® R i boP ur e T M K i t
( T he r m oF i s he r S c i e nt i f i c , #A M 1924) .
2 . 4 Q u a n t i t a t i v e r e a l - t i m e P C R
2.4.1 c D N A s y n t he s i s ( R e ve r s e - t r a ns c r i pt i on pol y m e r a s e c ha i n r e a c t i on, R T - P C R )
F i r s t - s t r a nd c D N A w a s s y n t he s i z e d f r om R N A us i ng t he us i ng P r i m e S r i pt ™ R T M a s t e r M i x
( T a ka r a , #R R 036A ) . T he r e ve r s e t r a ns c r i pt i on r e a c t i on w a s c om pos e d of R N A s a m pl e ( 500 ng ) ,
1 x M a s t e r M i x a nd R N a s e F r e e dH 2 O ( U p t o 25 µ L ) . T he r m a l c y c l e r pe r f or m e d on r e ve r s e -
t r a ns c r i pt i on r e a c t i on w a s 15 m i n a t 37° C ( R e ve r s e - t r a ns c r i pt i on) , f ol l ow i ng 5 s e c onds ( s ) a t
85℃ ( F or he a t i na c t i va t i on of r e ve r s e t r a ns c r i pt a s e ) a nd 4℃ f or hol di ng . S t or e r e a c t i on a t – 20° C
or pr oc e e d t o t he r e a l - t i m e P C R .
2.4.2 R e a l - T i m e P C R
19
T o qua nt i f y t he r e l a t i ve e xpr e s s i on of t ot a l C P Y 11A 1 , C Y P 11B 1 , C Y P 17A 1, C Y P 21A 2, a nd
H SD 3B 2, pr i m e r s w e r e us e d ( T a bl e 1) . T o de t e c t A c t i n e x pr e s s i on, A c t i n- F w d a nd A c t i n- R e v
pr i m e r s w e r e us e d ( T a bl e 1) . P r i m e r s w e r e pr e pa r e d by di s s ol vi ng i n nuc l e a s e - f r e e w a t e r t o m a ke
100 µ M s t oc k. A nd s t oc ks w e r e di l ut e d w i t h nuc l e a s e - f r e e w a t e r t o 5 µ M m i xt ur e of f or w a r d
pr i m e r a nd r e ve r s e pr i m e r w or ki ng s ol ut i on.
T ab l e 1. q P C R p r i m e r s
6 ng c D N A w a s us e d f or t he de t e c t i on of e a c h g e ne . T he c D N A s w e r e s ubj e c t e d t o r e a l - t i m e
P C R f or a c t i va t i on of A m pl i T a q ® D N A P ol y m e r a s e a t 95° C f or 2 m i n a nd 40 c y c l e s of 95° C f or
3 s , a nd 65° C f or 30 s us i ng a S Y B R ™ S e l e c t M a s t e r M i x ( T he r m oF i s he r S c i e nt i f i c ) w i t h 100
nM f or w a r d a nd r e ve r s e pr i m e r s ( I n t e gr a t e d D N A T e c hnol ogi e s ) . qR T - P C R w a s pe r f or m e d on a
C F X 384 T ouc h ™ R e a l - T i m e P C R D e t e c t i on S y s t e m ( B i o- R a d) a c c or di ng t o t he m a nuf a c t ur e r ’ s
G e ne P r i m e r F or w a r d P r i m e r R e ve r s e
C Y P 11A 1 C A T C T T C A G G G T C G A T G A C A A G T C C A C C T T C A C C A T G T C C
C Y P 11B 1 A G C C A G C A T C A G T G A A C A T C G G G A T G T G G T A G T T C T G A A G C
C Y P 17A 1 G T T G T T G G A C G C G A T G T C T A T T C G T A T G G G C A C C A A G A C T
C Y P 21A 2 A A G G A C A G G T C C G G G T A G T T C C A A G A G G A C C A T T G A G G A A
H SD 3B 2 C C A A G A G G A C C A T T G A G G A A T G G G T C T T A A C G C A C A A G T G T
A C T I N C C T T G C A C A T G C C G G A G G C A C A G A G C C T C G C C T T
H P R T C A T T A T G C T G A G G A T T T G G A A A G G C T T G A G C A C A C A G A G G G C T A C A
20
i ns t r uc t i ons . D a t a w e r e a na l y z e d us i ng t he r e l a t i ve qua nt i f i c a t i on ( Δ Δ C T ) m e t hod w i t h B i o- R a d
C F X M a e s t r o s of t w a r e a nd E xc e l . A l l r e a l - t i m e P C R r e s ul t s w e r e nor m a l i z e d w i t h t he e x pr e s s i on
of A c t i n, a nd t he r e l a t i ve t r a ns ge ne e x pr e s s i on l e ve l s w e r e s how n a s t he m e a n ± s t a nda r d e r r or s
( S E ) of t hr e e r e pe a t s i n e a c h e x pe r i m e nt a l g r oup.
2 . 5 B C A p r o t e i n a s s a y
B C A P r ot e i n A s s a y K i t ( T he r m o F i s he r S c i e nt i f i c , #23225) i s us e d t o m e a s ur e pr ot e i n
c onc e nt r a t i on. P r e pa r e t he di l ut e d A l bum i n ( B S A ) S t a nda r ds a s t he m a nuf a c t ur e ’ s i nt r oduc t i on.
A dd 25 µ L of t hos e s t a nda r ds a nd 25 µ L pr ot e i n s a m pl e s i n t he 96 w e l l pl a t e s . T r i pl i c a t e bot h
B S A s t a nda r d a nd pr ot e i n s a m pl e s . T he n pr e pa r e t he B C A W or ki ng R e a ge nt ( W R ) by m i xi ng
R e a ge nt A t o R e a ge nt B a s 50 t o 1. C ove r t he pl a t e a nd i nc uba t e a t 37℃ f or 30 m i n. M e a s ur e t he
a bs or ba nc e a t 562nm on a pl a t e r e a de r ( V I C T O R ) . T he c ount i ng t i m e i s 1 s . U s e E xc e l t o m a ke a
s t a nda r d c ur ve a nd t he n us e t hi s c ur ve t o c a l c ul a t e t he c onc e nt r a t i on of e a c h s a m pl e .
2 . 6 E L I S A a s s a y f o r c o r t i s o l ( C a y m a n C h e m i c a l , # 5 0 0 3 6 0 )
2.6.1 C e l l P r e pa r a t i on
T he H 295R c e l l s w e r e s e e de d 2 × 10
5
c e l l s / w e l l i n 24- w e l l pl a t e a nd c ul t ur e d w i t h t ot a l m e di um
f or 48 h. T he H 295R c e l l s w e r e t r a ns f e c t e d w i t h e i t he r of 20 nM ne ga t i ve c ont r ol s i R N A
( O r i ge ne , # S R 30004) , 20 nM H P R T pos i t i ve c ont r ol ( O r i ge ne , # S R 30003) or 6.6 nM
C Y P 21A 2( H um a n) s i R N A ( L o c us I D 1589, O r i ge ne , # S R 301121) us i ng s i T r a n 2.0 s i R N A
t r a ns f e c t i on r e a ge nt ( O r i ge ne , # T T 320001) a c c or di ng t o t he m a nuf a c t ur e r s ’ i ns t r uc t i ons . A f t e r
t r e a t m e nt w i t h s i R N A , H 295R c e l l s w e r e c ul t ur e d i n t ot a l m e di um . C ol l e c t m e di um 48 h a f t e r
t r a ns f e c t i on.
21
2.6.2 P r e f or m i ng t he A s s a y
F ol l ow t he pr ot oc ol t o di l ut e s t a nda r d w i t h c ul t ur e m e di um a nd a dd 50 μ L c ul t ur e m e di um i n t he
N S B a nd B 0 w e l l s . T he n a dd t he di l ut e d s t a nda r d a nd e a c h s a m pl e 50 μ L i n e a c h w e l l . A dd 50 μ L
C or t i s ol A C hE T r a c e r t o e a c h w e l l e x c e pt t he T A a nd B l k w e l l s . A dd 50 μ L C or t i s ol E L I S A
M onoc l ona l A nt i body t o e a c h w e l l e x c e pt t he T A , t he N S B , a nd t he B l k w e l l s . C ove r t he pl a t e
w i t h pl a s t i c f i l m a nd i nc uba t e a t 4℃ ove r ni ght on a n or bi t a l s ha ke r . R e c ons t i t ut e 250 dt n vi a l
E l l m a n ’ s R e a ge nt i nt o 50 m L U l t r a P ur e w a t e r a nd a voi d l i g ht . E m pt y t he w e l l s a nd us e w a s h
buf f e r t o r i ns e f or f i ve t i m e s . A dd 200 μ L of E l l m a n ’ s R e a ge nt t o e a c h w e l l . A dd 5 μ L of t r a c e r
t o t he T A w e l l s . C ove r t he pl a t e w i t h pl a s t i c f i l m . U s e or bi t a l s ha ke r t o s ha ke f or 120 m i n a nd
a voi d l i g ht . R e a d t he pl a t e a t a w a ve l e ngt h a t 420 nm . U s e E x c e l t o g e ne r a t e a s t a nda r d c ur ve
a nd us e t ha t c ur ve t o c a l c ul a t e t he a m ount of c or t i s ol pr oduc t i on pe r c e l l .
2 . 7 E L I S A a s s a y f o r a l d o s t e r o n e ( C a y m a n C h e m i c a l , # 5 0 1 0 9 0 )
2.7.1 C e l l P r e pa r a t i on
T he H 295R c e l l s w e r e s e e de d 2 × 10
5
c e l l s / w e l l i n 24- w e l l pl a t e a nd c ul t ur e d w i t h t ot a l m e di um
f or 48 h. T he H 295R c e l l s w e r e t r a ns f e c t e d w i t h e i t he r of 20 nM ne ga t i ve c ont r ol s i R N A
( O r i ge ne , # S R 30004) , 20 nM H P R T pos i t i ve c ont r ol ( O r i ge ne , # S R 30003) or 6.6 nM
C Y P 21A 2 ( H um a n) s i R N A ( L oc us I D 1589, O r i ge ne , # S R 301121) us i ng s i T r a n 2.0 s i R N A
t r a ns f e c t i on r e a ge nt ( O r i ge ne , # T T 320001) a c c or di ng t o t he m a nuf a c t ur e r s ’ i ns t r uc t i ons . A f t e r
t r e a t m e nt w i t h s i R N A , H 295R c e l l s w e r e c ul t ur e d i n t ot a l m e di um . C ol l e c t m e di um 48 h a f t e r
t r a ns f e c t i on.
22
2.7.2 P r e f or m i ng t he A s s a y
F ol l ow t he pr ot oc ol t o di l ut e s t a nda r d w i t h c ul t ur e m e di um a nd a dd 50 μ L c ul t ur e m e di um i n t he
N S B a nd B 0 w e l l s . T he n a dd t he di l ut e d s t a nda r d a nd e a c h s a m pl e 50 μ L i n e a c h w e l l . A dd 50 μ L
C or t i s ol A C hE T r a c e r t o e a c h w e l l e x c e pt t he T A a nd B l k w e l l s . A dd 50 μ L C or t i s ol E L I S A
M onoc l ona l A nt i body t o e a c h w e l l e x c e pt t he T A , t he N S B , a nd t he B l k w e l l s . C ove r t he pl a t e
w i t h pl a s t i c f i l m a nd i nc uba t e a t 4℃ f or 18 h on a n or bi t a l s ha ke r . R e c ons t i t ut e 250 dt n vi a l
E l l m a n ’ s R e a ge nt i nt o 50 m L U l t r a P ur e w a t e r a nd a voi d l i g ht . E m pt y t he w e l l s a nd us e w a s h
buf f e r t o r i ns e f or f i ve t i m e s . A dd 200 μ L of E l l m a n ’ s R e a ge nt t o e a c h w e l l . A dd 5 μ L of t r a c e r
t o t he T A w e l l s . C ove r t he pl a t e w i t h pl a s t i c f i l m . U s e or bi t a l s ha ke r t o s ha ke f or 120 m i n a nd
a voi d l i g ht . R e a d t he pl a t e a t a w a ve l e ngt h a t 420 nm . U s e E x c e l t o g e ne r a t e a s t a nda r d c ur ve
a nd us e t ha t c ur ve t o c a l c ul a t e t he a m ount of a l dos t e r one pr oduc t i on pe r c e l l .
23
C h a p t e r 3 R e s u l t s
3 . 1 T h e g e n e e x p r e s s i o n c h a n g e s i n H 2 9 5 R c e l l s a f t e r d o w n -
r e g u l a t i o n o f CYP21A2 g e n e
T o i nve s t i g a t e t he i nf l ue nc e of knoc ki ng dow n C Y P 21A 2 on ot he r s t e r oi doge ni c g e ne s , H 295R
c e l l s w e r e t r a ns f e c t e d w i t h s i R N A a nd us e R T - qP C R t o m e a s ur e t he c ha nge s of g e ne e x pr e s s i on.
F our c om bi na t i ons of s i R N A pr obe a nd t hr e e c onc e nt r a t i ons of s i R N A w e r e us e d f or t r a ns f e c t t o
g e t opt i m a l knoc kdow n e f f i c i e nc y .
A s s how n i n F i gur e 3A , H 295R c e l l s w e r e t r a ns f e c t e d w i t h A 1 pr obe , A 2 pr obe , A 3 pr obe a nd
t he m i xt ur e of a l l t hr e e pr obe s . T he A 1 pr obe a l one s how s no e f f e c t w hi l e ot he r c om bi na t i on
s how s t he a bi l i t y t o knoc k dow n C Y P 21A 2 e x pr e s s i on. T he m i xt ur e of t hr e e pr obe s ha s t he m os t
e f f e c t r e duc i ng C Y P 21A 2 e x pr e s s i on l e ve l s . T hi s c om bi na t i on w a s c onf i r m e d t o us e f or
t r a ns f e c t i on. A s s how n i n F i gur e 3B , 6.6 nM , 20 nM , a nd 60 nM s i R N A w e r e us e d t o t r a ns f e c t
H 295R c e l l s . A s a l l t hr e e g r oups de c r e a s e t he t a r ge t g e ne e x pr e s s i on, t he 6.6 nM g r oup s how s
t he be s t C Y P 21A 2 knoc kdow n e f f i c i e nc y a nd us e t he m i ni m um dos a ge of s i R N A .
A s s how n i n F i gur e 3C a nd F i gur e 3D , t he s i R N A t r a ns f e c t i on pr ovi de s a bout 70% knoc kdow n
of t a r ge t m R N A , C Y P 21A 2 , a nd t he H P R T pos i t i ve pr ovi de s 80% knoc kdow n e f f i c i e nc y . I t
s ug ge s t s t ha t t he m a t e r i a l s a nd ope r a t i ons r e qui r e d f or t r a ns f e c t i on a r e s ui t a bl e .
A s s how n i n F i gur e 3E - G , s t e r oi doge ni c r e l a t e d m R N A s e x pr e s s i on c ha nge d w hi l e C Y P 21A 2
w a s s i l e nc e d by s i R N A . T he e x pr e s s i on of C Y P 11B 1 ha s s l i g ht de c l i ne , but t he r e i s no s t a t i s t i c a l
24
di f f e r e nc e . T he r e i s a bout 50% knoc kdow n of C Y P 11B 2 m R N A , t he e x pr e s s i on s i g ni f i c a nt l y
de c r e a s e s . T he e x pr e s s i on of C Y P 17A 1 i nc r e a s e s s l i g ht l y w i t hout s t a t i s t i c a l di f f e r e nc e . T he up-
r e gul a t i on of H SD 3B 2 a r e s t a t i s t i c a l l y s i g ni f i c a nt . A c c or di ng t o t he pa t hw a y s how n i n F i gur e 1B ,
t he dow ns t r e a m g e ne s , C Y P 11B 1 a nd C Y P 11B 2 , w e r e i nhi bi t e d w he n C Y P 21A 2 g e ne i s s i l e nc e d.
A nd C Y P 17A 1 a nd H SD 3B 2 g e ne , e nc ode d e nz y m e s w or ki ng on ot he r pa t hw a y s , w e r e up-
r e gul a t e d w he n t he c or t i s ol , a l dos t e r one a nd c or t i c os t e r one s y n t he s i s pa t hw a y s w e r e bl oc ke d.
T hi s pa t t e r n of g e ne e x pr e s s i on c ha nge s i s c ons i s t e nt w i t h t he phe nom e non i n C A H .
F i gu r e 3
A B
25
C D
E F
26
G H
F i gu r e 3 G e n e e xp r e s s i on c h an ge af t e r R N A i n t e r f e r e n c e A s s ay
( A ) - ( H ) R T - qP C R a na l y s e s of s t e r oi doge ni c r e l a t e d g e ne e x pr e s s i on. ( A ) C Y P 21A 2 m R N A
e x pr e s s i on i n H 295R c e l l s a r e s i g ni f i c a nt l y dow n- r e gul a t e d w i t h di f f e r e nt c om bi na t i on of s i R N A
pr obe s . T he m i xt ur e of t hr e e pr obe s m os t e f f e c t i ve l y s i l e nc e s C Y P 21A 2 e x pr e s s i on. ( B ) H 295R
c e l l s w e r e t r a ns f e c t e d w i t h 6.6 nM , 20 nM , a nd 60 nM s i R N A . T he 6.6 nM g r oup s how s t he be s t
C Y P 21A 2 knoc kdow n e f f i c i e nc y . ( C ) T he s i R N A t r a ns f e c t i on pr ovi de s a bout 70% knoc kdow n of
t a r ge t m R N A , C Y P 21A 2 . ( D ) T he H P R T pos i t i ve pr ovi de s 80% knoc kdow n e f f i c i e nc y . ( E ) - ( H )
O t he r s t e r oi dog e ni c r e l a t e d m R N A s ’ e x pr e s s i on c ha nge w hi l e C Y P 21A 2 w a s s i l e nc e d by s i R N A .
T he e x pr e s s i on of C Y P 11B 1 ha s s l i g ht de c l i ne , but t he r e i s no s t a t i s t i c a l di f f e r e nc e . T he r e i s
a bout 50% knoc kdow n of C Y P 11B 2 m R N A , t he e x pr e s s i on s i g ni f i c a nt l y de c r e a s e s .
T he e x pr e s s i on of C Y P 17A 1 i nc r e a s e s s l i g ht l y w i t hout s t a t i s t i c a l di f f e r e nc e . T he up- r e gul a t i on of
H SD 3B 2 a r e s t a t i s t i c a l l y s i g ni f i c a nt .
T he l e ve l s of t he s t e r oi dog e ni c r e l a t e d m R N A s w e r e nor m a l i z e d w i t h t he A c t i n R N A . T he
m R N A l e ve l s of c e l l s t r a ns f e c t e d w i t h t he s c r a m bl e w e r e s e t a t 1, a nd r e l a t i ve m R N A l e ve l s a r e
i ndi c a t e d.
D a t a i n ( A ) - ( H ) a r e pr e s e nt e d a s m e a n ± S E M of 3 i nde pe nde nt e x pe r i m e nt s w i t h t r i pl i c a t e e a c h.
* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. S i g ni f i c a nt di f f e r e nc e s w e r e m e a s ur e d
w i t h pa i r e d a nd unpa i r e d S t ude nt ’ s t - T e s t .
27
3 . 2 S t e r o i d p r o d u c t i o n c h a n g e s i n H 2 9 5 R a f t e r k n o c k d o w n
C Y P 2 1 A 2 g e n e
T o i nve s t i g a t e w he t he r s i l e nc e of C Y P 21A 2 c a n a f f e c t c e l l s t e r oi doge ne s i s , H 295R c e l l s w e r e
t r e a t e d w i t h e i t he r s c r a m bl e d R N A ( 20 nM ) or s i R N A ( m i xt ur e of A 1, A 2, a nd A 3 pr obe s , 6.6
nM e a c h pr obe ) f or 48 h. A f t e r t ha t , c e l l s upe r na t a nt s w e r e c ol l e c t e d f or t he m e a s ur e m e nt of
a l dos t e r one a nd c or t i s ol pr oduc t i on.
A s s how n i n F i gur e 4A , i n H 295R c e l l , t he pr oduc t i on of a l dos t e r one i n t he e xpe r i m e nt a l g r oup
i s s l i g ht l y i nc r e a s e d c om pa r e d t o t he c ont r ol g r oup. T he di f f e r e nc e i s not s t a t i s t i c a l l y s i g ni f i c a nt .
T he t w o g r oups a r e c ons i de r e d t o ha ve t he s a m e l e ve l of a l dos t e r one pr oduc t i on.
A s s how n i n F i gur e 4B , i n H 295R c e l l , c om pa r e d t o t he c ont r ol g r oup, t he pr oduc t i on of c or t i s ol
i n e x pe r i m e nt a l g r oup i s i nc r e a s e d. H ow e ve r , t he r e i s no s t a t i s t i c a l l y s i g ni f i c a nt di f f e r e nc e . T he
a m ount of c or t i s ol pr oduc t i on i n t w o g r oups a r e c ons i de r e d t he s a m e .
T he s e r e s ul t s s ug ge s t e d t ha t t he s t e r oi d pr oduc t i on w a s not a f f e c t e d by t he s i R N A t r a ns f e c t i on.
U s i ng s i R N A t o s i l e nc e C Y P 21A 2 di d not c ha nge s t e r oi doge ne s i s , t houg h t he r e w e r e c ha nge s
e x i s t i ng i n g e ne e x pr e s s i on.
28
F i gu r e 4
A B
F i gu r e 4 E f f e c t of d ow n - r e gu l at e d C Y P 21A 2 on S t e r oi d P r od u c t i on i n H 295R C e l l
( A ) E L I S A a na l y s e s of a l dos t e r one pr oduc t i on i n H 295R c e l l s . A l dos t e r one pr oduc t i on w a s
s l i g ht l y i nc r e a s e d a f t e r s i l e nc i ng C Y P 21A 2. ( B ) E L I S A a na l y s e s of c or t i s ol pr oduc t i on i n
H 295R c e l l s . C or t i s ol pr oduc t i on w a s s l i g ht l y i nc r e a s e d a f t e r s i l e nc i ng C Y P 21A 2.
D a t a i n ( A ) - ( B ) a r e pr e s e nt e d a s m e a n ± S E M of 3 i nde pe nde nt e xpe r i m e nt s w i t h t r i pl i c a t e e a c h.
* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. S i g ni f i c a nt di f f e r e nc e s w e r e m e a s ur e d
w i t h pa i r e d a nd unpa i r e d S t ude nt ’ s t - T e s t .
29
C h a p t e r 4 D i s c u s s i o n
P r e vi ous s t udi e s de m ons t r a t e d t ha t t he e x pr e s s i on of C Y P 21A 2 w a s de c r e a s e d i n s t a r ve d H 295R
c e l l s ( U dha ne e t a l ., 2015) . T he y s ug ge s t e d t ha t unde r s t a r va t i on c ondi t i on, t he e nz y m e a c t i vi t i e s
of C Y P 17A 1 a nd H S D 3B 2 c ha nge d i n H 295R c e l l s , l e a di ng t o c ha nge s i n s t e r oi doge ne s i s .
A c c or di ng t o t he i r c om pa r i s on of m i c r oa r r a y e x pr e s s i on pr of i l i ng of nor m a l a nd s t a r ve d H 295R
c e l l s , H SD 3B 2, C Y P 21A 2 w e r e s i g ni f i c a nt l y dow n- r e gul a t e d unde r s t a r va t i on c ondi t i ons . I n our
e x pe r i m e nt , t he 48 h pos t - t r a ns f e c t i on pe r i od m a y c a us e t he c e l l s s t a r va t i on. T he n t he C Y P 21A 2
e x pr e s s i on i n bot h be t w e e n H 295R c e l l s t r a ns f e c t e d by s c r a m bl e R N A a nd H 295R c e l l s
t r a ns f e c t e d by s i R N A m a y s t a y i n a l ow e r l e ve l t ha n nor m a l H 295R c e l l s . T hus , i t i s ha r d t o
c om pa r e t he di f f e r e nc e s be t w e e n H 295R c e l l s t r a ns f e c t e d by s c r a m bl e R N A a nd H 295R c e l l s
t r a ns f e c t e d by s i R N A w hi l e bot h s how i ng C Y P 21A 2 e x pr e s s i on r e duc t i on. I t i s i m por t a nt t o
a voi d s t a r vi ng t he c e l l s a nd ke e p t he H 295R c e l l s i n a he a l t hy c ondi t i on. T o a voi d t he s t a r va t i on
s i t ua t i on, s e e di ng m or e c e l l s a nd onl y l e t i t g r ow f or one da y f r om t he be gi nni ng of s i R N A
t r a ns f e c t i on a s s a y m i g ht be he l pf ul .
G e ne e x pr e s s i on c ha nge s w he n s i l e nc i ng C Y P 21A 2 i n H 295R hum a n a dr e noc or t i c a l c a r c i nom a
c e l l s a r e c ons i s t e nt w i t h t he g e ne e x pr e s s i on c ha nge s r e por t e d i n C A H pa t i e nt s . H ow e ve r , t he
s t e r oi d pr oduc t i on di d not c ha nge a s e x pe c t e d. T he s i R N A s a r e t r a ns i e nt l y e xpr e s s e d i n c e l l s , a nd
t he C Y P 21A 2 s i l e nc i ng l a s t s f or a ve r y s hor t pe r i od of t i m e . I n t hi s s hor t pe r i od of t i m e , onl y
m R N A e x pr e s s i on ha s c ha nge d, not l ong e nough t o obs e r ve t he c ha nge s i n s t e r oi d s y n t he s i s .
F ur t he r m or e , i n C A H m os t of m ut a t i ons i n C Y P 21A 2 g e ne oc c ur on c hr om os om e 6 a nd t hus 21-
hy d r ox y l a s e de f i c i e nc y a l w a y s e x i s t s i n c e l l s . B e f or e t r e a t m e nt w i t h s i R N A , t he g e ne s a r e
nor m a l l y e x pr e s s e d i n c e l l s , a nd t he e nc ode d e nz y m e s w i l l not be e nt i r e l y c ons um e d be f or e
30
s i R N A de gr a da t i on. T hus , us i ng ot he r t ool s , e .g., s hR N A , t o pe r f or m a s t a bl e t r a ns f e c t i on f or
C Y P 21A 2 s i l e nc i ng m a y be ne e de d t o c a us e c ha nge s i n bot h pr ot e i n l e ve l s a nd s t e r o i d pr oduc t i on.
M or e ove r , a s t a bl e i nduc i bl e C Y P 21A 2 knoc kdow n H 295R c e l l l i ne m a y be a m or e us e f ul m ode l
t o s t udy t he c ha nge s i n c e l l ul a r phy s i ol ogy i nduc e d by C Y P 21A 2 unde r e x pr e s s i on a s t he g e ne
s us pe ns i on c oul d be r e ve r s i bl e a nd t hus f l e x i bl e .
S t udy i ng s t e r oi doge ne s i s i n a dr e na l c e l l t r e a t m e nt w i t h A C T H i s i ndi s pe ns a bl e t o a s s e s s t he
f unc t i on of t he c e l l s . A C T H , a l ong w i t h i t s r e c e pt or ( m e l a noc or t i n 2 r e c e pt or , M C 2R ) , i s t he
i ni t i a l hor m ona l m e di a t i ng t he a dr e na l c or t i s ol pr oduc t i on. T hough t he H 295R c e l l ha s pr ove n t o
be a us e f ul m ode l t o s t udy A ng I I r e gul a t e d a l dos t e r one pr oduc t i on ( N ogue i r a e t a l ., 2007) , t he
H 295R c e l l l i ne i s onl y m i l dl y r e s pons i ve t o A C T H ( P a r m a r e t a l ., 2008) . H A C 15 c e l l s a l s o
or i gi na t e f r om N C I - H 295 ( P a r m a r e t a l ., 2008) . T r a ns c r i pt s e nc odi ng S t A R , H S D 3B 2, a s w e l l a s
t he f i ve f or m s of c y t oc hr om e P 450 know n t o be i nvol ve d i n nor m a l a dr e na l s t e r oi dog e ne s i s
( C Y P 11A 1, C Y P 17, C Y P 21, C Y P 11B 2, a nd C Y P 11B 1 ) , a r e de t e c t a bl e i n t he H 295 c e l l l i ne s a nd
t he H A C 15 c l ona l c e l l l i ne . T hus , bot h H 295R c e l l l i ne s a nd t he H A C 15 c e l l l i ne ha ve t he s a m e
a bi l i t y t o pr oduc e s t e r oi ds . M or e ove r , H A C 15 c e l l s ha ve a hi g he r M C 2R g e ne e x pr e s s i on l e ve l
t ha n H 295R c e l l l i ne ( P a r m a r e t a l ., 2008) . W i t h m or e e x pr e s s i on of A C T H r e c e pt or e nc ode d by
M C 2R , t he A C T H t r e a t m e nt m a y r e c e i ve s t r ong r e s pons e i n H A C 15 c e l l l i ne . H A C 15 c e l l l i ne
s e e m s t o be a be t t e r m ode l t o s t udy t he r e s pons e t o A C T H s t i m ul a t i on. I n t he f ut ur e , e x pl or i ng
t he c ha nge s i n H A C 15 c e l l l i ne w i t h knoc kdow n C Y P 21A 2 c oul d he l p us be t t e r know t he
i nf l ue nc e of C Y P 21A 2 s i l e nc i ng on A C T H r e gul a t e d pa t hw a y i n t he a dr e na l c e l l l i ne .
31
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Abstract (if available)

Abstract Congenital adrenal hyperplasia is one of the most common genetic disorders. The clinical manifestations of CAH include adrenal insufficiency, genital ambiguity, short stature, and an increased risk of metabolic syndrome and infertility during adolescence and adulthood. CAH is a family of autosomal recessive hereditary diseases characterized by decreased adrenal steroid production resulting from mutations of genes encoding enzymes involved in steroidogenesis. The gene affected by mutations are: CYP21A, CYP11B, CYP17A1, HSD3B2, CYP11A1, and StAR. Diverse gene mutations interrupt synthesis pathway in different points, leading to several types of congenital adrenal hyperplasia. The most common CAH subtype is 21-hydroxylase deficiency (21OHD). 21OHD is divided into classic and non-classical forms. The classical form of 21OHD is further divided into “salt wasting” and “simple virilizing” subtypes. As 21-hydroxylase dysfunction caused by mutation of CYP21A2 gene could lead to severe CAH, we are interested in the changes of H295R cell activities and secretion that occur when the CYP21A2 gene is knocked down. To obtain further evidence regarding the role of CYP21A2 in the human H295R adrenocortical cancer cell line, the expression of CYP21A2 was silenced by siRNA transfection. When CYP21A2 gene is silenced, the expressions of downstream genes, CYP11B1 and CYP11B2, were inhibited. And CYP17A1 and HSD3B2 gene, encoded enzymes working on other pathways, were up-regulated when the cortisol, aldosterone and corticosterone synthesis pathways were blocked. This pattern of gene expression changes is consistent with the phenomenon in CAH.

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Asset Metadata

Creator Hu, Yiman (author)

Core Title Explore the influence of CYP21A2 silencing on H295R human adrenal cortical carcinoma cell gene expression function

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 04/25/2020

Defense Date 04/24/2020

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag congenital adrenal hyperplasia (CAH),CYP21A2,H295R,OAI-PMH Harvest,steroidogenesis

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Papadopoulos, Vassilios (committee chair), Haworth, Ian (committee member), Okamoto, Curtis (committee member)

Creator Email human95@126.com,yimanhu@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-290716

Unique identifier UC11666094

Identifier etd-HuYiman-8330.pdf (filename),usctheses-c89-290716 (legacy record id)

Legacy Identifier etd-HuYiman-8330.pdf

Dmrecord 290716

Document Type Thesis

Rights Hu, Yiman

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

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