The Clon ing of PS-2 Gene and Its Expression in Hepa tocellular Carc inoma

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1 ISSN CN gQ 16 1 Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 1, PS-2 1) 2) 3 (, 1), ; 2), ) m RNA, 14 N o rthern b lo t bp cdna,,, PS22 (p resen ilin22) 94% PS22, PS22,, PS22 Q 78, R The Clon ing of PS-2 Gene and Its Expression in Hepa tocellular Carc inoma L IU Yu2chuan, M IN G W en2yu, Y IN W ei, L IU L an2ying, M A J ian2quan 1), TAN G J ian 2), GU Jun (T he R esearch Center of M olecu lar M ed icine, S un Y at2s en U niversity of M ed ical S ciences, 1) D ep artm ent of B iochem istry, S un Y at2s en U niversity of M ed ical S ciences, Guang z hou ; 2) Institu te of Card iovascu lar R esearch, B eij ing M ed ical U niversity, B eij ing ) Abstract F luo rescen t differen tial disp lay (FDD ) w as u sed to study gene exp ression in hum an liv2 er, cirrho sis and hepatocellu lar carcinom a (HCC) respectively. 14 differen tially exp ressed cdna s w ere p icked and fu rther analyzed by N o rthern b lo ṫ Am ong them, 9 w ere confirm ed as differen tial2 ly exp ressed m RNA ṡ FDD and N o rthern b lo t analyses show ed that the am oun t of a 500 bp cdna fragm en t from the liver and cirrho sis tissues w as less than that from HCC tissue. T he se2 quence of th is cdna fragm en t shared 94% hom o logy w ith hum an p resen ilin22 (PS22) gene. T he m u tation of PS22 gene w as associated w ith the early on set fam iliar A lzheim er s disease (AD ), bu t the function of PS22 gene in hepatocarcinogenesis has no t been repo rted yeṫ U p 2regu lation of PS2 2 gene exp ression in HCC suggests its ro le in hepatocarcinogenesiṡ Key words H epatocellu lar carcinom a (HCC), F luo rescen t differen tial disp lay (FDD ), P resen ilin2 2 (PS22),, [ 1 ], ,., DNA [ 2 4 ], m RNA, 60% m RNA, [ 5 ] : 3 cdna, 3 2, m RNA, cdna 5 3 T el: (020) (PCR ) cdna ( 20 ) PS22 E2m ail: gujun38@ho tm ail. com,, ,, : , : PCR

2 2 16, M AN m o lgl dn T P, 4 l 2 m o lgl M 13 T 7, 4 l ( :,, 43, 014 l 5 U n itsgl T aq (Gibco BRL,, ) PCR PE480 PCR, ), (PE ) PCR PCR, N o rthen b lo t, 112 RNA : 110% (Gibco BRL ) RNA RNA, RNA N C (Sch leicher RN ase DN aseg (P rom ega ) RNA RNA A 260gA : (D IG DNA L abeling and D etection k it; Boeh ringer Genom yxl R Genom yxsc M annheim ) 20 h m o lgl L ic l, BECKM AN 215, - 20, 000 g (5 m RNA p rofile k it 1 fluo ro2dd k it), , 3 17 T 7 ; 5 16 M 13 3 ( tetram h, 2 SSC 1. 0% SD S ethylrhodam ine; TM R ) : RNA 015 ggl, 2 l RNA 2 l 2 m o lgl 3, ( 150 mm o lgl N ac l, 100 mm o lgl, ph m in, 2 l 250 m o lgl dn T P, 2 l 100 mm o lgl D T T, 4 l 5 30 m in,, 718 l RN ase, 012 l 200 U n itsgl 30 m in,, 1, 3% (SuperScrip t g R T enzym e, Gibco BRL ) 42 5 m in, m in, m in (100 mm o lgl T ris2hc l, ph 9. 5, 100 mm o lgl N a2, - 20 PCR : 0195 l, 1 l 10 PCR,,,, 115 l 25 mm o lgl M gc l2, 2 l 250 m o lgl dn T P, l 2 m o lgl 5, 017 l 5 m o lgl 3, 2 l, 011 l 5 U 2 n itsgl T aq (Gibco BRL ) PE480 PCR (PE ) : 95 2 m in; s, s, 72 2 m in, :, 50 l T E 37 1h 4 l M 13 T 7 PCR : 1819 l, 4 l 10 PCR, 115 l 25 mm o lgl M gc l2, 312 l Northern blot RNA :, TR IzoL 10 g RNA, 5 6 V gcm 3 h & Schuell), 20 SSC, 70 4 h,, : PCR, D IG 20 m in, 70% : SD S, 50 1 h,,, 2, 15 m in, 011 SSC 011% SD S 68 2, 15 m in 7. 5), 3% Tw een 20 5 m in, 1% Tw een 20 2, 15 m in 3 C l, 50 mm o lgl M gc l2) 114 N o rthern b lo t cdna, PCR 3 l 5 l 2, 1 l pgem 2T easy (P rom ega ), 1 l T 4 (P rom ega ), 4 E. coli DH 5 Α, 200 l ; s, s, 72 2 m in, 30 IPT G X2gel,, ; 72 7 m in; 4 PCR 1,,, E cor g BECK2 115

3 1 : PS22 3 Q IA rep Sp in M in ip rep k it (Q I2 A GEN ) : SP6,,, 18S [ Α2 32 P ] da T P,, 28S F ig. 2 N CB I BLA ST RNA, RNA, RNA RN ase DN aseg, 110%, RNA 212,, N : N o rm al hepatic tissue; F ig. 1 20,,, 113 kb, F ig. 2 N o rthern blo t analysis C: C irrho sis tissue; H: H epatocellular carcinom a tissue 214 cdna pgem 2T easy, bp, F ig. 3 F ig. 1 F luo rescent differential disp lay N : N o rm al hepatic tissue; C: C irrho sis tissue; H: H epatocellular carcinom a tissue; M : TM P2labeled mo lecular w eight DNA m arker The differential cdna band of N o. 20 clone w as m arked w ith arrow 213 Northern blot 14 N o rthern b lo t, 9, 60% 20 F ig. 3 A garo se gel electropho resis of restriction fragm ents M : DNA L adder M ix (M B I) 1: N o. 20 clonal p roduct digested by E cor g ; 2: pgem 2Teasy vecto r 215, cdna, F ig. 4 kb 113 kb, F ig kb N CB I B lastn, PS 22

4 4 16 F ig. 4 T he sequence of N o. 20 clone (STM 22) 94% 3 311, [ 11 ] Β2 (am ylo id2 70% [ 6 ], Βp recu rso r p ro tein; A PP ) Β2 (am y2 RNA lo id2β; A Β), A Β40 DNA,, A Β42 A Β42 [ 12, 13 RN ase DN aseg RNA, ] PCR, [ 14 PCR PS22 ], 14 N o rthern b lo t, 9 [ 15 PS22 ] 113 kb, 214 kb 113 kb,, 214 kb, 113 kb 314 PS-2 PS22, PS22 (early2on set fam ilial A lzheim er s disease; AD ) 113 kb m RNA PS22 sho rt (PS s), PS FDD PCR, (M et 298 2Ile 448 ), PS22 N CB I B lastn PS22s Α. [ 15 Fas ] PS22, Bcl2XL,, 20, c, F ig. 4, p resen ilin22 (PS22) [ 16 ] 94%, N o rthern b lo t 113 kb m RNA F ig. 1 F ig. 2,, p resen ilin s (PSs), N o tch, N o tch [ 17 21, ] 313 PS-2,, PSs N o tch l PS22 1 1q42. 1 [ 7 ] PS22 [ 22, ], 7, PS22 N o tch [ ] PS22, PS22 N o tch 448, m RNA, 214 kbps22, [ 9, ] PS22 PS22 PS22 AL G23,,, 214 kb, 113 kb [ 10 ] N o rthern b lo t PS kb, (References) 1 L i F P, Sh iang E L. Cancer mo rtality in Ch ina. J N atl Cancer Inst, 1980, 65: Pascale R M, Sim ile M M, Feo F. Genom ic abno rm alities in

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