ISSN 100727626 CN 1123870gQ 16 1 Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 1, 1 5 2000 2 PS-2 1) 2) 3 (, 1), 510089; 2), 100083) m RNA, 14 N o rthern b lo t 9 500 bp cdna,,, PS22 (p resen ilin22) 94% PS22, PS22,, PS22 Q 78, R 73517 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 510089; 2) Institu te of Card iovascu lar R esearch, B eij ing M ed ical U niversity, B eij ing 100083) 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 ], 250 000 1,., DNA [ 2 4 ], m RNA, 60% m RNA, [ 5 ] : 3 cdna, 3 2, m RNA, cdna 5 3 T el: (020) 87330837 3 (PCR ) cdna ( 20 ) PS22 E2m ail: gujun38@ho tm ail. com,, 1965 11,, : 1999208226, : 1999210229 PCR
2 16, M AN 1 111 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 11311 : 110% (Gibco BRL ) RNA RNA, RNA N C (Sch leicher RN ase DN aseg (P rom ega ) RNA RNA A 260gA 280 119 210 11212 : (D IG DNA L abeling and D etection k it; Boeh ringer Genom yxl R Genom yxsc M annheim ) 20 h 011 4 m o lgl L ic l, BECKM AN 215, - 20, 000 g (5 m RNA p rofile k it 1 fluo ro2dd k it), 12 3 20 5, 3 17 T 7 ; 5 16 M 13 3 ( tetram 2 50 20 h, 2 SSC 1. 0% SD S ethylrhodam ine; TM R ) : RNA 015 ggl, 2 l RNA 2 l 2 m o lgl 3, 70 5 1 ( 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, 50 50 m in, 70 15 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, 1. 75 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; 94 30 s, 50 40 s, 72 2 m in, 4 11213 :, 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 250 113 Northern blot 11211 RNA :, TR IzoL 10 g RNA, 5 6 V gcm 3 h & Schuell), 20 SSC, 70 4 h,, 5-70 11312 : PCR, D IG 20 m in, 70% 11313 : 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 ; 94 30 s, 60 40 s, 72 2 m in, 30 IPT G X2gel,, ; 72 7 m in; 4 PCR 1,,, E cor g BECK2 115
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 2 211 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, 20 520 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, 214 20 F ig. 4 kb 113 kb, F ig. 2 214 kb N CB I B lastn, PS 22
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 (PS2 312 2s), PS22 151 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 [ 8 6 7 ] 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 PS2 2 214 kb, (References) 1 L i F P, Sh iang E L. Cancer mo rtality in Ch ina. J N atl Cancer Inst, 1980, 65: 217 221 2 Pascale R M, Sim ile M M, Feo F. Genom ic abno rm alities in
1 : PS22 5 hepatocarcinogenesis. Imp lications fo r a chemop reventive strat2 egy. A nticancer R es, 1993, 13: 1341 1356 3 L apeyre J N, W alker M S, Becker F F. DNA m ethylation and m ethylase levels in no rm al and m alignant mouse hepatic tis2 sues. Carcinog enesis, 1981, 2: 873 878 4 Kautiainen T L, Jones P A. DNA m ethyltransferase levels in tumo rigenic and nontumo rigenic cells in culture. J B iol Chem, 1986, 261: 1594 1598 5 L iang P, Pardee A B. D ifferential disp lay of eukaryo tic m essen2 ger RNA by m eans of the po lym erase chain reaction. S cience, 1992, 257: 967 971 6 Sun Y, H egam yer G, Co lburn N H. M o lecular cloning of five m essenger RNA s differentially exp ressed in p reneop lastic o r neop lastic JB6 mouse ep iderm al cells: one is homo logous to hu2 m an tissue inhibito r of m etallop ro teinases23. Cancer R es, 1994, 54: 1139 1144 7 T akan T, Sahara N, Yamonouch Y, M o ri H. A ssignm ent of A lzheim er s p resenilin22 (PS22) gene to lq42. 1 by fluo rescence in situ hybridization. N eu rosci L ett, 1997, 221: 205 207 8 Kovacs D M, Fausett H J, Page K J, Kim T W, M o ir R D, M erriam D E, Ho llister R D, H allm ark O G,M ancini R, Felsen2 stein K M, H ym an B T, T anzi R E,W asco W. A lzheim er2asso2 ciated p resenilins 1 and 2: neuronal exp ression in brain and lo2 calization to intracellular m em branes in m amm alian cells. N at M ed, 1996, 2: 224 229 9 Rogaev E I, Sherrington R, Rogaeva E A, L evesque G, Ikeda M, L iang Y, Ch i H, L in C, Ho lm an K, T suda T,M ar L, So rbi S, N acm ias B, P lacentini S, Am aduccil, Chum akov I, Cohen D, L annfelt L, F raser P E, Romm ens J M, St Geo rge2h yslop P H. Fam ilial A lzheim er s disease in k indreds w ith m issense m uta2 tions in a gene on ch romo som e 1 related to the A lzheim er s dis2 ease type 3 gene. N atu re, 1995, 376: 775 778 10 V ito P, L acana E, D A dam io L. Interfering w ith apop to sis: Ca 2+ 2binding p ro tein AL G22 and A lzheim er s disease gene AL G23. S cience, 1996, 271: 521 525 11 L evy2l ahad E,W asco W, Poo rkaj P, Rom ano D M, O sh im a J, Pettingell W H, Yu C E, Jondro P D, Schm idt S D, W ang K, C row ley A C, Fu Y H, Guenette S Y, Galas D, N em ens E,W ijs2 m an E M, B ird T D, Schellenberg G D, T anzi R E. Candidate gene fo r the ch romo som e 1 fam ilial A lzheim er s disease locus. S cience, 1995, 269: 973 977 12 Jarrett J T, L ansbury P T J ṙ Seeding "one2dim ensional crystal2 lization" of am ylo id: a pathogenic m echanism in A lzheim er s disease and scrap ie?. Cell, 1993, 73: 1055 1058 13 Yankner B A. M echanism s of neuronal degeneration in A lzheim er s disease. N eu ron, 1996, 16: 921 932 14 W o lozin B, Iw asak i K, V ito P, Ganjei J K, L acan E, Sunderland T, Zhao B, Kusiak J W,W asco W, D A dam io L. Participation of p resenilin 2 in apop to sis: Enhanced basal activity conferred by an alzheim er m utation. S cience 1996, 274: 1710 1713 15 V ito P, Ghayur T, D A dam io L. Generation of anti2apop to tic p resenilin22 po lypep tides by alternative transcrip tion, p ro teo ly2 sis, and caspase23 cleavage. J B iol Chem, 1997, 272: 28315 28320 16 Passer B J, Pellegrini L, V ito P, Ganjei J K, D A dam io L. Inter2 action of A lzheim er s p resenilin21 and p resenilin22 w ith Bcl2XL. J B iol Chem, 1999, 274: 24007 24013 17 L evitan D, Greenw ald İ Effects of SEL 212 p resenilin on L IN 212 localization and function in caeno rhabditis elegans. D evelop 2 m ent, 1998, 125: 3599 3606 18 W einm aster G, Roberts V J,L em ke G. A homo log of dro soph ila no tch exp ressed during m amm alian developm enṫ D evelopm ent, 1991, 113: 199 205 19 H aass C. P resenilins: genes fo r life and death. N eu ron, 1997, 18: 687 690 20 A rtavanis2t sakonas S,M atsuno K, Fo rtinim E. N o tch signal2 ing. S cience, 1995, 268: 225 232 21 Struh l G, Greenw ald İ P resenilin is required fo r activity and nu2 clear access of N o tch in D ro soph ila. N atu re, 1999, 398: 522 525 22 Berezovska O, X ia M Q, Page K,W asco W, T anzi R E, H ym an B T. D evelopm ental regulation of p resenilin mrna exp ression parallels no tch exp ression. J N europ athol E xp N eurol, 1997, 56: 40 44