Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy. (o steoarth ritis, OA )

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1 ISSN CN gQ Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy 17 (2): ) 1), 3, 2), 1), 1), 1), 2) 2), ( 1), ; 2), ) ( rheum ato id arth ritis, RA ) (fib rob la st2like synovia l cells, FL S), (o steoarth ritis, OA ), RA,,, N o rthern RA OA, 150 RA, bp 8% (12g150), 400 bp 36. 7% (55g150), 400 bp, 23. 7% (13g55) 18, 12, IGF21 ( IGFBP) B 6, R ing2box 1 SON DNA IGFBP B N o rthern, RA, OA, ; IGF21 B RA,,, R : , : (N o ) (N o g ) 3 : Tel: , E2m ail: bioyao@ fmm u. edu. cn,, , Received: June 15, 2000; A ccep ted: A ugust 25, 2000 Screen ing of H igh-expressed Genes in F ibrobla st- like Synov ia l Cells of Rheuma to id Arthr itis Pa tien ts by an Im proved Subtractive Hybr id iza tion W AN G J i2cun 1), YAO L i2bo 1) 3,LU β Hou2shan 2),L IU X in2p ing 1),D EN G Yan2chun 1), N IE X iao2yan 1), SUN T ie2zheng 2), YAN T ai2qiang 2) ( 1) D ep artm ent of B iochem istry and M olecu lar B iology, F ou rth M ilitary M ed ical U niversity, X i an , Ch ina; 2) D ep artm ent of O rthop ed ics, P eop le s H osp ital, P ek ing U niversity, B eij ing , Ch ina) Abstract In o rder to study the m echan ism of hyperp lasy and invasiveness of fib rob last2like synovial cells (FL S) of rheum ato id arth ritis (RA ) patien ts, an im p roved sub tractive hyb ridization w as u sed to screen fo r the genes w h ich exp ression w as increased in RA FL S com pared w ith FL S of o steoarth ritis (OA ) patien tṡ T he cdna fragm en ts of h igh2exp ressed genes ob tained by Suppo rted by N ational N atural Science Foundation fo r distinguished scho lars (N o ), N ational Science Foundation fo r key p rogram (N o g ), and Foundation fo r U niversity Key T eacher by the M inistry of Education 3 Co rresponding autho r T el: , E2m ail: bioyao@fmm u. edu. cn

2 2 : 245 sub tractive hyb ridization w ere cloned in to p lasm id vecto rs and the false po sitive fragm en ts w ere rem oved by reverse do t2b lo tting hyb ridization. T hen the true po sitive fragm en ts w ere sequenced and the sequences w ere com pared w ith tho se genes in GenBank to iden tify w hether they w ere know n genes o r novel sequenceṡ A t last, N o rthern2b lo tting hyb ridization w as done to detect the exp ression level of som e h igh2exp ressed genes in several RA and OA patien ts FL S cellṡ T he resu lts show ed that, am ong the 150 cdna fragm en ts ob tained after sub tractive hyb ridization, 36. 7% (55g150)w ere longer than 400 bp, and abou t 8% (12g150) w ere longer than 1000 bp. am ong the 55 fragm en ts longer than 400 bp, false po sitive rate w as abou t 23. 7% (13g55). Am ong the 18 fragm en ts sequenced, 12 w ere cdna fragm en ts of know n genes including serine p ro tease ( IGF21 b inding p ro tein specific, IGFBP ), Cathep sin B and L am in in recep to r, and o ther six w ere novel sequences, tw o of w h ich w ere separately hom o logou s to R ing2box p ro tein 1 and SON DNA 2 b inding p ro tein. N o rthern2b lo tting hyb ridization resu lts show ed that IGF21 b inding p ro tein specific serine p ro tease, Cathep sin B and L am in in recep to r cou ld all be exp ressed in bo th RA and OA cells, bu t relatively m o re h igh ly exp ressed in RA FL S cells. T he resu lts suggest that the im p roved sub tractive hyb ridization is a sim p le and effective m ethod to screen differen tially exp ressed genes, and IGF21 b inding p ro tein specific serine p ro tease, Cathep sin B and L am in in recep to r m igh t be related to the hyperp lasy and invasiveness of FL S in RA patien tṡ Key words im p roved sub tractive hyb ridization, rheum ato id arth ritis (RA ), fib rob last2like synovial cells (FL S), h igh2exp ressed gene (rheum ato id arth ritis, RA ), (fib rob last2like synovial cells, P rom ega, Α2 32 P2dCT P FL S), PCR PE2400 PE,, PE310 PE, Pharm acia RA 112, RA OA [ 1 ],, cdna (o steoarth ritis, OA ) cdna s, RA (OA ) : cdna s, : 11 RA cdna s ; m RNA Po ly A (RA ) AA GCA GT GGTAA CAA CGCA GA GTA CT (30) N - 1 (OA ) N 23, cdna 3, m RNA Po ly A T ract System 1000 Sephacryl S , : 5 2AA GCA GT GGTAA ( Strep tavidin M agnespher R Param agnetic CAA CGCA GA GTA CGCGGG23, Particles, SA 2PM P s) P rom ega, cdna SU PER SCR IPT TM g RN ase H - R everse T ran s2 crip tase Gibco, L A T aq dn T P JM 109 pm D 182T T ar aka,, B io tin2du T P DNA, T ract System 1000 m RNA (RA ) (OA ) (2 10 5, ) m RNA RA m RNA,, 5 2, 5 GGG, PCR, 5 2AA GCA GT GGTAA CAA CGCA GA GT2 3 RNA H SU PER SCR IPT TM g OA m RNA, C lon tech,

3 OA OA, L CC > 0. 95, cdna s PCR, 2 (open reading du T P. 100 l, dn T P 20 nm o lgl, 102 m er 50 pm o lgl, o ligo dt (18) 50 pm o lgl, 2dU T P 0. 1 pm o lgl, cdna 1 l, T aq 1 U : s, 40 5 m in, 72 3 m in, RA cdna s OA ), RA cdna s 5 l OA 60 l, : 6 SSC, 0. 5% SD S, h , 4 RA Sephacryl S2400, dn T P 2 OA RNA 5 g DNA s l,, 0. 1 m l SA 2PM P s ( 0. 1 SSC ), 2 m in, 65 5 m in,, 0. 5% SD S 10, : m in, RA cdna ṡ PCR, 50 l : 40 l, dn T P 10 nm o lgl, L A T aq ( T aq ) 0. 5 U, 10 pm o lgl, 5 2 AA GCA GT GGTAA CAA CGC A GA GT 23, OA cdna s, : ( s, 68 5 m in ) 30, m in 11217,, (> RA cdna s bp bp ) C lon tech pm D 182T, JM 109, Am p + LB, bp (F ig. 1),,, E cor g H ind g 11218,,,, DNA Α2 32 P OA cdna, h, 11219, GenBank (BLA ST ),, (local com po sition com p lex ity, L CC) fram e finder, OR F finder), GenBank (BLA ST P),, (P rodom N o rthern IGFBP B, Α2 32 P2dCT P RNA,, h,, Β2actin, 211 (RA) cd NA s RA cdna s cdna s, OA cdna s, F ig. 1 PCR p roducts of the non2hybridized cdna fragm ents of RA FL S R: RA FL S; M : PCR m arker. The cdna fragm ents obtained by subtractive hybridization w ere from 300 bp to 4000 bp in length

4 2 : Am p + LB ,,, 12, 66. 7%,,, F ig. 2. S4, S23, S25 ( ribo som al p ro tein S4, S23,, 150, 55 ( 36. 7% ) 400 bp. 12 ( 8% ) (ferritin L chain), bp F ig. 2 Identification of the clones w ith inserted fragm ents by restriction enzym es digestion 1 11, 12 22: D ifferent clones,m : PCR m arker RD 30,, 213 RD 44 OA cdna s, RA 652 R ing2box F ig. 3, p ro tein 1, 56%,, 55, 13 (P rodom ), DNA,, 23. 7% R ing2box p ro tein 1 ; F ig. 3 Reverse do t2blo tting hybridization The do ts are cdna fragm ents of RA FL S fixed on N C m em brane. T he P robe is iso tope2labeled cdna PCR p roduct of OA FL S. T he do ts w ith po sitive signal (A 1, A 4, A 6, A 7, B2, B4, B8, C8, and D 2) are the cdna fragm ents that also exp ressed in OA FL S. T he negative do ts (o ther do ts) are the cdna fragm ents that are exp ressed o r high2exp ressed in RA FL S, but no t exp ressed o r low 2exp ressed in OA FL S 214 S25), ( in terferon2inducib le gene), ( lam in in recep to r), Β ( tran sfo rm ing grow th facto r, Β2induced), Χ ( tubu lin Χ2po lypep tide), B (cathep sin B ) g (p roalpha l (g ) co llagen), A 2raf (A 2raf21 oncogene) IGF21 ( IGF21 b inding p ro tein specific serine p ro teinase), 3 6, 33. 3% L CC, , ; OR F, RA 652 RB 82 RB 61 RB 16 ( ), 85, 45, , RD 30 RD 44 5 (135 ) 3 (75 ), RD 44 b reast carcinom a2associated p ro tein D F3 (55% ), RD 30 SON DNA b inding p ro tein (50% ), RB 61 HCG614 (39% ) RB 16 RB 82, GenBank (A ccession N o. ) T ab le Northern F ig. 4, N o rthern, RA OA IGFBP B, OA, 3 RA,

5 Table 1 Structure and function analysis of the novel sequences C lone N o. F ragm ent length Open reading fram e BLA ST P Dom ain A ccession N o. RA bp 85aa ( bp) R ing2box p ro tein 1 (56% ) P ro tein DNA 2 binding p robable A F RB bp 45 aa ( bp) no no A F RB bp 45 aa ( bp) no no A F RB bp 139 aa ( bp) HCG6. 4 (39% ) no A F RD bp 3 of ORF 75 aa ( bp) B reast carcinom a2associated p ro tein D F3 (55% ) no A F RD bp 5 of ORF 135 aa ( bp) SON DNA binding p ro tein (50% ) no A F F ig. 4 N o rthern hybridization w ith several k inds of h igh2 exp ressed gene fragm ents RA 1, RA 2, RA 3, RA 4, OA 1, OA 2: different RA o r OA patients FL S cells; IGFBP2specific p ro tease: hybridized w ith p ro tease fragm ent as p robe; IGFBP2specific Cathep sin B: hybridized w ith cathep sin B fragm ent as p robe; L am inin recep to r: hybridized w ith lam inin recep to r fragm ent as p robe; Β2actin: hybridized w ith Β2actin fragm ent as p robe [ 1 ], ; m RNA, 3,, m RNA 3 po ly (A ) 5 2GGG (b io tin2du T P) ; cdna s,, cdna s, [ 2 ] [ 3 ] : cdna s,, cdna s cdna ;,,,, 8% bp, 36. 7% 400 bp, 6, 23. 6% PCR,, 33. 3%, Β A 2raf,,,,, m RNA cdna cdna s : PCR, PCR ;, cdna 312 PCR, 31211

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