Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 6,

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1 ISSN CN gQ 16 6 Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 6, RNA 3 3 (, ) upa R RNA pu RA S M DA 2M B 2231, G418 N o rthern upa R RNA, R T 2PCR upa R, Boyden upa R RNA, upa R M DA 2M B 2231, upa R, RNA, RNA,, Q Inh ibition of Inva siveness of Human Brea st Cancer Cells by An tisen se RNA for Urok ina se Receptor 3 XU Shao2H ua,l IAO J in2h u i, ZHU Yun2Song 3 3 (D ep artm ent of M olecu lar Genetics, F ud an U niversity M ed ical Center, S hang hai , Ch ina) Abstract U rok inase recep to r an tisen se RNA exp ression p lasm id, pu RA S w as in troduced in to the h igh ly invasive hum an b reast cancer cell line M DA 2M B 2231 w ith lipofectin, and po sitive clones w ere screened ou t w ith G418. N o rthern b lo t m ethod w as app lied to detect the exp ression of an tisen se RNA, and R T 2PCR m ethod w as em p loyed to detect the exp ression of upa R. T he p las2 m inogen activity of cu ltu re supernatan t w as detected w ith m ilk p late m ethod. T he in v itro and in v ivo invasive ab ility of cancer cells w as defined w ith Boyden s cham ber m odel and nude m ice tran sp lan tation, respectively. O ne of the clonesm A S4 w as found to exp ress an tisen se RNA fo r u2 PA R. Its exp ression of upa R and p lasm inogen activity in cu ltu re supernatan t w ere sign ifican tly decreased. T he in v itro invasive ab ility of an tisen se clone w as sign ifican tly com p rom ised in com 2 parison w ith bo th paren tal cell and pcdna 3 vecto r2on ly tran sfected cell. T he in v ivo invasion ex2 perim en t on m ice show ed the tum o rigen icity, grow th and invasiveness of M A S4 had been sign ifi2 can tly inh ib ited. A n tisen se upa R sign ifican tly decreased the invasiveness bo th in v itro and in v i2 vo, as w ell as the tum o rigen icity and grow th of at least a certain k ind of hum an b reast canceṙ T he an tisen se techn ique is expected to becom e one effective m ethod in an ti2invasion therapy of canceṙ Key words U rok inase p lasm inogen activato r recep to r (upa R ), A n tisen se RNA, B reast cancer, N eop lasm invasiveness (upa R ) 3 ( ), upa, upa, 3 3 T el: (021) , Fax: (021) , [ 1, 2 ] upa R [ 3 ] [ 4, PKC N R T K ] upa R E2m ail: yszhu@ shm u. edu. cn,, , : , :

2 6 : RNA 815, actin PCR : : 5 2CCT CTA T GC CAA CA C A GT GC23 ; : 5 2GTA CTC, tpa upa, tpa CT G CT T GCT GA T CC23 T 7 : 5, upa, TAA TA C GA C TCA CTA TA G GG 3, SP6, : 5 2GCA T T T A GG T GA CA C TA T A GA upa, A TA GG23 upa R N o rthern, upa R N o rthern Boeh ringer [ 5, 6 ], M annheim D IG H igh P rim e L abeling and D e2, tection Starter K it 1 RNA R T 2PCR RNA T rizo l upa R, upa, cdna : 20 l RNA 1015 g 4 dn T P ( upa R RNA pu RA S 50 l, cdna [ 7 ], pcdna 3 Invitrogen, 4 l, 30 pm o l, 30 pm o l, 4 L ipofectam IN E G418 DM EM dn T P 200 m o lgl, 10 5 l, T aq Gibco BRL M DA 2M B 2231 Jake Gittlen Cancer R esearch In stitu te D anny W elch R T 2PCR m in P rom ega, T aq Sangon, up2 A R PCR, Boy2 upa den T ran sw ell Co star, upa, PA M atrigel D IG H igh P rim e L a2 beling and D etection Starter K it 1 Boeh ringer M annheim 4 5 BalbgC N ugn u [ 8 ] 24 T ran G418 L ipofect up2 A R RNA pu RA S M DA 2M B G m ggl, G m ggl pcdna 3 M DA 2M B 2231 ( gm l), 800 l DNA DNA N IH 23T 3, 37 5% CO upa R RNA M atrigel, A GC CCT GAA GAA CA G T GC CT, upa R cdna bp R T 2PCR upa R m RNA PCR, : : 5 2TA T AA G CT T CGC GA C A T G GGT CA C C23 : 5 2TA T GGA TCC GGT mm o lgl ) o ligo (dt ) 011 g RN asin 50 AM V 5 5 AM V 4 l, 42 1 h, 95 5 m in m in, 94 1 m in 60 1 m in 72 1 m in 30, upa upa PA, upa Boyden sw ell, 615 mm, 8 m 1414 g (M atrigel) 100 l DM EM, 37 2 h M atrigel 100 l 2 h,,, (400 ) (m i2 cropo rou s m em b rane), 6 : : BalbgC N ugn u, 4 5 M DA 2M B 2 CCA GA G GA G A GT G23 R T 2PCR Β2 231 (M PC)

3 (M A S4) 100%, 10 7 g (m. f. p. ), 6 6, M DA 2M B 2231 g 211 upar RNA (M PC) cm,,, G418 7 (M A S1 M A S7),, 4 m, H E DNA, pcd 2 Bou in s NA 3 T 7 SP h,, PCR, M A S4 [ 9, ], 620 bp (F ig. 1a) T rizo l 2 pu RA S M DA 2M B 2231, M A S4 RNA, upa R cd 2 NA bp N o rthern, upa R RNA (F ig. 1b) F ig. 1 Integration of pu RA S into the genom ic DNA of M DA 2M B2231 cells and the exp ression of upa R antisense RNA w ith N o rthern blo t (a)a fter transfected w ith pu RA S antisense RNA exp ression p lasm id fo r upa R,M DA 2M B2231 cells w ere screened w ith 500 m ggl of G418. Senven resistant clones (C lone 1 though 7)w ere obtained. Genom ic DNA s w ere extracted from these clones. PCR w ere perfo rm ed w ith p rim ers of T 7 and SP6 sequenses. Expected 620 bp band w as detected in C lone 4. (b)n o thern blo t w ere perfo rm ed as described in m ethod to detect the antisense RNA fo r upa R in C lone 4. L ane 1, parental M DA 2M B2231 cells; L ane 2, antisense clone 4 (M A S4) ; L ane 3, vecto r contro l(m PC) 212 upar RNA M A S4 Boyden R T 2PCR (M A S4) up2 M DA 2M B 2231 M PC (P < A R, M A S4 0105), 400 M PC M DA 2M B 2231 upa R :M A S ,M DA 2M B (F ig. 2) 213 upa upa, M PC (M A S4) PA upa M A S4, 3 (F ig. 3) , M PC (F ig. 4) 215 M DA 2M B 2231 M PC, M A S4

4 6 : RNA , 3 5, 1 6, 6 M A S4 (, P < 0105) : M PC 0159 cm g,m A S cm g, M PC (P < 01055) F ig. 4 T he in v itro invasiveness of parental cell, vecto r contro l(m PC) and antisense clone (M A S4) F ig. 2 Dow nregulation of upa R mrna in antisense clone (M A S4) T he sam e amount of RNA w ere app lied fo r RT 2PCR T he RNA s in lane 1 and 4 w ere from parental M DA 2M B2 231 cells. T he RNA s used in lane 2 and 5 w ere from vecto r contro l cells. T he RNA s used in lane 3 and 6 w ere from an2 tisense clone M A S4 cells. RT 2PCR in lane 1, 2 and 3 w ere perfo rm ed w ith p rim ers fo r internal contro l. RT2PCR in lane 4, 5 and 6 w ere perfo rm ed w ith p rim ers fo r upa R. M w as m arker, <X174gH aeg M PC M A S4 T ab le 1,M A S4 M PC (P = 01025), M PC, (F ig. 5A, C);, M A S4 (F ig. 5B,D ) (M PC) 3313% (2g6), (F ig. 5E) Table 1 Comparison of invasiveness of anti2sense clone to vecto r contro l cells Invasiveness M PC (n= 6) M A S4 (n= 5) N g 0 1 g 1 2 g 3 1 g 2 0 F ig. 3 T he activities of p lasm inogen activato r w ere detect2 ed w ith m ilk p late 1 7: tpa standard ( IU gm l) : 120, 60, 30, 15, 7. 5, 3. 7, 1. 8; 8 10: M DA 2M B2231; 11 13: A ntisense clone (M A S4 ) ; 14 16: V ecto r contro l(m PC) ; 8, 11, 14: A ntibody free; 9, 12, 15: A nti2tpa ; 10, 13, 16: A nti2upa 3 [ ],, upa R [ 11 ], upa R RNA Kook [ 12 ] Yu [ 13 ] upa R RNA H ep 3,, 10, Go upa R [ 9 ] RNA, 0 g g g g 95D, upa R

5 F ig. 5 T he invasiveness of vecto r contro l and anti2sense cells in nude m ice T he figures show vecto r contro l(m PC) cells have invaded to the ep iderm is (A )w h ile the anti2sense cells (M A S4) have no t in2 vaded derm is yet (B) (H E stain, 100 ). T he figures also show vecto r contro l (M PC) cells have invaded to the m uscle (C) w h ile the M A S4 cells rem ained in situ (D ) (H E stain, 100 ). T he angiogenesis of vecto r contro l(m PC) in nude m ice (E, H E stain, 200 ) RNA, ( ) [ 14, upa R RNA 4 ] : (1) M DA 2M B 2231 m RNA, M A S4 m RNA ; (2), RNA pu RA S., N o rthern M A S4 ; (3), RNA, m RNA upa R, H, RNA 2DNA M A S4, RNA ; (4), (M PC ) RNA GU C CU C,M A S4 RNA, RNA, RNA RNA [ 14, upa R ],,, upa R RNA m RNA RNA,

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