OPN antisense oligodeoxynucleotide inhibits p roliferation and apop tosis of human breast carcinoma cell line

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1 Basic & C linical M edicine August 2009 Vol. 29 No. 8 : ( ) , 2, 3, 4, 5 (11, ; 21, ; 31, ; 41, ; 51, ) : (OPN ) mrna (ASODN ) O PN, MCF27, O PN mrna ASODN, OPN MCF27; MTT ; ; RT2PCR O PN mrna ; O PN ASODN MCF27 ( P < 0105) ; 16 mol/l O PN ASODN 72 h, ; O PN mrna ; ( P < 0101) O PN ASODN MCF27 ; : ; ; ; ; : R 34 : A OPN antisense oligodeoxynucleotide inhibits p roliferation and apop tosis of human breast carcinoma cell line ZHOU Yun2song 13, W EN Xiao2hui 2, YANG L in2xi 3, CHEN Peng 4, 5 (11Medical School of Northwest University for Nationalities, Lanzhou ; 21Gansu College of Traditional Chinese Medicine, Lanzhou ; 31School of Basic Medical Sciences, Lanzhou University, Lanzhou ; 41School of L ife Sciences, Lanzhou University, Lanzhou ; 51Commercial Science and Technology Institute of Gansu Province, Lanzhou , China) Abstract: O bjective To investigate the effects on anti2exp ression of O steopontin (OPN ) by an antisense oligode2 oxynucleotide (ASODN) targeting to low free energy region of O PN mrna and its effects on the p roliferation and apop tosis of human breast carcinoma cell line MCF27. M ethods Designing and synthesizing an ASODN based on m inimum free energy algorithm in vitro, which targets to the low free energy region of O PN mrna. Transfecting it into breast carcinoma cell line MCF27 which exp resses OPN in high level. The cell p roliferation2inhibitory rate was determ ined by M TT method; The morphologic change was observed through transm ission electron m icroscope ( TEM ) ; The O PN mrna exp ression level was checked by RT2PCR method; Cell cycle and apop tosis rate were detected by flow cytometry ( FCM ) after transfection respectively. Results O PN ASODN inhibited the p roliferation of the cells by a time and concentration dependent manner ( P < 0105). Apop tosis specific changes was observed after incubated with 16 mol/l O PN ASODN for 72 h. O PN mrna exp ression in ASODN group s were notably : : ( correspond ing author) : zys887799@126. com

2 (8) Basic & ClinicalMedicine 851 decreased. There was a significant high apop tosis rate in ASODN group s ( P < 0101). Conclusion OPN ASODN transfection significantly inhibits the p roliferation of hum an breast carcinom a MCF27 cells and induces cell apop tosis in vitro. Itπs a reliable method to design ASODN based on m inim um free energy algorithm. Key words: osteopontin; antisense oligodeoxynucleotide; free energy; breast carcinoma; apop tosis (osteopontin, OPN), Senger [ 1 ] 1979,, OPN, [ 2 ] OPN,, [ 3 ] sirna GC9811, O PN mrna [ 4 ] RNA, [ 5 ], OPN [ 6 ],, RPM I 1640 ( ), (MTT) RNA RT2 PCR ( ) MCF MCF U /ml 100 g/l 10% RPM I 1640, 37 5% CO 2, (ASODN ) (MSODN ) ( ) ( ASODN) O PN mrna : 5 2GCAGCCTTCTCAG 113 ASODN, RNA structure ( v413 ) O PN mrna ASODN, 5 2GGTCTGTGGGGCTAGG AGAT23, 5 2GTGAGGCTGAGGATGTTG GC23,, Genbank 114 M TT MCF L , 200 L, 50% 60%,, RPM I 1640, mol/l, 12 h 200 L, h MTT ( 5 g/l ) 50 L /, 4 h, DMSO 150 L /, 10 m in, 490 nm (A ) h ( L - 1 ), PBS 2, 3% 48 h, 1% 90 m in (4 ), Epon 812, (JEM 21230, ), Gaton CCD h ( L - 1 ), PBS 2, 70%, PBS 2, RNaseA ( 0105 g/l) 30 m in, 4, ( P I, 50 g/l ) 30 m in,, 117 ( RT2PCR) O PN m RNA, RNA RNA, AMV RT2PCR CCAAAC23, : 5 2GGTTGCTGGCAGGTC CG23, O PN 430 bp; 2actin mrna : 5 2AGCGGGAAATCGTGCGTG23,

3 852 Basic & ClinicalMedicine (8) : 5 2CAGGGTACATGGTGGTGCC23, 2actin 309 bp 2%, (BTS220M, Uvitec ) 118 SPSS1010, x s, O PN ASOD N M CF27 O PN ASODN MCF27 ; ASODN 72 h ASODN MCF27 ( P < 0101) ;,MSODN ( 1) 16 mol/l 72 h 212 O PN ASOD N M CF27,,,, ;,, ( 1) 213 O PN ASOD N M CF27 DNA, O PN ASODN,, G 0 /G 1 (7517% ), S, (1617% ), G 0 /G 1 ; ( 2, 2) 214 O PN ASOD N M CF27 O PN m RNA 430 bp 1 M TT O PN ASOD N M CF27 Table 1 M TT reduction a ssay detected inh ib ition ra tio of M CF27 cell tran sfected by O PN ASOD N a t d ifferen t tim e( x s, n = 4) group ( mol/l) 24 h 48 h 72 h 96 h control MSODN ASODN ASODN ASODN ASODN P < 0105, P < 0101 compared with control 1 16 m ol/l AS2OD N 72 h M CF27 F ig 1 M orpholog ica l changes of M CF27 cells, exam ined by electronm icroscope in concen tra tion of 16 m ol/l of ASOD N in 72 h A1control( 6 000) ; B1MSODN ( ) ; C1ASODN ( 6 000)

4 (8) Basic & ClinicalMedicine Table 2 The percen tage of the d ifferen t groups cell cycle( x s, %, n = 3) group s G 0 /G 1 S G 2 /M Sub2G 1 control ASODN MSODN P < 0101 compared with control 2 FCM M CF27 D NA F ig 2 The percen tage of M CF27 cell D NA and cycle d iv ision m ea sured by FCM A1control; B1MSODN; C1ASODN O PN, Band Leader V1310, O PN 2actin O PN mrna / 2actin mrna ( 430 bp ) = 8611% ; O PN mrna / 2actin mrna (309 bp) = 9517%,, ASODN O PN ( 3) 3 M CF27 O PN m RNA RT2PCR ig 3 RT2PCR detection of O PN m RNA expression in M CF27 cell lines M1DNA marker; 11M SODN; 21ASODN; 31control 3 MCF27 O PN mrna, OPN,, ASODN,, 16 mol/l, 72 h, 96 h, MSODN O PN ASODN MCF27, RT2 PCR ASODN O PN mrna O PN mrna,, OPN,, ASODN mrna ( accessible site) RNA structure O PN mrna,, ASODN, ASODN,

5 854 Basic & ClinicalMedicine (8) : [ 1 ] Senger DR, W irth DF, Hynes RO. Transformed mammali2 an cells secrete specific p roteins and phosphop roteins [ J ]. Cell, 1979, 16 (4) : [ 2 ],,. 65 [ J ]., 2007, 13 (1) : [ 3 ] B ramwell VH, Doig GS, Tuck AB, et al. Serial p lasma os2 teopontin levels have p rognostic value in metastatic breast cancer [ J ]. Clin Cancer Res, 2006, 12 (11 Pt 1) : [ 4 ],,,. sirna GC9811 [ J ]., 2005, 26 (3) : [ 5 ],,,. RNA [ J ]., 2008, 7 (2) : 1-2. [ 6 ],. : [ J ]., 2006, 27 (12) : BBC (BBC NEW S) ,,,, (perfluorinated chem icals, PFCs), 12 Aarhus Jorn O lsen,, 75% PFCs,,, O lsen, ; PFC O lsen 1 240, 4 14 ; 12, , O lsen, 4, 4 2 PFCs : PFOS(, perfluorooctane sulfonate) PFOA (, perfluorooctanoate) ; PFOS, 3 PFOS, 12 ;, PFOS PFOA, ;, PFOA 12 PFOA 2 O lsen,,, O lsen,, O lsen, 10,,,, O lsen,,, Human Rep roduction