Experimental Research Chin J Curr Adv Gen Surg Survivin sirna 253 1 2 1 3 1 1 1 1 266003 266003 : survivin sirna survivin sirna BXPC- 3 : BXPC- 3 BXPC- 3 4 survivin sirna survivin sirna RT- PCR survivin mrna Western blot survivin MTT : 72 h survivin sirna survivin mrna 3 P<0.05 survivin sirna 3 P<0.05 :survivin sirna BXPC- 3 survivin mrna BX- PC- 3 RNA Survivin R735.9 A doi 10.3969/j.issn.1009-9905.2012.04.001 1009-9905 2012 04-0253- 05 Inhibitory effect of small interference RNA targeting survivin nanospheres on human pancreatic carcinoma BXPC-3 cell growth PAN Xin- ting 1 ZHU Qing- yun 2 WU Li- qun 1 ZHAO Zhi- hui 3 CAO Jing- yu 1 WANG Zu- sen 1 HU Wei- yu 1 HAN Bing 1 1 Department of Hepatobiliary Surgery 2 Department of Ultrasound the Affiliated Hospital of Medical College Qingdao University Qingdao 266003 China 3 Nano New Material Key Laboratories of Qingdao University Qingdao 266003 China [ ] (2011M500697) [ ] (1974-09~ ) : Tel:15806559602 E-mail:0536pxt@163.com [ ] (1958-10~ ) : Tel:13589268689 E-mail:wulq5810@126.com
2012 4 15 4 254 ABSTRACT Objective: To used nanotechnology and gene interference to block survivin expression in pancreatic carcinoma cell and to study the inhibitory effect of small interference RNA targeting survivin nanospheres on the proliferation and apoptosis of human pancreatic cancer BXPC- 3 cells. Methods: Human pancreatic cancer BXPC- 3 cells cultured in vitro were assigned into four groups: saline gene- free nanospheres sirna- survivin and sirna- survivin nanospheres groups. Survivin mrna expression was detected by RT- PCR; Survivin protein expression was detected by Western blot; Apoptosis of BXPC- 3 cells was determined by flow cytometry; BXPC- 3 cells growth was examined by MTT. Results: At 72 hours after treatment both survivin mrna expression and survivin protein expression in the sirna- survivin nanospheres group were significantly less than other three groups P<0.05. BXPC- 3 cell growth was remarkably inhibited in the sirna- survivin nanospheres group but apoptotic rate was significantly greater than other three groups P<0.05. Conclusion: sirna- survivin nanospheres can specifically reduce both survivin mrna and survivin protein expressions in human pancreatic cancer BXPC- 3 cells significantly increase tumor cell apoptosis and remarkably inhibit BXPC- 3 cell proliferation. KEY WORDS Nanocarrier RNA interference Survivin Cell proliferation Cell apoptosis Pancreatic neoplasms [1-2] (survivin) (in- PLGA/poloxamer : hibitors of apoptosis proteiniap) PLGA poloxamer 2 ml 500 μg sirna ( RNA (RNA interferencernai) PLGA poloxamer 1%~2%) 200 μl 25 ml 21 25 ml 10 min 30 15 8 000 r/min 1 h survivin RNAi 15% 100 U 100 mg/l RPMI-1640 37 5% CO 2 1 0.25% 1.1 BXPC-3 ( 70%~80% );survivin RNA(siRNA 1.2.3 );RNA RT-PCR RPMI-1640 1 (Takara );Western blot MTT Annexin V/PI ( Invitrigon ); survivin sirna ( );RPMI-1640 survivin sirna 6 ( Gibco ); survivin ( survivin sirna 0.8 μmol/l 37 Santa ); - 5% CO 2 72 h [poly (lactic -co -glycolic acid)plga] 1.2.4 RT -PCR survivin mrna (poloxamer) F68( ) Trizol RNA cdna PCR 1.2 1.2.1 survivin sirna 1.2.2 BXPC-3 10 5 / 96 4 survivin GAPDH :94 5 min94 30 s 58 35 s 72
.Survivin sirna 50 s 30 72 10 min PCR 2% 72 h sur- Quantity One vivin mrna survivin sirna mrna 255 1.2.5 Western blot 4 (P<0.05) PBS survivin sirna survivin mrna 3 50 μg 8% SDS-PAGE (P<0.05 2 3) 5% 2 h M 1 2 3 4 5 6 7 8 1% 4 37 1 h 1.2.6 MTT 96 200 μl ( 4 000 / );37 5% CO 2 6 12 24 36 48 72 h 5 g/l MTT 20 2 RT-PCR BXPC-3 μl 4 h survivin mrna 200 μl 10 min 595 nm 2.2 sirna survivin mrna sirna ;78:survivin sirna 1.2.7 (Annexin-V/PI 1 2 3 4 ) 0.25% PBS 3 A 1 10 9 L -1 FITC Annexin_V 10 μl 30 min PI 5 μl 5 B min 3 Western blot BXPC-3 1.3 SPSS13.0 survivin x±s (One-way A:1 ;2 ;3survivin sirna ;4survivin ANOVA) t P 0.05 sirna ;B:β-actin 2.3 MTT 12 h 2 survivin sirna survivin sirna 2.1 survivin sirna survivin sirna 160~220 nm 3 (P<0.05 4) ( 1) 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 survivin sirna ( 20 000) M:Marker;12: ;34: ;56:survivin 4 12 24 36 48 60 72 (h) MTT BXPC-3 A: ; B: ;C:survivin sirna ;D:survivin sirna 2.4 survivin sirna BXPC-3 sur-
2012 4 15 4 256 vivin sirna survivin sirna sirna survivin sirna (4.21±0.32)% (4.38±0.0.29)% (15.72± BXPC-3 survivin sirna 3.12)% (27.57±4.25)% survivin sirna survivin mrna 3 (P< sirna mrna 0.05 5) survivin sirna (P<0.05) 35 5 (%) 30 25 20 15 10 5 0 A B C D BXPC-3 A: ; B: ;C:survivin sirna ; D:survivin sirna 3 survivin survivin sirna BXPC-3 survivin caspase caspase-3caspase-7 [11-12] MTT survivin (P<0.05); survivin sirna (P<0.05) survivin survivin survivin BXPC-3 survivin mrna BXPC-3 BXPC-3 ; survivin survivin survivin [3-5] survivin [1] Parajó Y d'angelo I Horváth A et al. PLGA: poloxamer blend micro-and nanoparticles as controlled release systems for synthetic RNAi RNA proangiogenic factors[j]. Eur J Pharm Sci 201041(5):644-649. RNA Dicer [2] Luo G Jin C Long J et al. RNA interference of MBD1 in BxPC-3 sirna human pancreatic cancer cells delivered by PLGA -poloxamer mrna nanoparticles[j]. Cancer Biol Ther 20098(7):594-598. [6-7] RNAi [3] Sarela AI Verbeke CS Ramsdale J et al. Expression of survivin a novel inhibitor of apoptosis and cell cycle regulatory protein in pancreatic adenocarcinoma[j]. Br J Cancer 200286(6):886-892. Uprichard [8] [4] Lee MA Park GS Lee HJ et al. Survivin expression and its clinical sirna RNA significance in pancreatic cancer[j]. BMC Cancer 20055:127. sirna [5] Guan HT Xue XH Dai ZJ et al. Down-regulation of survivin expression by small interfering RNA induces pancreatic cancer cell apoptosis and enhances its radiosensitivity[j]. World J Gastroenterol 200612(18):2901-2907. [6] Baker M. RNA interference: Homing in on delivery [J]. Nature 2010464(7292):1225-1228. [7] Song H Zhang Z Wang L. Small interference RNA against PTP-1B reduces hypoxia/reoxygenation induced apoptosis of rat cardiomyocytes[j]. Apoptosis 200813(3):383-393. [9-10] [8] Uprichard SL Boyd B Althage A et al. Clearance of hepatitis B H 2 O CO 2 virus from the liver of transgenic mice by short hairpin RNAs[J]. Proc Natl Acad Sci USA 2005102(3):773-778. [9] Blanco E Hsiao A Mann AP et al. Nanomedicine in cancer therapy: innovative trends and prospects[j]. Cancer Sci 2011102 (7): survivin 1247-1252.
.Survivin sirna [10] McNeeley KM Karathanasis E Annapragada AV et al. Masking and triggered unmasking of targeting ligands on nanocarriers to improve drug delivery to brain tumors [J]. Biomaterials 200930 (23-24):3986-3995. [11] Cheung CH Chen HH Kuo CC et al. Survivin counteracts the therapeutic effect of microtubule de -stabilizers by stabilizing tubulin polymers[j]. Mol Cancer 20098:43. [12] Kappler M Taubert H Bartel F et al. Radiosensitization after a combined treatment of survivin sirna and irradiation is correlated with the activation of caspases 3 and 7 in a wt-p53 sarcoma cell line but not in a mt-p53 sarcoma cell line[j]. Oncol Rep 200513 (1):167-172. ( :2011-12-22) ( : ) 257 1009-9905 2012 04-0257- 01 doi 10.3969/j.issn.1009-9905.2012.04.002 23 112000 21 2 20 TMC 1 1 15 d [1] 1~4 (thyroid mi- 3 crocarcinomatmc) TMC 4%~7% 50% 2006 8 2011 8 23 [1] TMC 2006 8 1 1.1 2006 8 2011 8 1783 TMC 23 1.29%(23/1783) 4 19 1 4.7; 37~71 47 23 [2] 13 10 1~3 [3] 23 [4-5] 10% ( ) 13 5 TMC 1.2 ( ) ( ) + 20 5 mm 79.2% TMC [6-7] 3 TMC 1 mm TMC TMC [8] 3 1 ;1 1 TMC ;1 TMC 2 TMC TMC TMC 5 mm 12 (52.2%)>5 mm 11 (47.8%) 21 2 TMC 23 1.29% 23 286