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河腾禹
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1 Chinese Journal of Cell Biology 216, 38(1): DOI: /cjcb /STAT3 ( 1,, 332; 2, 336; 3, 336) (interleukin-6, )/STAT3(signal transducer and activator of transcription 3, STAT3), ELISA (human umbilical cord-derived mesenchymal stem cells, huc-mscs) Saos-2-6 Western blot Saos-2 STAT3 p-stat3 RT-PCR PCNA CyclinD1 Survivin STAT3 CCK-8 Saos-2 Transwell Saos-2, huc-mscs Saos-2 ; huc-mscs (conditioned medium, ) STAT3, huc-mscs- ; STAT3, Saos-2 PCNA CyclinD1 Survivin mrna,,, STAT3 ; ; 6; STAT3; ; huc-mscs Promote the Proliferation and Migration of Osteosarcoma Cells through /STAT3 Signaling Hu Wenlong 1,2,3, Yin Changchang 1, Wu Pingping 2, Wu Yahua 1, Wang Lili 1, Ke Xiumei 1 ( 1 Jiujiang Key Laboratory of Translational Medicine, Basic Medical College, Jiujiang University, Jiujiang 332, China; 2 Medicine Graduate School, Nanchang University, Nanchang 336, China; 3 Department of Orthopedics, the Second Affi liated Hospital of Nanchang University, Nanchang 336, China) Abstract This work was aimed to study the effects of /STAT3 signaling on the proliferation and migration of osteosarcoma cells. The expressions of interleukin-6 () in human umbilical cord-derived mesenchymal stem cells (huc-mscs) and Saos-2 cells were assayed by ELISA. The protein levels of STAT3 and p-stat3 in extracts from Saos-2 cells were determined by Western blot. RT-PCR was used to determine the transcription levels of PCNA, CyclinD1, Survivin and STAT3 genes. The proliferation of Saos-2 cells was measured by cytometry and CCK-8 assay, and migration potential by wound-healing assay and Transwell assay. The results demonstrated that the expression of in huc-mscs was obviously higher than that in Saos-2 cells. STAT3 was activated by conditioned medium from huc-mscs (huc-mscs-) and and the activation could be : : ( : 2142BBG718) Tel: , yinchangchang112@163.com Received: September 1, 215 Accepted: December 15, 215 This work was supported by the Foundation of the Science and Technology Planning Project of Jiangxi Province (Grant No.2142BBG718) Corresponding author. Tel: , yinchangchang112@163.com : :54:16 URL:

2 : /STAT3 25 attenuated by a neutralization antibody against. The inhibition of STAT3 in Saos-2 cells by neutralizing antibody or down-regulated the levels of mrnas of PCNA, CyclinD1 and Survivin proliferation related genes related to proliferation, decreased cell proliferation and migration. The results proved that STAT3 activation by from huc-mscs might promote the proliferation and migration of osteosarcoma cells in vitro. Keywords umbilical cord-derived mesenchymal stem cells; osteosarcoma; interleukin-6; STAT3; proliferation; migration,,,, [1-3] (human umbilical cord-derived mesenchymal stem cells, huc-mscs),, [4], [5] [6], huc-mscs [7],,, -6(interleukin-6, ) [8],,, [9] α (R) β (gp13) gp13,, 2(janus kinase 2, JAK2)/ 3(signal transducer and activator of transcription 3, STAT3) [1] STAT3 [11], STAT3, [12] Ryu [13], STAT3, p-stat3,, huc- MSCs, huc-mscs JAK2/STAT3 huc-mscs, huc-mscs JAK2/STAT ,,, 1.2 α-mem.25% -EDTA Gibco -6(recombinant human interleukin-6, rh) Peprotech Selleckchem.1% STAT3 p-stat3 Cell Signaling Teachnology GAPDH Abcam HiFi-MMLV cdna -8(cell counting kit-8, CCK-8) GREENspin RNA 2 Taq Master Mix 25 cm Transwell 24 Corning Nikon BioTek huc-mscs huc-mscs 1% FBS α-mem huc-mscs, 8%~9%, 3 1 5, huscs CD19 CD29 CD9 CD15

3 huc-mscs (conditioned medium, ) 5 huc-mscs, /ml, 3 ml T25, α-mem, 24 h, 3 r/min 2 min,.22 μm, 8 C (enzyme-linked immunosorbent assay, ELISA) Saos-2 huc-mscs, ELISA Saos-2 huc-mscs RNA RT-PCR Saos / 6, 6 (1) : ; (2) : 2 μg/l rh ; (3) : 4% huc-mscs- ; (4) : 2 mg/l 4% huc-mscs- ; (5) : 5 μmol/l 4% huc-mscs- ; (6) : 5 μmol/l 24 h, RNA, cdna PCR, 1% RNA PCR β-actin, : PCNA CyclinD1 Survivin STAT3, Western blot STAT3 Saos-2, 8%, 3 min,, BCA SDS-PAGE ( 3~5 μg),, 4 C, 2 h, X-, GAPDH, STAT3 p-stat CCK-8 : Saos / 6, 6, 1.3.4, 2 ml, 4, CCK-8 : Saos-2 1 / 96, 6,, 4, 2 μl 6 d 9 d 1, 1 μl CCK-8 37 C 2 h, 45 nm (D) Transwell Saos-2 6, 8%, 48 h,, α-mem, /ml 1 μl, 6 μl 1% FBS α-mem, 37 C 12 h 1%, Saos-2 6, 8%, 6, 2 μl,, 37 C 5% CO 2, 24 h 1.4 3, ± Genes β-actin PCNA CyclinD1 Survivin STAT3 1 Table 1 Sequence of primers and product length Sequences of primers Forward: 5 -CGG GAA ATC GTG CGT GAC-3 Reverse: 5 -TGG AAG GTG GAC AGC GAG G-3 Forward: 5 -CGG TTA GAA GGG GTT A-3 Reverse: 5 -GAC GGT CTC GGT GTG T-3 Forward: 5 -GTGCTGCGAAGTGGAAACC-3 Reverse: 5 - ATC CAG GTG GCG ACG ATC T-3 Forward: 5 -AGG ACC ACC GCA TCT CTA CAT-3 Reverse: 5 - AAG TCT GGC TCG TTC TCA GTG-3 Forward: 5 -CCT GAA GCT GAC CCA GGT AG-3 Reverse: 5 -TTC CAA ACT GCA TCA ATG AAT C-3 Product length 443 bp 36 bp 174 bp 118 bp 133 bp

: /STAT3 27 A B C 2 μm D 2 μm 2 μm 2 μm A: huc-mscs 6 d; B: huc-mscs 14 d; C: 1 huc-mscs 2 d; D: 5 huc-mscs 3 d A: huc-mscs at primary cultured for 6 days; B: huc-mscs at primary cultured for 14

1 huc-mscs 1~14 d,,,, ( 1), CD29 CD9 CD15(>95%), CD19(.1%), 2.2 huc-mscs- Saos-2 STAT3 p-stat3 2 μg/l rh 4% huc-msc- Saos-2 3 min, p-stat3 STAT3, ELISA, huc- MSCs- (1 84.8±152.2) ng/l, Saos-2 (17.

4 : /STAT3 27 A B C 2 μm D 2 μm 2 μm 2 μm A: huc-mscs 6 d; B: huc-mscs 14 d; C: 1 huc-mscs 2 d; D: 5 huc-mscs 3 d A: huc-mscs at primary cultured for 6 days; B: huc-mscs at primary cultured for 14 days; C: huc-mscs at passage 1 cultured for 2 days; D: huc- MSCs at passage 5 cultured for 3 days. 1 huc-mscs Fig.1 Morphology observation of huc-mscs, SPSS 19., Scheffe,, P< huc-mscs 1~14 d,,,, ( 1), CD29 CD9 CD15(>95%), CD19(.1%), 2.2 huc-mscs- Saos-2 STAT3 p-stat3 2 μg/l rh 4% huc-msc- Saos-2 3 min, p-stat3 STAT3, ELISA, huc- MSCs- (1 84.8±152.2) ng/l, Saos-2 (17.1±22.5) ng/l ( 2), p-stat3, STAT3, JAK2, Saos-2 STAT3, Concentration of (ng/l) Saos huc-mscs Saos-2 3 ml DMEM 24 h, ELISA n=4, P<.5, Saos huc-mscs and Saos-2 cells were grown in 3 ml serum-free DMEM for 24 h, and the level in the supernatant was measured by ELISA. n=4, P<.5 vs Saos-2 cell group. 2 ELISA Saos-2 huc-mscs Fig.2 The expressions of in Saos-2 and huc-mscs detected by ELISA STAT3, (P<.5) STAT3 ( 3) 2.3 STAT3 Saos-2 RT-PCR, PCNA CyclinD1 Survivin, huc-mscs

28 GAPDH β-actin PCNA STAT3 Survivin p-stat3 CyclinD1 Relative proten expression 1..8.6.4.2 STAT3 p-stat3 Relative mrna expression.8.6.4.2 PCNA Survivin CyclinD1 STAT3 STAT3 Western blot Saos-2 3 min STAT3 p-stat3 n=3, P<.

5 vs group. 3 huc-mscs- Saos-2 STAT3 p-stat3 Fig.

5 28 GAPDH β-actin PCNA STAT3 Survivin p-stat3 CyclinD1 Relative proten expression STAT3 p-stat3 Relative mrna expression PCNA Survivin CyclinD1 STAT3 STAT3 Western blot Saos-2 3 min STAT3 p-stat3 n=3, P<.5, ; P<.5, ; P<.5, The expressions of STAT3 and p-stat3 protein in the different groups of Saos-2 cells cultured for 3 min were analyzed by Western blot. n=3, P<.5 vs control group; P<.5 vs group; P<.5 vs group. 3 huc-mscs- Saos-2 STAT3 p-stat3 Fig.3 Effects of and huc-mscs- on the expressions of STAT3 and p-stat3 protein in Saos-2 cells Saos-2 24 h, RNA RT-PCR PCNA CyclinD1 Survivin STAT3 mrna n=3, P<.5, ; P<.5, ; P<.5, Total RNA was extracted from different groups of Saos-2 cells cultured for 24 h, and the expressions of PCNA, CyclinD1, Survivin and STAT3 mrna were detected by RT-PCR. n=3, P<.5 vs control group; P<.5 vs group; P<.5 vs group. 4 STAT3 Saos-2 Fig.4 Effects of and STAT3 on the expressions of proliferation related genes in Saos-2 cells (A) Cells/well ( 1 4 ) (B) D (A) CCK-8 (B) 6 d 9 d Saos-2 n=4, P<.5, ; P<.5, ; P<.5, The proliferation of Saos-2 cells in different groups were measured at 6 d and 9 d with cytometry (A) and CCK-8 assay (B). n=4, P<.5 vs control group; P<.5 vs group; P<.5 vs group. 5 STAT3 Saos-2 Fig.5 Effects of and STAT3 on the proliferation of Saos-2 cells

: /STAT3 29 (A) (B) 8 Cells/field 6 4 2 (C) A: Saos-2 24 h,, Transwell, 12 h ( =5 μm); B: Transwell ; C: ( =2 μm) n=5, P<.5, ; P<.

6 : /STAT3 29 (A) (B) 8 Cells/field (C) A: Saos-2 24 h,, Transwell, 12 h ( =5 μm); B: Transwell ; C: ( =2 μm) n=5, P<.5, ; P<.5, ; P<.5, A: Saos-2 cells in different groups were cultured for 24 h, then cells were suspended in complete medium and plated onto upper chamber of a transwell plate for 12 h. Cells migrated to the undersurface were fixed, stained and counted (Scale bars=5 μm). B: the number of migratory cells. C: woundhealing assay (Scale bars=2 μm). n=5, P<.5 vs control group; P<.5 vs group; P<.5 vs group. 6 STAT3 Saos-2 Fig.6 Effects of and STAT3 on the migration of Saos-2 cells, STAT3 ( 3), Saos-2 PCNA CyclinD1 Survivin mrna, rh, (P<.5) STAT3 24 h, ( 4) 2.4 STAT3 Saos-2 CCK-8, huc-mscs- Saos-2, D Saos-2,, (P<.5)( 5) 2.5 STAT3 Saos-2 2 μg/l rh 4% huc-msc- Saos-2 24 h, (P<.5);,, (P<.5); (P<.5)( 6A 6B), huc-msc- Saos-2,,, Transwell Saos-2 JAK2/ STAT3, huc-msc-,

7 3 ( 6C) 3 Chang [14],, huc-mscs, Saos-2 1, huc- MSCs, CCK-8 Transwell, huc-mscs- Saos-2,,, huc-mscs, JAK2/STAT3 [1], STAT3, CyclinD1 Survivin Cdc2 Bcl-2 HIF1α Hsp9 VEGF(vascular endothelial growth factor) [15], STAT3 mir-2c, [16], STAT3, huc-mscs- Saos-2 STAT3,, STAT3 huc-mscs-, STAT3, huc-mscs Saos-2 STAT3 ; +anti- p-stat3 huc- MSCs- STAT3 JAK2, STAT3, Lck Lyn Btk Syk Src [17], JAK2/STAT3 [18] STAT3 huc-mscs, STAT3,, huc-mscs, Saos-2 STAT3, STAT3 Bid [19], STAT3 LLL12 (VEGF) -9(matrix metalloproteinase-9, MMP-9) -1(fibroblast growth factor-1, FGF-1), Onimoe [2] LLL12 STAT3,,, huc-mscs- Saos-2 PCNA CyclinD1 Survivin, huc-mscs Saos-2 huc-mscs- Saos-2 ( )STAT3, PCNA CyclinD1 Survivin, huc-mscs STAT3 Saos-2 huc-mscs- STAT3,,, JAK2/STAT3, Saos-2 STAT3, rh STAT3 Saos-2 ; STAT3, Saos-2, JAK2/STAT3 huc-mscs Saos-2, (P>.5),,, Saos-2, +anti-il6 Saos-2, huc-mscs, huc-mscs Saos-2,, JAK2/STAT3 huc-mscs, STAT3

8 : /STAT3 31, huc-mscs, huc-mscs, huc-mscs (References) 1 Uchibori R, Tsukahara T, Ohmine K, Ozawa K. Cancer gene therapy using mesenchymal stem cells. Int J Hematol 214; 99(4): Bolontrade MF, Sganga L, Piaggio E, Viale DL, Sorrentino MA, Robinson A, et al. A specific subpopulation of mesenchymal stromal cell carriers overrides melanoma resistance to an oncolytic adenovirus. Stem Cells Dev 212; 21(14): Ryu CH, Park SH, Park SA, Kim SM, Lim JY, Jeong CH, et al. Gene therapy of intracranial glioma using interleukin 12- secreting human umbilical cord blood-derived mesenchymal stem cells. Hum Gene Ther 211; 22(6): Petsa A, Gargani S, Felesakis A, Grigoriadis N, Grigoriadis I. Effectiveness of protocol for the isolation of Wharton s Jelly stem cells in large-scale applications. In Vitro Cell Dev Biol Anim 29; 45(1): Prasanna SJ, Gopalakrishnan D, Shankar SR, Vasandan AB. Proinflammatory cytokines, IFNgamma and TNFalpha, influence immune properties of human bone marrow and Wharton jelly mesenchymal stem cells differentially. PLoS One 21; 5(2): e Baksh D, Yao R, Tuan RS. Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells 27; 25(6): Friedman R, Betancur M, Boissel L, Tuncer H, Cetrulo C, Klingemann H. Umbilical cord mesenchymal stem cells: Adjuvants for human cell transplantation. Biol Blood Marrow Transplant 27; 13(12): Candido J, Hagemann T. Cancer-related inflammation. J Clin Immunol 213; 33 Suppl 1: S79-S84. 9 Liu X, Wang J, Wang H, Yin G, Liu Y, Lei X, et al. REG3A accelerates pancreatic cancer cell growth under -associated inflammatory condition: Involvement of a REG3A-JAK2/STAT3 positive feedback loop. Cancer Lett 215; 362(1): Garbers C, Aparicio-Siegmund S, Rose-John S. The / gp13/stat3 signaling axis: Recent advances towards specific inhibition. Curr Opin Immunol 215; 34: Mali SB. Review of STAT3 (Signal Transducers and Activators of Transcription) in head and neck cancer. Oral Oncol 215; 51(6): Salas S, Jiguet-Jiglaire C, Campion L, Bartoli C, Frassineti F, Deville J L, et al. Correlation between ERK1 and STAT3 expression and chemoresistance in patients with conventional osteosarcoma. BMC Cancer 214; 14(66): Ryu K, Choy E, Yang C,Susa M, Hornicek FJ, Mankin H, et al. Activation of signal transducer and activator of transcription 3 (Stat3) pathway in osteosarcoma cells and overexpression of phosphorylated-stat3 correlates with poor prognosis. J Orthop Res 21; 28(7): Chang Q, Daly L, Bromberg J. The feed-forward loop: A driver of tumorigenesis. Semin Immunol 214; 26(1): Bournazou E, Bromberg J. Targeting the tumor microenvironment: JAK-STAT3 signaling. JAKSTAT 213; 2(2): e Rokavec M, Wu W, Luo JL. IL6-mediated suppression of mir- 2c directs constitutive activation of inflammatory signaling circuit driving transformation and tumorigenesis. Mol Cell 212; 45(6): Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia E, Lapidot Z, et al. Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor. Nature 1996; 379(6566): Teng Y, Ghoshal P, Ngoka L, Mei Y, Cowell JK. Critical role of the WASF3 gene in JAK2/STAT3 regulation of cancer cell motility. Carcinogenesis 213; 34(9): Bid HK, Oswald D, Li C, London CA, Lin J, Houghton PJ. Antiangiogenic activity of a small molecule STAT3 inhibitor LLL12. PLoS One 212; 7(4): e Onimoe GI, Liu A, Lin L, Wei CC, Schwartz EB, Bhasin D, et al. Small molecules, LLL12 and FLLL32, inhibit STAT3 and exhibit potent growth suppressive activity in osteosarcoma cells and tumor growth in mice. Invest New Drugs 212; 3(3):

932 SUZHOU UVIVERSITY JOURNAL OF MEDICAL SCIENCE E1A inhibite H22 ascitic hepatoma obviously and almost has no side effect on immune functio

932 SUZHOU UVIVERSITY JOURNAL OF MEDICAL SCIENCE E1A inhibite H22 ascitic hepatoma obviously and almost has no side effect on immune functio 2010 30 5 931 Ad-E1A 215123 Ad-E1A BALB /c Ad-E1A RT-PCR E1A H22 BALB /c H22 PBS Ad-E1A 5-Fu 7 10 9 pfu /ml Ad-E1A Ad-E1A 5-Fu 66. 7% 68. 8% P > 0. 05 Ad-E1A PBS 5-Fu P < 0. 01 P < 0. 05 Ad-E1A PBS 5-Fu