http / /cjbmb. bjmu. edu. cn Chinese Journal of Biochemistry and Molecular Biology A431 . Western aza-dC FUT4-siRNA

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1 ISSN CN / Q http / /cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 842 DOI /j cnki cjbmb FUT4-siRNA 5-aza-dC 1 3 * * IV fucosyltransferase IV FUT4 FUT4-siRNA aza-dC 5-aza-dC FUT4-siRNA A431 SCC12 5-aza-dC FUT4-siRNA MTT 5-aza-dC A431 SCC12 4 d 18% 20% P < 0 05 G 1 S Western A431 FUT4 SCC12 5-aza-dC SCC12 FUT4 A431 FUT4-siRNA FUT4-siRNA FUT4 5-azadC FUT4-siRNA A431 SCC12 54% 60% P < aza-dC FUT4-siRNA 5-aza-dC 5-aza-dC S FUT4-siRNA 5-aza-dC aza-dC FUT4-siRNA Q291 R34 R73-36 FUT4- sirna Increased 5-aza-dC Induced Inhibition on Proliferation and Migration in Squamous Carcinoma Cells 1 LI Hong-Yan 3 * TONG Shao-Ming 1 ZOU Wei 1 3 YAN Qiu 2 * 1 College of Life Science Liaoning Normal University Dalian China 2 Department of Biochemistry and Molecular Biology Dalian Medical University Dalian China 3 Key Laboratory of Biotechnology and Molecular Pharmacy of Liaoning Province Liaoning Normal University Dalian China Abstract Fucosyltransferase IV FUT4 is a key enzyme for the synthesis of fucosylated oligosaccharides RNAi of FUT4 has been reported to suppress the proliferation of squamous carcinoma cells 5-aza-dC is a common drug for squamous carcinoma chemotherapy However it is unknown whether FUT4-siRNA could be used in combination with 5-aza-dC treatment In this study the effect of 5-aza-dC combined FUT4-siRNA treatment on squamous carcinoma proliferation and migration were investigated in A431 and SCC12 cells The results of MTT and flowcytometry assays showed that 18% and 20% of growth inhibition were observed in A431 and SCC12 cells treated with 5 μmol /L 5-aza-dC for 4 days P < 0 05 respectively The cell percentage were decreased in G 1 phase and increased in S phase Western blotting showed that the FUT4 expression was lower but more significantly increased with 5-aza-dC treatment in SCC12 than in A431 cells The results from trypan blue staining and wound * Tel l-hongyan@ 163 com yanqiu63@ 126 com Received December Accepted May * Corresponding author Tel L-hongyan@ 163 com yanqiu63@ 126 com

2 8 FUT4-siRNA 5-aza-dC 837 healing assays indicated that FUT4-siRNA transfection decreased FUT4 expression and amplified the 5- aza-dc inhibition of cell proliferation 54% and 60% in A431 and SCC12 and migration These suggested that FUT4-siRNA increased the proliferation inhibition by 5-aza-dC treatments in squamous carcinoma cells which involved in cell cycle arrest at S phase Key words 5-aza-dC FUT4-siRNA squamous carcinoma cells proliferation migration 5-aza-dC 1 DNA Pliml Stoma 5-aza-dC DNA 1 1 DNA DMEM /F12 5-aza-dC Gibco MTT 5-aza-dC DMSO Sigma Trizol Invitrogen PCR 8 5-aza-dC FUT4 β-actin Santa Cruz FUT4-siRNA A431 ATCC SCC12 Lewis Dr James Lewis a Lea slewis a Rheinwald 10% 100 slea Lewis b Leb Lewis X LeX slewis X U /ml 50 μg /ml DMEM /F12 slex Lewis Y LeY 5% CO 2 37 C fucose 1 2 MTT fucosyltransferase FUT / FUT μmol /L 5-aza-dC FUT4 LeY LeY 1 MTT A Fuc1 2Galβ1 4 Fuc1 3 GlcNAcβ1 R 9 6 d A = A - LeY A / A FUT4 1 3 FUT4 10 cm A μmol /L 5-aza-dC 4 d FUT4 5-aza-dC 80% FUT4 5-azadC FUT4 1 4 FUT4-siRNA 5-aza-dC aza-dc 60% ~ 80% FUT4 FUT4 24 h 5-aza-dC Lipofectamine 2000 FUT4-siRNA 5-aza-dC 6 h FUT4-siRNA FUT4 5 μmol /L 5-aza-dC d 5-aza-dC 2 d3 d4 d FUT4 1

3 RT-PCR Trizol RNA cdna PCR FUT4 F 5'- 1 8 CGGCGTCTTTGTGCCTTAT-3' R 5'-CGAGGAAAAG CAGGTACGAG-3' β-actin F 5'- ATCTGGCACCACACCTTCTACAATGAGCTGCG-3' R 5'-CGTCATACTCCTGCCTGCTGATCCACATCTG C-3' 94 4 min s s s min PCR 5-aza-dC % 6 d MTT d aza-dC A431 SCC12 5-aza-dC 3 d A aza-dC 5-aza-dC FUT4-siRNA P < 0 05 Fig 1 24 h 3 t P < 0 05 P < aza-dC 5 μmol /L 5-aza-dC 6% SCC12 8% 5-azadC 4 d A431 18% SCC12 20% Fig 1 Effect of 5-aza-dC on A431 and SCC12 cell proliferation A431 and SCC12 cells were treated by 5 μmol /L 5- aza-dc for 6 days Cell proliferation was detected by MTT assay with measurement of absorbance at 490 nm every day * 0 05 compared with no-treatment group P < aza-dC S 5-aza-dC FUT4 5-aza-dC 2 Western FUT4 5-aza-dC 2 FUT4 A % ± 1 85% G 0 /G % ± 0 63% S Fig 2 A 5-aza-dC A431 G 0 / G % ± 1 37% S Fig 3A 54 24% ± 0 45% Fig 2 C SCC12 FUT4-siRNA FUT % ± 2 11% G 0 /G % ± 0 98% S Fig 2 B 5-aza-dC FUT4 2 SCC12 G 0 /G % ± 1 49% S 56 79% ± 1 16% Fig 2 D aza-dC FUT4-siRNA FUT4-siRNA 5-aza-dC A431 SCC12 Fig 3A 5 μmol /L 5-aza-dC SCC12 FUT4 A431 FUT4 RT-PCR Western FUT4-siRNA FUT4-siRNA FUT4 mrna Fig 3B FUT4 Fig 3C A431 SCC12 5-aza-dC

4 8 FUT4-siRNA 5-aza-dC 839 Fig 2 Analysis of cell cycle distribution in A431 and SCC12 cells treated by 5-aza-dC A431 and SCC12 cells were treated by 5 μmol /L 5-aza-dC for 4 days The cells were collected and then fixed by cold ethanol for 24 hours The cell cycle distribution was analyzed by flow cytometry assay after RNAse incubation for 30 minutes A Cell cycle distribution in A431 cells B Cell cycle distribution in SCC12 cells C Cell cycle distribution in A431 cells treated by 5-aza-dC D Cell cycle distribution in SCC12 cells treated by 5-aza-dC FUT4-siRNA 5-aza-dC G aza-dC 5-aza-dC FUT4-siRNA S Yuan 15 54% 60% P < 0 05 Fig 3D E aza-dC FUT4-siRNA FUT4-siRNA 5-aza-dC 5-aza- dc FUT4-siRNA DNA aza-dC 5-aza aza- dc FUT4-siRNA 2 dc IL-13Rα aza-dC Fig 4 3 A431 SCC12 5-aza-dC aza-dC Ptch FUT4 FUT4 A431 FUT4-siRNA 5-aza-dC 1 A aza-dC 5-aza-dC FUT4-siRNA

5 Fig 3 Effect of 5-aza-dC combined FUT4-siRNA treatment on the cell proliferation A The cells were treated by 5 μmol / L 5-aza-dC for 48 hours and cell lysates were prepared The proteins in lysates were separated by 10% SDS-PAGE After transferring onto the membrane the blots were probed with anti-fut4 and anti-β-actin antibodies B The cells were plated into 6 well plate and cultured to 80% confluence FUT4-siRNA were transiently transfected into cells Total RNA of cells was extracted and cdnas were obtained by reverse transcription after transfection for 48 hours FUT4 expression was analyzed by PCR C FUT4 expression was analyzed in SCC12 cells by Western blot D E The cells were plated into 24 well plate and cultured to 60% confluence and then transiently transfected with FUT4-siRNA The cells were cultured in medium with 5 μmol / L 5-aza-dC for 3 days and counted by hemocytometer after being dyed by trypan blue everyday β-actin was used as an internal control * P < 0 05 compared with no-treatment group # P < 0 05 compared with treatment group by 5-aza-dC Fig 4 Effect of 5-aza-dC combined FUT4-siRNA treatment on A431 and SCC12 cell migration The cells were treated with 5 μmol / L 5-aza-dC and FUT4-siRNA transfection as described above and cultured to 100% confluence The scratch was made by pipette tip and the cell migration distance was analyzed by taking photograph after 24 hours FUT4 FUT4 FUT4 5-aza-dC SCC12 5-aza-dC HaCaT FUT FUT4 SCC12 5-aza-dC 5-aza-dC FUT4-siRNA

6 8 FUT4-siRNA 5-aza-dC aza-dC SCC12 FUT4-siRNA 5-aza-dC A431 FUT4 9 FUT4-siRNA 5-aza-dC 2 A431 SCC12 Fig 3A A431 FUT4 SCC12 FUT4 A431 5-aza-dC SCC12 FUT4 5-aza-dC FUT4 5-aza-dC FUT4-siRNA FUT4 References 1 Lin J Lai M Huang Q et al Reactivation of IGFBP7 by DNA demethylation inhibits human colon cancer cell growth in vitro J Cancer Biol Ther Xiong H Chen Z F Liang Q C et al Inhibition of DNA methyltransferase induces G2 cell cycle arrest and apoptosis in human colorectal cancer cells via inhibition of JAK2 / STAT3 / STAT5 signalling J J Cell Mol Med B Sun D Toan X Zhang Y et al Mammalian target of rapamycin pathway inhibition enhances the effects of 5-aza-dC on suppressing cell proliferation in human gastric cancer cell lines J Sci China C Life Sci Fang J Y Yang L Zhu H Y et al 5-Aza-2'-deoxycitydine induces demethylation and up-regulates transcription of p16ink4a gene in human gastric cancer cell lines J Chin Med J Valdez B C Li Y Murray D et al 5-Aza-2'-deoxycytidine sensitizes busulfan-resistant myeloid leukemia cells by regulating expression of genes involved in cell cycle checkpoint and apoptosis J Leuk Res Fan H Zhao Z J Cheng Y C et al Gene induction and apoptosis in human hepatocellular carcinoma cells SMMC-7721 exposed to 5-aza-2'-deoxycytidine J Chin Med J Singh K P Treas J Tyagi T et al DNA demethylation by 5-aza-2- deoxycytidine treatment abrogates 17 beta-estradiol-induced cell growth and restores expression of DNA repair genes in human breast cancer cells J Cancer Lett Zhou X Q Huang S Y Zhang D S et al Effects of 5-aza-2' deoxycytidine on RECK gene expression and tumor invasion in salivary adenoid cystic carcinoma J Braz J Med Biol Res Yang X Zhang Z Jia S et al Overexpression of fucosyltransferase IV in A431 cell line increases cell proliferation J Int J Biochem Cell Biol Zhang Z B Sun P Liu J et al Suppression of FUT1 / FUT4 expression by sirna inhibits tumor growth J Biochim Biophys Acta FUT4 J Yang X S Liu S Yan Q Structural analysis of human FUT4 promoter J Chin J Biochem Mol Biol FUT4 J Liu S Fan L X Yang X S et al Overexpression of fucosyltransferase Ⅳ promotes embryonic cells to adhere to uterine cells J Chin J Biochem Mol Biol Li H Tong S Liu J et al Differential fucosyltransferase IV expression in squamous carcinoma cells is regulated by promoter methylation J Cell Mol Biol Lett HaCaT FUT4 J Li H Y Tong S M Yan Q Correlation between FUT4 expression and its promoter methylation in HaCat cells J Hereditas Yuan B Z Jefferson A M Popescu N C et al Aberrant gene expression in human non small cell lung carcinoma cells exposed to demethylating agent 5-aza-2'-deoxycytidine J Neoplasia Patties I Jahns J Hildebrandt G et al Additive effects of 5-aza- 2'-deoxycytidine and irradiation on clonogenic survival of human medulloblastoma cell lines J Strahlenther Onkol Marino A M Frijhoff J Calero R et al Effects of epigenetic modificators in combination with small molecule inhibitors of receptor tyrosine kinases on medulloblastoma growth J Biochem Biophys Res Commun Kim J G Bae J H Kim J A et al Combination effect of epigenetic regulation and ionizing radiation in colorectal cancer cells J PLoS One e Ecke I Petry F Rosenberger A et al Antitumor effects of a combined 5-aza-2' deoxycytidine and valproic acid treatment on rhabdomyosarcoma and medulloblastoma in Ptch mutant mice J Cancer Res Luszczek W Cheriyath V Mekhail T M et al Combinations of DNA methyltransferase and histone deacetylase inhibitors induce DNA damage in small cell lung cancer cells correlation of resistance with IFN-stimulated gene expression J Mol Cancer Ther

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