Chinese Journal of Biochemistry and Molecular Biology. psilencer 2. 0-FucT Ⅶ 2

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1 ISSN CN / Q Chinese Journal of Biochemistry and Molecular Biology ~ 294 RNA FucT VII HT-29 HUVECs * RNA RNAi Ⅶ FucT Ⅶ HT-29 HUVEC 3 FucT Ⅶ RNAi HT-29 Western FucT Ⅶ slex PCR FucT Ⅶ mrna RNAi HT-29 HUVECs 3 FucT Ⅶ sirna HT-29 FucT Ⅶ mrna psilencer 2 0-FucT Ⅶ 2 psilencer 2 0-FucT Ⅶ HT-29 slex psilencer 2 0-FucT Ⅶ 2 RNA FucT Ⅶ HT-29 HUVEC RNAi HT-29 FucT Ⅶ slex HT- 29 HUVECs RNA Q78 Knock-down of FucT Ⅶ Expression with RNAi Inhibits the Adhesion of Human Colon Carcinoma HT-29 Cells to HUVEC Cells 1 YUE Li-Ling 2 FAN Li 1 NIU Ying-Cai 1 LIU Ji-Cheng 1 * 1 Department of Biological Heredity Institute of Medicine Qiqihar Medical College Qiqihar 2 Heilongjiang University of Chinese Medicine Harbin China China Abstract In this study the effect of FucT Ⅶ expression inhibition with RNA interference RNAi on the adhesion of human colon carcinoma HT-29 cells to HUVECs was investigated Three pairs of sirna were cloned into psilencer 2 0 vectors and transfected into human colon carcinoma HT-29 cells The FucT Ⅶ mrna levels were tested by real-time PCR The adhesion of transfected HT-29 cells to HUVECs was assayed by the Rose-Bengal method The slex protein levels downstream of FucT Ⅶ were assayed by Western blot The results shown that three sirna pairs effectively inhibited the FucT Ⅶ and the slex expression especially with the psilencer 2 0-FucT Ⅶ 2 The RNAi-mediated down-regulation of FucT Ⅶ significantly inhibited the ability of HT-29 cells to adhere to HUVECs Key words RNA interference RNAi fucosyltransferase sialyl-lewis X slex cell adhesion No No QC08C28 * Tel qyybliu@ 126 com Received September Accepted December Supported by National Natural Science Foundation of China No and Youth Science Foundation of Heilongjiang Province No QC08C28 * Corresponding author Tel qyybliu@ 126 com

2 3 RNA FucT Ⅶ HT-29 HUVECs DNA 3 Ⅶ DNA psilencer 2 0-U6 sirna fucosyltransferase Ⅶ FucT Ⅶ BamH Ⅰ Hind Ⅲ DH5α 5 ~ 6 sialyl-lewis X slex 1 3 sirna HT-29 4 slex HT % McCoy's FucT Ⅶ 5A 24 h cell / 6 80% 5 FucT Ⅶ sirna HT-29 Lipofectamine TM 2000 RNAi HT-29 FucT Ⅶ mrna shrna psilencer HT-29 RNA 2 0-FucT Ⅶ 1 psilencer 2 0-FucT Ⅶ 2 FucT Ⅶ psilencer 2 0-FucT Ⅶ psilencer 2 0-U6 sirna expression Vector 1 4 PCR FucT Ⅶ mrna Ambion HT h Trizol reagent human umbilical endothelial cells HUVEC ATCC DH5α cdna 4 μl Omega cdna PCR PCR LineSilence RNAi Kit Allele Lipofectamine2000 Trizol Invitrogen TAC CT-3 5 -GCC TGA AAC CAA CCC BamHⅠ HindⅢ TaKaRa Gibco McCoy's 5A F12K Rose Bengal Sigma RT-PCR slex 226 bp PCR s 95 Calbiochem FucT Ⅶ 5 s s 40 HRP Ig Santa TNF-α R D 1 5 Western FucT Ⅶ slex 48 h HT sirna PBS psilencer 2 0-U6 sirna 20 min g 5 min sirna 6 GenBank 3 BCA 20 μg FucTⅦ NM FucT Ⅶ 1 5 -GCTGAGCACAGTACATCT TCA-3 FucT Ⅶ 2 5 -CGCGACTACATTACGG AGAAA-3 FucT AATCT-3 21 Ⅶ 3 5 -GCTGCCGTGATTTGAG min / 7 CTCTTGA h TBST 3 10 TTTTTT min ECL X β BamHⅠ Hind Ⅲ blast Lipofectamine TM nmol /l 4 h 48 h RNA 20 μl RT 1 μg RNA FucT Ⅶ 5 -GCC CAG TAC CGC TTC TCA A bp GAPDH 5 - GAA GGT GAA GGT CGG AGT C GAA GAT GGT GAT GGG ATT TC-3 ABI7300 PCR PCR SDS-PAGE 5 % 2 h FucT Ⅶ /slex TBST 3 10

3 A FucT Ⅶ /β-actin = A HT-29 HUVEC A 570 / A % HUVEC /100 μl 96 HUVEC 5 SPSS 13 0 ng /ml TNF-α 37 4 h 7 x珋 s HT /200 μl 37 one-way ANOVA P < r /min 20 min PBS μl 0 25% 5 min 2 10% McCoy's 5A 2 95% PBS μl 2 1 FucT Ⅶ -sirna 30 min A A 570 = psilencer 2 0-U6 Fig 1 A A 570 sirna = A 570 / A % 1 7 Fig 1 Identification of recombinant plasmid digested by BamH Ⅰ and Hind Ⅲ The data shown a successfull recombination 1 RNAiⅠ 2 RNAiⅠ / BamHⅠ + Hind Ⅲ 3 DL70 DNA marker 4 RNAiⅡ 5 RNAiⅡ / BamHⅠ + Hind Ⅲ 6 RNAi Ⅲ 7 RNAi Ⅲ / BamHⅠ + Hind Ⅲ 8 DL70 DNA marker 9 Negative control sirna 10 Negative control sirna / BamHⅠ + Hind Ⅲ 2 2 FucT Ⅶ mrna PCR RNAi psilencer 2 0-FucT Ⅶ 1 psilencer 2 0- FucT Ⅶ 2 psilencer 2 0-FucT Ⅶ 3 FucT Ⅶ mrna 2 -ΔΔCt P < 0 05 psilencer 2 0-FucT Ⅶ 3 HT-29 FucT Ⅶ 2 Fig FucT Ⅶ sirna FucT Ⅶ slex Fig 2 Expression of FucT Ⅶ mrna detected by realtime PCR Total RNA were extracted respectively from Western HT-29 FucT Ⅶ slex Fig 3 β HT-29 cells after transfection for 48 hours cdna was A FucT Ⅶ /β-actin synthesized with total RNA FucT Ⅶ mrna was recovered psilencer 2 0-FucT by real-time PCR Comparison with untreated group Ⅶ FucT Ⅶ slex expression of FucT Ⅶ on HT-29 cell line in experimental P < 0 05 psilencer 2 0-FucT Ⅶ 2 group was dominantly decreased particularly psilencer 2 0- RNAi FucT Ⅶ slex FucT Ⅶ 2 most apparently * P < 0 05 vs untransfected and P > 0 05 control sirna respectively

4 3 RNA FucT Ⅶ HT-29 HUVECs 293 Fig 3 Western blot analysis for FucT Ⅶ and slex protein in transfecting HT-29 cells with sirna Cell lysates were prepared The proteins in lysates were separated by 10% SDS-PAGE After transfering onto the membrane the blots were probed with anti-fuct Ⅶ and anti-slex antibodies β-actin was used as an internal control 1 Blank contorl group 2 Control sirna group 3 RNAi I group 4 RNAi Ⅱgroup 5 RNAi Ⅲ group * P < 0 05 # P < 0 05 vs untransfected and control sirna respectively 2 4 FucT Ⅶ HT-29 HUVEC RNAi FucT Ⅶ HT-29 HUVEC RNAi 2 6% psilencer 2 0-FucT Ⅶ % psilencer 2 0-FucT Ⅶ % psilencer 2 0-FucT Ⅶ % P < Fig 4 Analysis of the adhesion ability of HT-29 cells to 0 05 Fig 4 human umbilical endothelial cells HUVECs After FucT Ⅶ gene expression in HT-29 cells inhibited by FucT 3 FucT Ⅶ suppressed HUVECs were incubated with TNF-α 5 ng / fucosyltransferase FucT 8 ml for 4 hours before the assay Control wells were FucT consisted of HUVEC only Ⅶ * P < 0 05 vs untransfected and control sirna respectively Ⅱ Ⅰ FucT Ⅶ sirna FucT Ⅶ HT- 29 slex GlcNAc slex Lewis 9 slex slea selectin FucT Ⅶ cdna E- Eselectin 12 FucT Ⅶ slex E-selectin slex slea FucT Ⅶ 10 slex slea 13 FucT Ⅶ Hiraiwa E- P-selectin 11 Jurkat FucT Ⅶ slex FucT Ⅶ-siRNA the adhesion of HT-29 cells to HUVECs was 14 RNAi Ⅶ slex E- 15 slex

5 FucT Ⅶ shrna RNA HT-29 FucT Ⅶ PCR Western psilencer 2 0-FucT Ⅶ 3 HT-29 FucT Ⅶ FucT Ⅶ slex 3 psilencer 2 0-FucT Ⅶ HT-29 slex psilencer 2 0-FucT Ⅶ 2 HT-29 FucT Ⅶ slex HT- 29 psilencer 2 0-FucT Ⅶ Glycobiology FucT Ⅶ 10 Jeschke U Mylonas I Shabani N HT-29 HUVECs X sialyl Lewis A RNAi FucT Ⅶ slex E-selectin situ slex 11 Hiraiwa N Hiraiwa M Kannagi R RNA References 1 Hoon DB Ng S K Ramshaw I A Analysis of mammary tumour cell metastasis and release of bound n-acetylneuraminic acid J Br J Cancer Azab A K Kleinstern J Srebnik M et al The metastatic stagedependent mucosal expression of sialic acid is a potential marker for targeting colon cancer with cationic polymers J Pharm Res Contessa J N Bhojani M S Freeze H H et al Inhibition of N- linked glycosylation disrupts receptor tyrosine kinase signaling in tumor cells J Cancer Res Miyashiro M Furuya S Fujishige K et al Highly sensitive cellbased assay system to monitor the sialyl Lewis X biosynthesis mediated by α1-3 fucosyltransferase-Ⅶ J Biochem Biophys Res Commun Guo P Zhang Y Zhang X Y et al Analysis of lewis antigens on cell surface and alpha1 3 fucosyltransferase subtypes in H7721 human hepatocarcinoma cells J J Exp Clin Cancer Res Tuschl T Expanding small RNA interference J Nat Biotechnol Zeisiga R Stahnb R Wenzeld K et al Effect of sialyl Lewis X- glycoliposomes on the inhibition of E-selectin-mediated tumour cell adhesion in vitro J Biochim Biophys Acta Reguigne-Arnould I Wolfe J Hornigold N et al Fucosyltransferase genes are dispersed in the genome FUT7 is located on 9q34 3 distal to D9S1830 J C R Acad Sci Ⅲ Britten C J van den Eijnden D H McDowell W et al Acceptor specificity of the human leukocyte alpha3 fucosyltransferase role of FucT-Ⅶ in the generation of selectin ligands J et al Expression of sialyl lewis E-cadherin and cathepsin-d in human breast cancer immunohistochemical analysis in mammary carcinoma in invasive carcinomas and their lymph node metastasis J Anticancer Res A Human T-cell leukemia virus-1 encoded Tax protein transactivates alpha 1--> 3 fucosyltransferase Fuc-T Ⅶ which synthesizes sialyl Lewis X a selectin ligand expressed on adult T-cell leukemia cells J Biochem Biophys Res Commun Wagers A J Lowe J B Kansas G S An important role for the alpha 1 3 fucosyltransferase FucT-Ⅶ in leukocyte adhesion to E-selectin J Blood Weninger W Ulfman L H Cheng G et al Specialized contributions by α 1 3 -fucosyltransferase-iv and FucT-Ⅶ during leukocyte rolling in dermal microvessels J Immunity Martinez M Joffraud M Giraud S et al Regulation of PSGL-1 interactions with L-selectin P-selectin and E-selectin J J Biol Chem RNA J Cui Y X Liu M Li X et al Silencing of carcino-embryonic antigen by RNA interference inhibit proliferation of colorectal carcinoma cells J Chin J Biochem Mol Biol