5). sulfatide. In the early stage of sulfatide stimulation, phosphorylation of Erk as well as c-Src was noted, and inhibition of Erk activation with either U0126 or PD98059 significantly suppressed Sp1 phosphorylation and integrin V expression. We demonstrated that GNE-6640 sulfatide regulated integrin V expression and cell adhesion, which was associated with Erk activation. activity. Hep3B cells overexpressing that produces sulfatide significantly promotes the metastasis behaviors in nude mice. Apart from (is also observed to be involved in tumor metastasis (16). Both genes encode galactose-3-expressed an elevated level of integrin V and intensively adhered to vitronectin, the ligand of integrin V3 (15, 18). However, the mechanism by which the is involved in regulation of integrin V and cell adhesion is not fully understood. The relationship between the promotion mechanism of cancer cells and elevated expression of sulfatide remains unknown (19). A recent study showed that sulfatide can serve as a laminin-binding glycolipid and can anchor laminin-1 and laminin-2 to the Schwann cell surface, form a laminin-associated complex, and enable basement membrane assembly to initiate c-Src activation (9). Sulfatide was also identified as an interacting partner of P-selectin and promoted a P-selectin-mediated metastasis process in colon cancer cells (20). Sulfatide and P-selectin interactions led to subsequent platelet aggregation (21) and played an important role in the formation of cancer embolus. Our previous study (15, 18) revealed that hepatoma cells expressed sulfatide after transfection. The enzyme in HCC can only catalyze the production of sulfatide, which acts as the endogenous sulfated cerebroside. We thus hypothesize that the enzyme product sulfatide is responsible for the regulation of the integrin V subunit and involves the metastasis process. To test this, we investigated, in this study, the regulatory effect of both exogenous and endogenous sulfatide, the product of overexpressed HCC cells mainly produced lactosyl sulfatide. The cells were maintained in RPMI 1640 medium supplemented with 10% newborn bovine serum (PAA, Austria) at 37C under a 5% CO2 atmosphere. For the treatment, cells were cultured in RPMI 1640 medium containing 2 M sulfatide, lactocerebroside, or galactocerebroside added from stock solution in DMSO. An equal amount of DMSO (0.1% v/v) was added to control group. Plasmid construction The short hairpin sequences, including 5-AGGAGUUGGUGGCAAUAAU-3 and 5-UAUUAGGCAUCACUCCAGG-3, which specifically interfered and targeted Sp1 mRNA, were designed according GNE-6640 to the protocol from Ambion (24). The synthesized 55 bp forward and reverse oligonucleotides containing the siRNA sequence were annealed and ligated to the pSilencer 4.1 vector. The pcDNA3.0-Sp1 expression plasmid was kindly provided by Dr. Jian-Hai Jiang (Fudan University, P.R. China). A human cDNA expression plasmid was previously constructed (15, 18). The integrin V promoter fragments from ?1295 to +207 bp, ?795 to +207 bp, ?309 to +207 bp, and ?16 to +207 bp were obtained by PCR from the genomic DNA of SMMC-7721 cells. The following primers were used: integrin V/Kpn I ?1295: 5- CCCGGTACGGTCCACACAATGCACTTAAA-3, integrin V/Kpn I ?795: 5- AAAGGTACGCAAGAGGCTATGCTGGC-3, integrin V/Kpn I ?309: 5- AAAGGTACGCCTCCTTCCAGGTCTCC-3, integrin V/Kpn I ?16: 5- AAAGGTACGTGGGGCGGGGGGAGGT-3, integrin V/Xho I +207: 5-CCCGTCGAGAGAAATCCACGGCGAA-3. The PCR products were inserted into the Xho I/Kpn I sites of the pGL3-basic vector (Promega, Madison, MI) and designated as pGL3- integrin V. All of the constructs were verified by sequencing. RT-PCR and real-time PCR Total RNA was extracted from cells with the Trizol reagent according to the manufacturer’s instructions and was used as the template for cDNA synthesis. Reverse transcription was then carried out by M-MLV. The following primer sets were used for RT-PCR and real-time PCR: integrin V subunit (sense 5-GACAGTCCTGCCGAGTA-3, anti-sense 5-CTGGGTGGTGTTTGC-3); Sp1 (sense 5-TCACAAGCCAGTTCCAGCTCC-3, anti-sense 5-GGGTGCACCTGGATTCCTGAA-3); Sp3v1 (sense 5-GAAATGGCTGCCTTGGACG-3, anti-sense 5-AGCGGTGACGGCTGAGTGT-3); Sp3v2 (sense 5-ACCCCCTCCCCCTGTCTCCCTC-3, anti-sense 5-CTCCCATCGGTTTGGTGCTCCT-3); ETS (sense 5-TCACAAGCCAGTTCCAGCTCC-3, anti-sense 5-GGGTGCACCTGGATTCCTGAA3); AP2 (sense 5-GCTGGGCACTGTAGGTC-3, anti-sense 5-ACTTGGACAGGGACACG-3); EGR1 (sense 5-CAGCAGCCTTCGCTAACC-3, anti-sense 5-CATCGCTCCTGGCAAACT-3); EGR2 (sense GNE-6640 5-CAGCCTCATCCAGCGTCAC-3, anti-sense 5-TCGTCAGAGCGGGAGAACC-3); -actin (sense 5-CTCCATCCTGGCCTCGCTGT-3, anti-sense 5-GCTGTCACCTTCACCGTTCC-3); CST (sense 5-CAAGTTCGCCTTCCCTAA-3, anti-sense 5-CACAGCAGGTCCTTCAG-3). The PCR amplifications were performed, and the products were analyzed by 2% agarose gel electrophoresis. -actin was used as an internal control. Real-time PCR was performed using a BIO-RAD IQ5 real-time PCR system (Bio-Rad). Transfection For transient transfection, SMMC-7721 cells were seeded in 6-well plates at 5 105 cells/well. The cells were then transfected with Rabbit Polyclonal to PPM1L GNE-6640 Sp1 expression vectors for 24 h and then treated with Lacto-Cer.