Effect Of Methylation On Fucosyltransferase IV Gene Expression And Cells Proliferation In Squamous Carcinoma Cells

I. Objective and purposeGlycoproteins and glycolipids on the cells surface play an important role in cellssignal transduction through physiological and pathological progression. Oligosac-charides, such as Lewis a (Lea), sLewis a (sLea), Lewis b (Leb), Lewis X (LeX),sLewis X (sLeX) and Lewis Y (LeY), are important component for the function ofglycoproteins and glycolipids. LeY, a difucosylated oligosaccharide, is elevated incarcinomas of breast, ovary, pancreas and lung and correlates with carcinomaproliferation, invasion and metastasis.Fucosyltransferases (FUTs) are considered key enzymes that regulate the synthesisof fucosylated oligosaccharides. FUT4, one of key enzymes for the synthesis of LeY, islocated in Golgi apparatus and catalyzes Fuc residues transferred from GDP-Fuc to[Fuc1â†'2Galβ1â†'4GlcNAc1â†'R] in α-1,3linkage. FUT4is expressed in a variety oftissues. FUT4expression is elevated in tumor tissues such as colon, ovary, gastric, lungand in acute myeloid leukemia. Previous studies in our and other groups show that cellproliferation could be controlled by regulating FUT4and LeY expression. FUT4expression is different significantly in tumor tissues and normal tissues. Although theimpact of FUT4expression on cell proliferation has been studied, little information isavailable on how FUT4expression is regulated.Accumulating evidence suggests that tumor progression is related to the abnormalmethylation alteration of genes. DNA methylation is a common epigenetic modificationin mammalian genome. Hypomethylation of genes, such as R-Ras, S100A4, PRAMEand p-Cadherin, play an important role on tumor growth, invasion and metastasis due toincreasing gene expression. However, it is not clear whether FUT4expression is controlled by the methylationstatus of its promoter. The effect of promoter methylation on FUT4expression wasinvestigated in this study. The effect of5-aza-dC on the proliferation and migration ofsquamous carcinoma cells was also analyzed.II. Methods1. The proliferation capability of A431and SCC12was comparative analyzed byMTT assay and Western blot.2. FUTs expression in A431and SCC12cells was detected by Real-timequantitative PCR and protein fucosylation was detected by Lectin blot.3. FUT4expression in A431and SCC12was detected by RT-PCR, Western blot,Immunofluorescence staining and Flow cytometry assay.4. LeY expression in A431and SCC12was detected by Western blot, Flowcytometry assay and Immunofluorescence staining.5. CpG islands in FUT4promoter were predicted by a program software,MethPrimer (http//www.urogene.org//methprimer/indexl.html).6. The methylation status of CpG island in FUT4promoter was analyzed byMethylation-specific PCR (MSP) and Bisulfite sequencing PCR (BSP).7. The methylation status of CpG island in FUT4promoter was analyzed by BSPin cells treated with5-aza-dC.8. The expression of FUT4was detected by Real-time quantitative PCR, RT-PCRand Western blot in cells treated with5-aza-dC.9. The expression of LeY was detected by Immunofluorescence staining and Flowcytometry assay in cells treated by5-aza-dC.10. The effect of5-aza-dC on cells proliferation was detected by MTT assay.11. Cell cycle was analyzed by Flow cytometry assay in cells treated with5-aza-dC.12. The proliferation of cells treated by5-aza-dC combination with FUT4-siRNAwas analyzed by cell counting with a hemocytometer.13. Cells scratch assay was used to detect the effect of5-aza-dC and FUT4-siRNAon cells migration.III. Results(I) The comparison of proliferation capability, FUTs expression and proteinfucosylation in A431and SCC12cells1. A431cells were higher proliferated than SCC12cells. 2. FUTs were expressed in both squamous carcinoma cell lines. The expressionlevel of FUTs was higher in A431cells than that in SCC12cells.3. Protein fucosylation level was higher in A431cells than SCC12cells.(II) Effect of methylation on FUT4gene expression1. The results from Real-time quantitative PCR, RT-PCR, Western blot,Immunofluorescence staining and Flow cytometry assay showed that the expression ofFUT4was higher in A431cells than SCC12cells.2. The results from Western blot, Immunofluorescence staining and Flowcytometry assay showed that the expression of LeY was higher in A431cells thanSCC12cells.3. There were two CpG islands in FUT4promoter region.4. The results from MSP and BSP indicated methylation degree of CpG island inFUT4promoter was higher in SCC12cells than that in A431cells.5. Methylation degree was decreased and unmethylation degree was increased inSCC12cells treated by5-aza-dC. There was no obvious alteration in A431cells.6. FUT4and LeY expression was increased significantly by treatment with5-aza-dC in SCC12cells, but no obvious change was seen in A431cells.(III) Effect of5-aza-dC on the proliferation and migration of A431andSCC12cells1. The proliferation of A431and SCC12cells was inhibited significantly bytreatment with5-aza-dC through arresting cells at S phase.2. The proliferation of A431and SCC12cells was inhibited significantly bytransfection of FUT4-siRNA into cells.3. The inhibition of5-aza-dC on cells proliferation was enhanced by concurrentlyknocking down FUT4expression.4. The cells migration was inhibited by5-aza-dC.5. The cells migration was inhibited significantly by transfection of FUT4-siRNAinto cells.6. The inhibition of5-aza-dC on cells migration was enhanced by concurrentlyknocking down FUT4expression.IV. Conclusions(I) The FUTs and protein fucosylation level was higher in A431cells with a higherproliferative capability and lower in SCC12cells with a lower proliferative capability,which suggested FUTs expression and protein fucosylation level was correlated to the proliferative capability of squamous carcinoma cells.(II) FUT4expression was higher in A431cells than that in SCC12cells, andmethylation level of FUT4promoter was higher in SCC12cells than that in A431cells.Methylation level was decreased and FUT4expression was increased in SCC12cellstreated by5-aza-dC. These results suggested different methylation level of FUT4promoter played a critical role to regulate FUT4expression, which further affected cellprolierative capability in squamous carcinoma cells.(III) A431and SCC12cells proliferation and migration was inhibited by5-aza-dC,and also prevented by FUT4-siRNA. The inhibition of5-aza-dC on cell proliferationand migration was enhanced by concurrently knocking down FUT4expression. Theseresults suggested that5-aza-dC and FUT4-siRNA could be used combinatively to curesquamous carcinomas, especially in which FUT4expressed highly.