| Object: Lewis antigens, elevated in many carcinomas , are the glyco-chain components of the cell surface glycoconjugates (glycoproteins, glycolipids and proteoglycans). Their expression level correlates closely with the proliferation, infiltration, metastasis, clinical stage and the treatment of carcinoma. Studies demonstrate that the overexpression of Lewis X(LeX) and Lewis Y(LeY) antigens on the cell surface of bladder, gastric, large intestine and cutaneous cancers et al. The analysis of expression of LeX and LeY is significant in the diagnosis, treatment and evaluation of the prognosis of the carcinoma. The reports show the malignancy of the tumor cells is decreased by blocking LeY. Whereas, suppressing the expression of the fucosyltransferases with RNAi technology to inhibit the synthesis of oligosaccharide antigens in order to decrease the proliferation, infiltration and metastasis of carcinoma is clearly elucidated now. In this study we investigated the mechanism of molecular glycol-pathology in order to find new ideas and methods of targeted tumor gene therapy.Mothods: 1. SiRNA plasmids of FUT1 and FUT4 were transiently transfected into A431 cell mediated by lipidosome. 2. The expression rates of LeX and LeY were detected by flow cytometry. 3. The inhibition of cell growth was analyzed by colony-forming unit assay. 4. Cell cycle analysis was performed on flow cytometry. 5. The apoptosis of A431 cells was assessed by flow cytometry. 6. The expression of Bcl-2 and Bax was detected by flow cytometry.Results: 1. After FUT1 and FUT4 SiRNA plasmids were transiently transfected into A431 cells, the expression ratios of LeX wereFUT1-1(51.29±0.98)%, FUT1-2(47.30±0.93)%, FUT4-1 (44.59±1.99)%, FUT4-2 (43.82±0.48)%, were significantly lower than control group (92.09±2.49)% and vector group (84.57±1.38)% (p<0.01), the expression ratios of LeY were FUT1-1 (28.34±1.01)%, FUT1-2(29.33±0.99)%, FUT4-1(35.11±1.57)%, FUT4-2(41.50±1.03)%, were significantly lower than control group (81.36±1.07)% and vector group (74.84±0.84)% (p<0.01).2. The colony-forming unit assay revealed that the ratios of transfected groups were FUT1-1(11.73±0.62)%, FUT1-2(13.33±0.61)%, FUT4-1(8.53±0.61)%, FUT4-2(6.80±0.80)%, were significantly lower than control group (22.80±0.80)% and vector group (18.93±1.29)% (p<0.01).3. The results of cell cycle analysis revealed ratios of G0/G1 phase were FUT1-1(40.83±0.03)%, FUT1-2(37.20±0.63)%, FUT4-1(40.68±0.04)%, FUT4-2(42.79±0.45)%, were significantly lower than control group (50.69±0.39)%(p<0.01), but compared with vector group (42.82±1.62)% had no significant discrepancy. The ratios of S phase were FUT1-1(57.49±0.08)%, FUT1-2(61.89±0.50)%, FUT4-1(58.38±0.17)%, FUT4-2(55.73±0.60)%, were significantly higher than control group (34.11±0.61)% and vector group (41.85±0.24)% (p<0.01). The ratios of G2/M phase were FUT1-1(1.67±0.11)%, FUT1-2(0.91±0.25)%, FUT4-1(0.94±0.19)%, FUT4-2(1.49±0.49)%, were significantly lower than control group (15.20±0.29)% and vector group (16.26±0.99)% (p<0.01).4. The apoptosis rates of transfected groups were FUT1-1(43.82±0.47)%, FUT1-2(30.11±0.56)%, FUT4-1(49.35±0.47)%, FUT4-2(60.76±0.57)%, were significantly higher than control group (4.99±0.07)% and vector group (7.99±3.76)% (p<0.01).5. The expression rates of Bcl-2 were FUT1-1(1.98±0.26)%, FUT1-2(3.16±0.29)%, FUT4-1(1.98±0.14)%, FUT4-2(1.46±0.07)%, were significantly lower than control group (11.64±1.01)% and vector group (7.44±1.23)% (p<0.01), Bax were FUT1-1(20.36±0.88)%, FUT1-2(13.60±2.25)%, FUT4-1(21.43±0.97)%, FUT4-2(18.47±0.79)%, were significantly higher than control group (5.93±0.25)% and vector group (7.41±1.23)%(p<0.01).Conclusion: FUT1 and FUT4 SiRNA plasmids could down regulate the expression of LeX and LeY on the surface of A431 cell. The cell proliferation was decrease and the S phase of cell cycle was arrested. A431cell was induced to apoptosis. Meanwhile, the expression of Bcl-2 was down-regulated and Bax was up-regulated. |
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