Effects Of ST6Gal I SiRNA And ASO -mediated Gene Silencing On Metastasis Ability In Cervical Carcinoma Cells And Colon Carcinoma Cells

ObjectivesMetastasis was one of the most important characteristics of malignant tumors. It was also the major reason that leads to the death of the tumor sufferers. Metastasis is multi-phases and complicated process, the aberrant expression of cell surface carbohydrates and in particular that of terminal sialylation is closely associated with metastasis of tumor cells. In N-linked glycoprotein, sialic acids are attached to Galβ1,4GlcNaC with alpha2,6 linkages by the action of alpha2,6- sialyltransferase (ST6Galâ… ) and become the important receptors between cancer cells and extracellular matrix(ECM). The gene which codes ST6Gal I is overexpressed in cervical carcinoma cells and colon carcinoma cells and it is considered one of the main reasons which caused the high metastasis in cervical carcinoma and colon carcinoma.According to the base complementation, the principle antisense nucleic acids technology is the sequence specific binding of complementary antisense nucleic acids or their chemical modification to the nucleic acids in cells, resulting in the suppression or prevention of gene translation. RNA interference (RNAi) and antisense oligodeoxynucleotide (ASO) technology are two popular and hot antisense nucleic acids techniques to research gene expression and regulation in recent years. These two kinds of technology have their advantages and disadvantages respectively.Our study was through the combinatorial effects of small interfering RNA (siRNA) and its homologous ASO₁, nonhomologous ASO₂, targeting ST6Gal I on the cell adhesion and invasiveness in human cervical carcinoma cell line, HeLa and colon carcinoma cell line, SW480, which both over-expressed ST6Gal I. We hope to establish an experimental base for mechanism and apply of anti-tumor metastasis with technology of RNAi and ASO.Methods(1) siRrA and ASO targeting ST6Gal I were designed and synthesized, then transfected them into HeLa cells and SW480 cells by lipofectmine 2000. HeLa cells and SW480 cells were cultured and divided into four control groups (blank control group, liposome group, non-specific siRNA group and non-specific ASO group) and five experimental groups (siRNA group transfected with ST6Gal I siRNA, ASO₁ group transfected with ST6Gal I ASO whose target site is same as siRNA, ASO₂ group transfected with ST6Gal I ASO whose target site is different with siRNA, siRNA+ASO₁ group transfected with siRNA and its homologous. ASO₁, siRNA+ASO₂ group transfected with siRNA and its nonhomologous ASO₂). RT-PCR was used to examine the ST6Gal I mRNA expression. Amplified products were electrophoresed in 2% agarose containing ethidium bromide. Density measurements were analyzed by Gene Genius. ST6Gal I mRNA expression was discurbed by the density ratio of purposing gene ST6Gal I and housekeeping geneβ-actin. The more higher the ratio, the more higher the ST6Gal I mRNA expression.(2) Fluorescein isothiocyanate labelled sambucus nigra agglutinin (FITC-SNA) which could recognize sialic acid receptor specially was used in cells of each group. Flow cytometry was used to examine the amount ofα2, 6-sialylation on the HeLa cell surface and SW480 cell surface. Fluorescence intensity (FI) was used to disturb the expression ofα2, 6-sialylation on cell surface. The more higher the FI, the more higher amount ofα2, 6-sialylation on cell surface. At the same time, the expression ofα2, 6-sialylation on each group cell surface was observed under the fluorescence microscope by immunofluorescene technology.(3) The HeLa cells and SW480 cells adhesion to extracellular matrix (ECM) were detected by using CytoMatrix^TM cell adhesion assay kit. The absorbance at 490 nm (A₄₉₀) was used to assess the ability of cell adhesion to ECM, the more higher the A₄₉₀, the more stronger ability of cell adhesion to ECM.(4) The HeLa cell and SW480 cell invasiveness to ECM were detected by using CytoMatrix^TM cell invasion assay kit. The number of transwell cells was used to assess the ability of cell invasion to ECM, the more higher the number of transwell cells, the more stronger ability of cell invasiveness to ECM.Results(1) After tranfected 48h, the expressions of ST6Gal I mRNA in HeLa cells were 0.93±0.05, 0.89±0.05, 0.86±0.06, 0.88±0.04 in blank control group, liposome group, non-specific siRNA group, non-specific ASO group respectively and 0.55±0.08, 0.56±0.07, 0.59±0.07, 0.54±0.09, 0.33±0.09 in siRNA group, ASO₁ group, ASO₂ group, siRNA+ASO₁ group, siRNA+ASO₂ group respectively. The expressions of ST6Gal I mRNA in SW480 cells were 1.01±0.11, 0.93±0.09, 0.97±0.15, 0.99±0.14 in blank control group, liposome group, non-specific siRNA group, non-specific ASO group respectively and 0.41±0.06, 0.65±0.07, 0.61±0.09, 0.43±0.03, 0.23±0.09 in siRNA group, ASO₁ group, ASO₂ group, siRNA+ASO₁ group, siRNA+ASO₂ group respectively. The expression of ST6Gal I mRNA in experimental groups, all significantly lower than those of the control groups (all P＜0.05), especially in siRNA+ASO₂ group. The difference was significant between siRNA+ASO₂ group and siRNA group (all P＜0.05). No significant difference was observed between siRNA+ASO₁ group and siRNA group (all P＞0.05). (2) After tranfected 72h, FI in HeLa cells were 64.17±8.33, 62.20±7.54, 68.73±5.95, 69.47±5.97 in blank control group, liposome group, non-specific siRNA group and non-specific ASO group respectively and 43.30±8.42, 39.27±4.80, 42.30±7.95, 40.07±9.66, 10.47±1.70 in siRNA group, ASO₁ group, ASO₂ group, siRNA+ASO₁ group, siRNA+ASO₂ group respectively. The FI in SW480 cells were 98.77±14.68, 95.03±13.95, 87.80±11.04, 92.97±14.40 in blank control group, liposome group, non-specific siRNA group and non-specific ASO group respectively and 36.23±4.91, 59.10±10.43, 56.73±9.02, 37.00±9.95, 15.53±5.16 in siRNA group, ASO₁ group, ASO₂ group, siRNA+ASO₁ group, siRNA+ASO₂ group respectively. The FI in experimental groups, all significantly lower than those of the control groups (all P＜0.05), especially in siRNA+ASO₂ group. The difference was significant between siRNA+ASO₂ group and siRNA group (all P＜0.05). No significant difference was observed between siRNA+ASO₁ group and siRNA group (all P＞0.05). Through the fluorescence microscope, the fluorescence intensity ofα-2,6-sialylation on cell surface in experimental groups were significantly weaker than that of the control groups, especially in siRNA+ASO₂ group. No significant difference was observed between siRNA+ASO₁ group and siRNA group.(3) After tranfected 72h, the A₄₉₀ in HeLa cells were 0.62±0.09, 0.66±0.06, 0.67±0.05, 0.64±0.07 in blank control group, liposome group, non-specific siRNA group and non-specific ASO group respectively and 0.44±0.09, 0.43±0.08, 0.46±0.12, 0.42±0.10, 0.27±0.05 in siRNA group, ASO₁ group, ASO₂ group, siRNA+ASO₁ group, siRNA+ASO₂ group respectively. The A₄₉₀ in SW480 cells were 0.81±0.07, 0.84±0.05, 0.79±0.02, 0.83±0.07 in blank control group, liposome group, non-specific siRNA group and non-specific ASO group respectively and 0.39±0.05, 0.56±0.03, 0.53±0.09, 0.40±0.08, 0.26±0.05 in siRNA group, ASO₁ group, ASO₂ group, siRNA+ASO₁ group, siRNA+ASO₂ group respectively. The A₄₉₀ in experimental groups, all significantly lower than those of the control groups (all P＜0.05), especially in siRNA+ASO₂ group. The difference was significant between siRNA+ASO₂ group and siRNA group (all P＜0.05). No significant difference was observed between siRNA+ASO₁ group and siRNA group (all P＞0.05).(4) After tranfected 72h, the number of transwell cells in HeLa cells were 33.80±6.38, 35.40±5.77, 34.00±6.78, 36.20±5.50 in blank control group, liposome group, non-specific siRNA group and non-specific ASO group respectively and 23.60±6.23, 24.80±3.35, 25.20±4.09, 22.80±6.57, 12.40±2.30 in siRNA group, ASO₁ group, ASO₂ group, siRNA+ASO₁ group, siRNA+ASO₂ group respectively. The number oftranswell cells in SW480 cells were 120.40±15.53, 123.00±11.98, 126.60±13.61, 122.40±12.32 in blank control group, liposome group, non-specific siRNA group and non-specific ASO group respectively and 61.20±9.60, 82.20±8.93, 76.60±4.16, 59.40±10.01, 30.80±9.73 in siRNA group, ASO₁ group, ASO₂ group, siRNA+ASO₁ group, siRNA+ASO₂ group respectively. The number oftranswell cells in experimental groups, all significantly lower than those of the control groups (all P＜0.05), especially in siRNA+ASO₂ group. The difference was significant between siRNA+ASO₂ group and siRNA group (all P＜0.05). No significant difference was observed between siRNA+ASO₁ group and siRNA group (all P＞0.05).Conclusions(1) Chemically synthesized specific siRNA and ASO targeting ST6Gal I both effectively inhibit the expression of ST6Gal I mRNA in HeLa cells and SW480 cells. Surpression of the expression of ST6Gal I mRNA in siRNA with its nonhomologous ASO group was stronger than that in siRNA group alone, while no significant difference was observed between siRNA with its nonhomologous ASO group and siRNA group.(2) Chemically synthesized specific siRNA and ASO targeting ST6Gal I both effectively inhibit the amount ofα2, 6-sialylation on the HeLa cell surface and SW480 cell surface. Surpression of the amount ofα2,6-sialylation on cell surface in siRNA with its nonhomologous ASO group was stronger than that in siRNA group alone, while no significant difference was observed between siRNA with its nonhomologous ASO group and siRNA group.(3) Chemically synthesized specific siRNA and ASO targeting ST6Gal I both effectively inhibit the cell adhesion to ECM in HeLa cells and SW480 cells. Surpression of the cell adhesion to ECM in siRNA with its nonhomologous ASO group was stronger than that in siRNA group alone, while no significant difference was observed between siRNA with its nonhomologous ASO group and siRNA group.(4) Chemically synthesized specific siRNA and ASO targeting ST6Gal I both effectively inhibit the cell invasiveness to ECM in HeLa cells and SW480 cells. Surpression of the cell invasiveness to ECM in siRNA with its nonhomologous ASO group was stronger than that in siRNA group alone, while no significant difference was observed between siRNA with its nonhomologous ASO group and siRNA group.