| Objective Esophageal cancer is a common digestive system, accounting for 2% of all malignant tumors. Esophageal cancer is a high incidence in China, the death rate of which in the second, however, its mechanism remains unkonow. Tumor is a long-term multi-factor, multi-step complex process, including cell proliferation and differentiation, apoptosis, cell adhesion, circumventing the immune system, angiogenesis, tumor invasion, metastasis and so on. Galectin-3 protein as one of the lectin family of galactose is suggested that widely expressed in normal tissue and tumor tissue, and tumor development, metastasis and invasion are closely relatedRNA interference (RNA interference, RNAi) is an effective tool to prevent nucleic acid sequence-specific gene function that can efficiently and specifically inhibits the expression of target genes. Studies have found that using RNAi technology to prevent tumor-related gene expression can effectively inhibit tumor cell proliferation. We design and synthesis small interfering RNA (short interfering RNA, siRNA) expression vector specifically on Galectin-3 gene, and then make Galectin-3-siRNA to interfer on esophageal cancer cell line Eca-109, which in order to study the interference on Galectin-3, CyclinD1 protein expression, cell proliferation and cell cycle, and to explore the effection of the Galectin-3 protein on the esophageal cancer cell line Eca-109 cell proliferation and cell cycle kinetics and the relationship with the CyclinD1, so as to provide data for the mechanism and clinical treatment of esophageal cancer.Methods Galectin-3 siRNA was transfected into esophageal cancer Eca-109 cells. The experiment was divided into transfection group, blank control group, positive control group and negative control group. MTT assay used in each group of esophageal cancer cell line Eca-109 proliferation, to detect the optical density (OD) on behalf of the group, proliferation, and inhibition rate of each group; detected by immunocytochemistry in each group esophageal cancer cell line Eca-109 in the Galectin-3, CyclinD1 protein expression, the use of HIPAS-2000-based computer image acquisition system,×200 magnification in the images in the collection, use Imageproplus5.0 (IPP) measured integrated optical density of positive cells value of the IOD, each slice select five horizons and find their average. Were detected by flow cytometry esophageal cancer cell line Eca-109 cell cycle, the results to be used and the software ModFit cell cycle analysis. All data between groups by analysis of variance compared mean to p <0.05 indicated significant difference. Results 1. Transfection efficiency of detection: application of FAM labeled N-egative siRNA transfected esophageal cancer cell Eca-109 6h detected by flow cytometry after the transfection efficiency, transfection efficiency was 69.60%.2.Inverted phase contrast microscope for cytological observation, G- alectin-3-siRNA transfected cells after 48h obvious morphological changes, cells transfe cted with negative control group and blank control group were smaller than the cell volume and tend to uniformity, the majority of cells tr- end from the spindle an d round, smaller than the nuclear plasma, tumor gi ant cells decreased compared with the control group cells grew slowly, so me cells die and fall off.3. MTT results showed that: Galectin-3-siRNA into the cells 48h after transfection, inhibition of the esophageal cancer Eca-109 cell line growth in the group transfected with the positive control group absorbance at 490 nm wave length values were 0.63±0.13, 0.58±0.19, 0.76±0.09, 0.77±0.18, significantly less than control group 1.43±0.12 and the negative control group 1.65±0.14, transfection, positive control group and blank con trol group and negative control group, the difference are There was signifi cant (P <0.05), the cells were transfected with the inhibitory rates were 5 5.85%, 59.71%, 46.17%. 4. Immunocytochemistry showed that: The Galectin-3-siRNA transfe cted esophageal cancer cell line Eca-109 protein after 48h Galectin-3 expr ession decreased significantly lighter immunocytochemistry, application IPP image analysis system in the detection of the transfected group IOD values were 20.45±0.15, 20.33±0.24, 20.36±0.22, control group IOD valu e of 58.15±0.22, IOD value of the negative control group 57.65±0.43, po- sitive control group IOD value of 19.85±0.67; transfection group, positive control group and blank control group and negative control group compare d to the differences were statistically significant (p <0.05).Application of Galectin-3-siRNA transfected esophageal cancer cell line Eca-109 protein after 48h cyclinD1 decreased significantly lighter imm unocytochemistry, transfected IOD values of each group were 39.98±0.05, 39.53±0.82, 40.86±0.85, control group IOD value of 61.83±0.53, IOD value of the negative control group 62.28±0.57, positive control group IO- D value of 40.42±0.60; transfection, positive control group and blank control group and negative control group compared The difference was statistically significant (p <0.05).According to the results of immunohistochemistry analysis to IOD as a semi-quantitative data, 48h after transfection, transfection of the group and the positive control group CyclinD1 Galectin-3 and the IOD values were decreased, statistical correlation analysis showed that Galectin-3 and the correlation CyclinD1 Resistance (r = 0.806, p = 0.000), with Galectin-3 expression is reduced, CyclinD1 reduced accordingly.5.Flow cytometry showed that: Galectin-3-siRNA transfected esophageal cancer cell line Eca-109 48h after transfection group in each group, positive control group sub-G1 phase cells compared hundreds of blank control group and negative control group increased, Sphase cells compared hundreds of hours the control group and negative control group were significantly reduced. Transfected in each group, the positive control group the percentage of G1 phase cells, respectively 65.76±0.45, 66.42±0.17, 65.10±0.67, 65.44±0.42, negative control group and blank control group G1 phase cells were 36.76±0.15, 35.63±0.36, transfection, positive control group and blank control group and negative control group, the difference was significant (p<0.05). Transfected in each group, the positive control group the percentage of S phase cells were 23.13±1.34, 23.19±1.28, 24.40±2.17, 24.32±1.73, negative control group and blank control group the number of S phase cells were 46.79±0.76 , 45.33±0.72, transfection, positive control group and blank control group and negative control group, the difference was significant (p <0.05).Conclusions Being transfected specific Galectin-3 siRNA into esophageal cancer cell line Eca-109, after 48h the cell growth was significantly inhibited; expression of Galectin-3 protein and Cyclin D1 protein in esophageal cancer cell line Eca-109 reduced; the cell growth was arrested in G1; the number of cells in transfection group was significantly higher than that of non-transfection group and negative control group. Galectin-3 was closely related with Cyclin D1 protein in the proliferation of esophageal cancer cell line Eca-109. Using RNAi technology can prevent tumor-related gene expression and effectively inhibit tumor cell growth, which possibly provides an important method in diagnosis and treatment of tumors. |
PDF Full Text Request