| Objective:The experiment use the human colorectal cancer SW620 cell line as research object, using RNA interference technology, RT-PCR, Annexin V FITC/PI double staining method and PI single staining at the cell level to study of shRNA silencing ARD1 gene effects of oxaliplatin chemical treatment sensitive in colorectal cancer cells, so as to explore the ARD1 gene in colorectal cancer chemotherapy with oxaliplatin effect and mechanism. Provide theoretical basis for chemical therapy and molecular targeted therapy of colorectal cancer.Method:1.Recovery and cultivation of SW620 cells line^ shRNA-NC SW620 cell line and shRNA-ARD1 SW620 cell line of human colorectal cancer;2. The ARD1 mRNA expression level of SW620 cell line、shRNA-NC SW620 cell line、shRNA-ARD1 SW620 cell line were tested by RT-PCR, the silence rate of shRNA-ARD1 SW620 cell line was calculated.;3 Experimental group:human colorectal cancer SW620 cell line (blank control group), colorectal cancer shRNA-NC SW620 cell line (negative control group), colorectal cancer shRNA-ARD1 SW620 cell line (experimental group);4. Methyl Thiazolyl Tetrazolium (MTT) method was used to detect OD value of different concentrations oxaliplatin treatment in each group in different time, and calculate each cell proliferation inhibition rate and IC50 values.5. The ARD1 mRNA expression levels after different concentrations oxaliplatin treatment in each group were detected by RT-PCR, calculate each cell inhibition rate;6. Flow cytometry technique (PI staining) to detect cycle changes of cells in each group with different concentrations oxaliplatin treatment;7. Flow cytometry technique (Annexin V FITC/PI staining method) to detect different concentrations oxaliplatin treatment in each group cells total apoptosis rate and early apoptosis rate.8. Statistical analysis:The SPSS 17.0 statistical software package was used for all analyses. The comparison of two groups of mean analyzed using t test; Multiple sets of mean analyzed using the ANVOA.The date were expressed by mean ± standard deviation (x±s).Significant level of a=0.05Result:1. The colorectal cancer SW620 cells、the colorectal cancer SW620 shRNA-NC cells and the colorectal cancer SW620 shRNA-ARD1 cells were cultured and the growth was state;2. PT-PCR was uesed to detect the cell ARDlmRNA expression:the ARD1 mRNA of shRNA-ARD1 cell group relative expression amount was lower than that in SW620 cells group, NC SW620 cells (P< 0.05), shRNA-ARD1 in SW620 48h relative ARD1 mRNA relative expression inhibition rate up to 54.8%.3. MTT was used to detect OD value of different concentrations (0,2,4,10,16, 32,64ug/ml) L-OHP treatment cells in each group after 48h,calculate inhibitory rate. Cells proliferation inhibition rate increased with the increase of L-OHP concentration (P<0.05), experimental group inhibition rate was higher than the negative control group and blank control group (P<0.05), and experimental group IC50 (12±0.7) ug/ml lower than the negative control group (15.5±0.68) ug/ml and control group (15±0.56) ug/ml(P<0.05), in 16ug/ml, MTT was used to detect OD value of L-OHP trertment SW620 cells at different time, calculate the inhibition rate of each groups of SW620 cells, inhibition rate of each groups with L-OHP function time increased, inhibition rates of experimental group was higher than the negative control group and blank control group (P<0.05), negative control group and blank the control group no statistical significance (P>0.05). shRNA interference technology to silence ARD1 expression can improve the sensitivity of L-OHP chemotherapy in SW620 cells, and the inhibition effect of cell proliferation is obviously enhanced,dependence of the concentration and time;4. The each groups of ARD1 mRNA relative expression was detected by RT-PCR after different concentrations (0,7.5,15,30ug/ml) L-OHP treatment, and the relative expression of ARD1 mRNA decreased (p<0.05) as the increase of L-OHP concentration. The relative expression of mRNA in the experimental group was lower than that in the control group and the negative control group (P<0.05), and there was no significant difference between the control group and the negative control group (P>0.05), ARD1 mRNA expression is inhibition after deal with oxaliplatin At the cellular level and with L-OHP concentrations increased ARD1 mRNA in each groups cells relative expression inhibited degree increasing.5. Flow cytometry technique (PI single staining) was uesed to detect each cell groups cycle in different concentrations of L-OHP treatment (0,5,15ug/ml) after cultured 48 hours:under the low concentration (5ug/ml) treatment the cells in each group of cells in G2/M was higher than no drug treatment (Oug/ml group) (P< 0.05). The experimental group G2/M phase cells increased greater than that in the blank control group and negative control group (P< 0.05), blank control group and negative control group no significant difference (P> 0.05). Sub-G1 peak appeared in the high concentration (15ug/ml) of L-OHP, the cells in sub-G1 phase was increased significantly than the lower concentrations of oxaliplatin and no L-OHP group (P< 0.01), sub-G1 phase cell percentage which in the experimental group a greater extent increased than blank control group and negative control group (P< 0.05). At low concentrations of L-OHP was arrested G2/M after shRNA silencing ARD1 and the apoptosis of SW620 cells was promoted at high concentrations of L-OHP6. Flow cytometry technique (AnneinV FITC/PI double staining) was used to detect apoptosis of SW620 cell line cells in different concentrations of L-OHP (0,5, 15ug/ml) treatment 48h:low concentration of L-OHP (5ug/ml) of each cell apoptosis rate than those without drugs (Oug/ml) treatment group increased (P< 0.05), the early apoptosis rate had no significant difference (P> 0.05). In the experimental group, the total cell apoptosis increased compared with blank control group and negative control group was significantly (P< 0.05). In high concentration of L-OHP treatment groups early apoptosis rate and apoptosis rate were increased (both P< 0.05). In the experimental group, the total apoptosis rate compared with the blank control group and negative control group cells group increased significantly (P< 0.01). ShRNA silencing ARD1 gene can promote the SW620 cells line apoptosis, and increase the sensitivity of L-OHP chemotherapy in colorectal cancer SW620 cells line;Conclusion:1. shRNA silencing ARD1 gene can enhance the sensitivity of human colorectal cancer cells to oxaliplatin chemotherapy, Proliferation of colorectal cancer cells was inhibited;2. The expression of oxaliplatin can inhibit ARD1 mRNA of colorectal cancer cell at the cellular level;3. Combined shRNA silencing ARD1 gene with low concentrations of L-OHP chemotherapy can arrest the colorectal cancer cell cycle in G2/M phase, and high concentrations of L-OHP chemotherapy can promote apoptosis of the colorectal cancer cell;4. Combined shRNA silencing ARD1 gene with L-OHP chemotherapy can promote the apoptosis of cells. |
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