The Effect Of RNA Interfering With ARD1on Human Colorectal Adenocarcinoma Cell Strains

Objective:In this experiment, We choose LS-174T SW620and HCT-8CRC cell strains to be the research objects and use siRNA which can effectively inhibit the expression of ARD1to transfect the three cell strains and detecte the expression of ARD1from the gene and protein levels by RT-PCR and Western blot.Finally we detect the changes of apoptosis in three colorectal cancer cell strains with Flow Cytometry to reveal the biological function and role of hARDl in colorectal cancer and we will provide a theoretical basis for hARD1as a tumor marker in the early diagnosis, treatment and prevention of colorectal cancer.Methods:1. Cultivation of LS-174T SW620and HCT-8CRC cell strains;2.The design and synthesis of siRNA;3.Transfect three cell strains by siRNA;4. Total RNA extraction and quality control;5.Detect the transfection rate by RT-PCR after transfecting the three cell strains for24hours and detect the transfection rate by Western blot after transfecting the three cell strains for48hours;6. Stain three groups of three colorectal cancer cell strains with Annexin V-FITC/PI and Hoechst33342/PI;7. Detect the apoptosis in three colorectal cancer cell strains with Flow Cytometry.Results:1. S-174T HCT-8and SW620cells are cultivated in vitro successfully and both grow well;2. The quality inspection results of the total RNA sample of each group are28S/18S>0.7, which means that all are eligible sample;3. SiRNA can effectively inhibits the expression of ARD1. RT-PCR results shows that the transfection rate of three cell strains LS-174T SW620and HCT-8is64%,53.03%, 55.28%. Statistical analysis shows that the difference between the group which was transfected before an after has a statistical significance in gene expression level (P <0.05).It shows that siRNA has a significant inhibitory effect on the expression of ARD1mRNA in three cell strains after being transfected for24hours. Western blot results shows that the transfection rate of three cell strains LS-174T SW620and HCT-8is86.22%,85.89%,85.73%. Statistical analysis shows that the difference between the group which was transfected before and after has a statistical significance at the protein level (P<0.05). It shows that siRNA has a significant inhibitory effect on the expression of ARD1mRNA in three cell strains after being transfected for48hours;4. Flow Cytometry results shows that the apoptosis and the early apoptosis rate of three cell strains are increased after being transfected for2448and72hours. Statistical analysis shows that the difference between the Negative control group and the blank group has a statistical significance(P<0.05). Statistical analysis shows that the difference between the transfection group and the Negative control group has a statistical significance(P<0.05).Conclusion:1. This experiment verifies that RNA interference technology can effectively inhibits the expression of ARD1from gene level and protein level. It shows that this approach has great possibility for tumor gene therapy;2. This experiment verifies that inhibiting the expression of ARD1in three cell strains can promote the apoptosis of colorectal cancer cells.The results suggest that hARDl may be a new target for the treatment of colorectal cancer and may provide basis for the gene therapy of the treatment cycle and the course of treatment.