Study On Biological Functions And Radio Sensitivity Of DDX46 In Colorectal Cancer

Part one:The expression of DDX46 in colorectal cancer（CRC）tissues and its clinical significanceObjective:Investigate the clinical pathology and prognosis mechanism of DDX46 in CRC through the study on expression of DDX46 in colorectal cancer tissues and non-dysplastic tissues.Methods:1.Detected the expression of DDX46 protein in 149 colorectal cancer tissue samples and 45 non-dysplastic tissue samples through immunohistochemical staining method.2.Studied the relation between protein expression level and the age,gender,tumor differentiation,lymph node metastasis,TNM stage and lymphatic invasion of CRC patient.3.Observed the correlation between DDX46 protein expression level and that of C-erbB-2,KI-67,P53 and nm23.The relation between protein expression level and prognosis was also been observed.Results:Of the 149 CRC tissue samples,50（33.6%）showed low expression of DDX46 and 99（66.4%）showed high expression.Of the 45 pericarcinous tissue samples,37（82.2%）showed low expression of DDX46,and 8（18.8%）showed high expression.There existed significant difference between DDX46 expression in cancer tissue and peri-cancer tissue,the former much higher（P<0.05）.22 of the 29 mucinous adenocarcinoma tissue samples（75.8%）showed low DDX46 expression,and 24 of the 114 tubular adenoma tissue samples showed low DDX46 expression.Significant difference existed（P<0.01）.Statistical analysis showed that significant correlations were noted between DDX46 expression and lymph node status（P<0.05）,TNM stage（P<0.05）and differentiation（P<0.01）of CRC patients.DDX46 expression was significantly correlated with ki67,p53,nm23（P<0.05）,and the expression of DDX46 had positive correlation with ki67 and p53.72 of the 99 CRC patients with high DDX expression had MST of 32.2 months and 4-YSR of 67.7%,significantly lower compared with the remaining 27 patients with low DDX expression（MST>42.7months,4-YSR=79.4%;P<0.001）.Conclusions:DDX46 expression was high in CRC tissues.DDX46 cells mainly located at the epithelial cell nucleus of cancer tissues.Its expression had positive relation with Ki-67 and p53 while negative relation with nm23.Higher DDX46 expression was induced by lower differentiation degree and later TNM stage,having no relations with age,gender or location.Lower DDX46 expression led to longer survival time.DDX46 could be regarded as potential signal mark for the diagnosis and prognosis of CRC.Part two:Study on the biological function of DDX46 in colorectal cancer（CRC）cellsObjective:To investigate the biological function of DDX46 in colorectal cancer cells.Methods:The short hairpin RNA（shRNA）sequence targeting DDX46 and non-silencing control sequence were designed and synthesized by GeneChem（Shanghai,China）according to the sequence encoding human DDX46 gene（NM₀14829）.Built lentiviral DDX46-RNAi-LV silent RKO and SW480 cell,and tested transfection effect with RT-PCR and Western blot assay.MTT and colongenic formation experiment were used to test the influence of silent DDX46 on the growth and proliferation of CRC cell.MTT was used to test the influence of silent DDX46 on the survival rate of CRC cell.Western blot were used to detect the protein expression.Cell cycle analysis was performed using flow cytometry.Immunohistochemical staining was performed to investigate the expression and localization of DDX46 in CRC cell.Results:Compared with the control group,the DDX46 mRNA level was separately reduced by 77.0%（P<0.05）in RKO cells and 83%（P<0.05）in SW480 cells after lentiviral infection.Similarly,the DDX46 protein level also significantly decreased in the DDX46-RNAi-LV infected RKO and SW480 cells.The number of GFP-labeled cells in the DDX46-RNAi-LV group was greatly reduced compared to the Control-LV group through 5-day observation under fluorescence microscope.The cell viability of DDX46-RNAi-LV infected RKO and SW480 cells were significantly inhibited in a time-dependent manner.Inhibition levels reached as high as（27.1 ± 3.1）%in RKO cells and（44.0 ± 11.1）%in SW480 cells separately in day 5（P<0.05）.DDX46 inhibition resulted in a marked decrease in the number and size of RKO and SW480 cell colonies.The number of colonies in DDX46-RNAi-LV infected RKO and SW480 cells was reduced to 61%and 80%compared to corresponding Control-LV group（P<0.05）.The average apoptosis rate of DDX46-RNAi-LV infected RKO cells was 9.14%,significantly higher than the Control-LV group（6.12%,P<0.05）.Similarly,DDX46-RNAi-LV infected SW480 cells showed higher apoptosis rate than control group.Western blot assay revealed an increase in the level of caspase-3 and PARP-1 in DDX46-RNAi-LV infected SW480 cells.There was a slight increase in the number of DDX46-RNAi-LV infected RKO and SW480 cells in G0/G1 phase.Conclusion:DDX46 knockdown significantly inhibited cell growth and suppressed colony formation in CRC cells,which induced G0/G1 cell cycle arrest.DDX46 knockdown induced apoptosis in CRC cells,which may be caused by the increase of activated caspase-3 and PARP-1.Part three:Study on DDX46 influence on ionizing radiation sensitivity of colorectal cancer（CRC）cellsObjective:To investigate the effect and mechanism of ionizing radiation sensitivity of DDX46 in colorectal cancer cells.Methods:Lentiviral DDX46 CRC SW480 and Control-LV SW480 were designed to receive radiotherapy respectively with 0 Gy X ray and 4 Gy X ray,and cell vitality would be detected respectively in 0 h and in 24 h.Detect the number of y-H2 AX foci and the expression of Key protein in DNA damage repair pathways 24 hours after irradiation to determine the relationship between DDX46 and radiation induced DNA damage repair.Results:After 24 hours,the cell vitality of 4Gy X-ray combined DDX46-RNAi-LV group decreased（17.2±3.7）%（P<0.05）compared with DDX46-RNAi-LV group and decreased（26.3 ±1.2）%（P<0.05）compared with the 4Gy X-ray combined control-LV group.There was no significant difference between Control-LV group and 4Gy X-ray combined control-LV group.24h after irradiation,compared with the Control-LV group,the ATM protein expression increased significantly in SW480 cell of DDX46-RNAi-LV group（P<0.05）,while the protein levels of p-ATM and Rad50 were significantly reduced（P<0.05）.And protein expression of y-H2AX increased to a certain degree.DNA-PK in the two groups showed few changes.Conclusion:DDX46 silence can improve the radiation sensitivity of SW480 colon cancer cell.Its mechanism could be that silencing DDX46 gene can inhibit ATM activation in SW480,thus inhibit the DNA repair induced by radiation,which finally improves SW480 radiation sensitivity.