Effects Of Silencing Livin Gene By RNA Interference Mediated By Lentiviral Vector On Colorectal Cancer HT-29Cell Xenograft Growth And Sensitivity To Chemoradiotherapy In Nude Mice

Objective:The expression of Livin gene in HT-29cells could be downregulated efficientlyand stably by lentiviral vector-mediated RNA inteference in vitro experimental of ourstudy. When the Livin gene in HT-29cells was inhibited, its proliferation capacitywas weakened, its apoptosis rate increased, and the cell cycle redistribution.SilencingLivin gene could enhance the anti-tumor effect of5-FU, oxaliplatin and irinotecan.Now our further study would conduct to explore the effects of silencing Livin geneby RNA interference mediated by lentiviral vector on colorectal cancer HT-29cell xenograft growth and sensitivity to chemoradiotherapy in nude mice,preliminary clarify whether Livin gene is a new target in the treatment ofcolorectal cancer.Methods:1. Three different BALB/C nude mice models were established bysubcutaneously inoculating differently treated HT-29cells into three nude micegroups: the blank control group was inoculated with normal cultured HT-29cells,while the negative group was inoculated with cells transfected withlentivirus-carried unrelated sequence fragments shRNA, the experimental groupwas inoculated with cells transfected with lentivirus-carried Livin shRNA. Thenthe growth of tumors was observed, and the volume of tumors, the weight of thenude mice were measured at different time points.42days later, mice weresacrificed, and removed the tumors, Livin gene expression in the tumor tissueswas determined by RT-PCR and Inmunohistochemistry. Cell apoptosis of tumortissues was detected by TUNEL.2. The tumor tissue blocks of the first generation of HT-29cells xenograftwas subcutaneously inoculated to nude mice. When the tumors grew to a certainvolume, divided the nude mice into three groups.The blank group wasintratumoral injection with normal saline, and the negative group was intratumoral injection with lentivirus carried unrelated sequences, while theexperimental group was intratumoral injection with lentivirus carried LivinshRNA. Then, all the nude mice were divided into three groups again andintraperitoneal injected with5-Fu, oxaliplatin and irinotecan respectively.Eventually, there were nine groups in this part of experiment. Observed the miceweight and the tumor volume at15points for70days，twice a week. The volumeof tumors and the weight of the nude mice were measured at different time points,drew the weight and tumor growth curves.3. The tumor tissue blocks of the first generation of HT-29cells xenograftwas subcutaneously inoculated to nude mice.When the tumors grew to a certainvolume, divided the nude mice into three groups. The blank group wasintratumoral injection with normal saline, and the negative group wasintratumoral injection with lentivirus carried unrelated sequences, while theexperimental group was intratumoral injection with lentivirus carried LivinshRNA. Then all the nude mice were given the6MV X-ray irradiation,2Gy/F,ata total of10Gy. Observed the mice weight and the tumor volume at24points for100days, twice a week. The volume of tumors and the weight of the nude micewere measured at different time points, drew the weight and tumor growthcurves.4. Adopted SPSS17.0software for statistical analysis, the measurementdata between groups were compared using single factor analysis of variance, theenumeration data between groups were compared using the χ2test, the data oftumor volume were compared using repeated measures ANOVA. p<0.05represents significant difference, p<0.01represents statistically significantdifference.Results:1. Direct inhibition of tumor growth through silencing Livin gene by RNAinterference mediated by lentiviral vector: three groups of nude mice all grewtumors, tumorigenicity was100%. Observed the mice for42days, then the micewere sacrificed, there was no significant difference between the weight of nudemice (F=0.352, p=0.709). There was significant difference between each period and groups in tumor volume (F=217.178, p<0.001; F=18.103, p<0.001). Therewas interaction between period and group(F=14.237, p<0.001). The inhibitionrate of tumor volume was (50.04±0.07)%，the tumor weight was lighter inexperimental group as compared to the blank and negative groups(F=4.847,p=0.024), the inhibition rate of tumor weight was （50.27±0.17）%. Livin mRNAexpression level in experimental group was (17.75±0.08)%, significantly lowerthan the blank group (67.60±0.05)%and the negative group (68.54±0.03)%(F=89.971, p<0.001). Livin protein expression level was also significantly lowerthan the blank and the negative groups [(36.00±3.40)%versus (85±3.15)%,(80.33±3.08)%, F=107.321, p<0.001]. The apoptosis rate in the experimentalgroup was significantly higher than the blank and the negative groups[(23.67±2.25)%versu (5±1.50)%,(8.33±1.82)%, F=56.936, p<0.001].2. The antitumor effect of silencing Livin gene by RNA interferencemediated by lentiviral vector combined with chemotherapy: There wassignificant difference between the weight of nude mice at different time pointsand groups (F=3.086, p=0.002; F=11.739, p<0.001). There was significantdifference between each period and groups in tumor volume (F=64.993, p<0.001;F=8.610, p<0.001). The tumor volume in5-Fu, oxaliplatin and irinotecanexperimental groups were all significantly smaller than those in negative controlgroups (p=0.001, p=0.049, p=0.001).3. The antitumor effect of silencing Livin gene by RNA interferencemediated by lentiviral vector combined with radiotherapy: There were nosignificant difference between the weight of nude mice at different time pointsand groups(F=1.1015, p=0.330; F=1.022, p=0.383). There was significantdifference between time period and grouos in tumor volume (F=71.250, p<0.001;F=10.702, p=0.001), the tumor volume in experimental group were significantlysmaller than those in negative group and blank group (p=0.001, p=0.001).Conclusions:Silencing Livin gene expression by lentiviral vector-mediated RNAinterferences could inhibit colorectal HT-29cells xenograft growth, increase the sensitivity of the transplanted tumors to chemoradiotherapy. Livin gene wasexpected to become a new target for treatment of colorectal cancer, lentiviralvector-mediated RNA interference technology was expected to be an effectivetool for gene therapy.