Lentivirus-Mediated RNA Interference Of Human Telomerase Reverse Transcriptase In Cervical Cancer And Functional Analysis In The Caski Cell

Chapter 1 Construction and Identification of a plasmid expressing small hairpin RNA against hTERT GeneObjective:To construct a plasmid expressing a small hairpin RNA (shRNA) to knockdown expression of human telomerase reverse transcriptase (hTERT), and to observe its effects on hTERT expression in caski cervical cancer cells.Methods:Designer3.0 software (Genepharma) was used to design an shRNA targeting hTERT. The shRNA was cloned into the pGLV3/Hi/GFP+Puro vector and confirmed by sequencing. Cells were divided into blank control group, negative control group and hTERT interference group. The resulting plasmid was transfected into caski cervical cancer cells and expression of the GFP reporter was analyzed. In addition, The expression of hTERT gene (described as relative ratio with GAPDH) was examined by real-time polymerase chain reaction(real-time PCR).Results:A plasmid expressing an shRNA targeting hTERT was constructed and used to generate recombinant lentivirus. Lentiviral infection of caski cells led to lower expression of hTERT mRNA than controls (P<0.01)Conclusion:A plasmid expressing shRNA targeting hTERT can effectively knockdown endogenous hTERT expression. This reagent may prove useful for understanding the role of telomerase in the pathogenesis of cervical cancer.Chapter 2 Biological effect of the hTERT gene inhibition by lentivirus-mediated small interfering RNA on cervical cancer caski cells acted in experimental study in vitroObjective:To explore the feasibility of lentivirus mediated small interfering RNA(siRNA) silencing the hTERT gene and its biological influence on the caski cell.Methods:The expression vector LV-shhTERT was established and packaged and the lentivirus particles containing hTERT-shRNA was collected for use. The experiment was divided into blank group, green fluorescent protein (GFP)-NC-LV group (control group)and si-hTERT-LV group (interference group). Cell proliferation in the presence and absence of shRNA was measured using the cck-8 assay. The cell cycle and apoptosis were detected by flow cytometry respectively. The hTERT expression, apoptosis rate and cell cycle in three groups were compared using one-way analysis of variance (ANOVA) and t-test.Results:The cell growth speed and monoclonal ability of hTERT gene in the interference group was significantly lower compared with the control group and the blank group. There were significant differences (P<0.05). The G1 phase cell ratio in the interference group was significantly increased compared with that in the blank group and the control group and the difference was statistically significant (P<0.05).The S phase cell ratio in the interference group was significantly decreased compared with that in the blank group and the control group and the difference was statistically significant (P<0.05). The cell apoptotic rate in the interference group was significantly higher than that in the control group and the blank control group and the difference was statistically significant (P<0.05)Conclusions:The lentivirus-mediated siRNA expression vector can effectively lead to cell growth inhibition and apoptosis in vitro,the cell cycle arrest in G0/G1 phase.at the same time hTERT gene subdued cervical cancer caski cell monoclonal ability,...