Experiement On Inhibitory Effect Of RNA Interference Targeting Of HTERT Gene Of Large Intestine Cancer HT-29 Cells

Objective To provid the theory evidence of gene therapy for large intestine cancer cells by the special inhibitory effect of RNAi on hTERT gene of large intestine cancer cells .Methods 1.Cultivation and passage of large intestine cancer HT-29 cells.2.Small interfering RNA (siRNA)targeting of hTERT gene was synthesized by chemistry,and siRNA was transfected into HT-29 cells by using liposome.3.Dividing into four groups: Control group(untransfected HT-29 cells group),Lipofectamine group,N-hTERT group(Negative group)and sihTERT group(RNA interference group).4.The telomerase activity , hTERT mRNA and hTERT ptotein was detected by telomerase repeat amplification protocol (TRAP), reverse transcriptase polymerase chain reaction(RT-PCR) and Western blot respectively.Flow cytometry was used to observe the proliferation and apoptosis of HT-29 cells induced by siRNA.Results 1. The fluorescence photos showed that the transfection efficiency of HT-29 cells was 60%.2. The telomerase activity of Control group,N-hTERT group,Lipofectamine group and sihTERT group was 1.14±0.21,1.10±0.19,1.17±0.22,0.73±0.14;Compared with the other groups, the telomerase activity of sihTERT group was inhibited significantly(p<0.01).3. The hTERT mRNA of Control group,N-hTERT group,Lipofectamine group and sihTERT group was 0.87±0.15, 0.91±0.16, 0.85±0.14, 0.47±0.08;Compared with the other groups, the expression level of the hTERT mRNA of sihTERT group was declined remarkably (p<0.01).4. The hTERT protein of Control group,N-hTERT group,Lipofectamine group and sihTERT group was 0.62±0.12, 0.67±0.13,0.59±0.12, 0.37±0.07;Compared with the other groups, the expression level of the hTERT protein of sihTERT group was declined remarkably (p<0.01).5. The apoptosis rate of Control group,N-hTERT group,Lipofectamine group and sihTERT group was (7.5±0.21)%,(8.6±0.24)%,(8.3±0.15)%和(49.5± 0.26)%;Compared with the other groups, the apoptosis rate of sihTERT group was elevated remarkably (p<0.01).Conclusion RNAi can specificly scilence the hTERT gene of large intestine cancer cells by down-regulation the expression level of the hTERT mRNA and its protein, depressing the telomerase activity.Therefore, RNAi can inhibit the proliferation of the cancer cells and induce their apoptosis. The theory evidence of gene therapy for large intestine cancer cells might be expected.