Influence On SGC-7901 Cell Proliferation And Telomerase Activity By Stably RNAi HTERT And TRAP Gene

Blocking oncogenes highly specific in malignant cells by RNAinterference is a new hope for cure cancer. Previous studies have shownthat more than 85% of malignant tumor cells demonstrate telomeraseactivity, whereas most normal cells do not express this enzyme. Humantelomerase reverse transcriptase (hTERT), a major components of humantelomerase, is the limiting component of telomerase activity, hTERT isexpressed in 80%-90%tumors and play an important role in theimmortality of tumor cells. Therefore, hTERT might serve as a goodtarget for cancer therapy. The regulationg the expression of hTERTfalls into two major classes: activation of the promoter of hTERT bytranscription factor and level regulation after transcription.However, thelong time lag which between telomerase inhibition and the arrest of cellproliferation demand long-term of telomerase inhibittion to achievebetter anti-cancer effect. In this study, we utilize the characteristic thatRNA interference have long-term gene silencing response induced byshRNA in cells to investigate anticancer effects of regulation the hTERTgene at the two classes. So, we have done these works hereinafter: 1 Constructed an shRNA expressing vector direct to hTERTand TRAP gene.63 nt oligos were chemically synthesized, which is targeted totelomerase hTERT gene or TRAP gene. After annealed, oligos wereligated into the psilencer2.1-U6 neo siRNA expression vector by usingT4 DNA ligase. Recombinant psi-hTERT, psi-TRAP vector wasidentified by digesting with BamHI and HindⅢand confirmed thecorrect insertion by sequencing. The results demonstrated that 63bp hadbeen inserted the expected site. Furthermore, the insertion sequence wereexactly correct. So we think that psi-hTERT, psi-TRAP vector expressingRNAi system has been constructed successfully.2 Effects of psi-hTERT on proliferation and telomeraseactivity of SGC-7901 Cellpsi-hTERT were introduced it into SGC-7901 cells. A population ofcells that stably expressed the shRNA were selected by G418 andcontinuously cultured in a medium with half the antibiotic concentrationfor 3 months.Then,real-time PCR demonstrated that level of hTERTmRNA was knocked down 75.1% in psi-hTERT1,85.9% in psi-hTERT2;western blot demonstrated that level of hTERT protein was knockeddown 68.6% in psi-hTERT1,80.4% in psi-hTERT2. Assayed byTRAP-ELISA, the activity of telomerase was inhibited,and knockeddown 36.4% in psi-hTERT1, 51.2% in psi-hTERT2. MTT cellproliferation curve showed that the proliferation of SGC-7901transfected with psi-hTERT1 or psi-hTERT2 was inhibited. The results indicated that psi-hTERT could significantly downregulating hTERTexpression both in mRNA and protein level, inhibit cell proliferation andincrease cell apoptosis.3 Effects of psi-TRAP on proliferation and telomerase activityof SGC-7901 Cellpsi-TRAP were introduced it into SGC-7901 cells, the stablyexpressing shRNA cells were selected by G418 and continuouslycultured in half the antibiotic concentration for 3 months. Then,real-timePCR demonstrated that level of TRAP mRNA was knocked down 85.0%in psi-TRAP1,54.6% in psi-TRAP2, level of hTERT mRNA psi-TRAP1was 0.136±0.02. psi-TRAP2 was 0.315±0.03, distinctly lower thanthe level of psi-control.Western blot demonstrated that level of hTERTprotein was 0.463±0.01 in psi-TRAP1,1.35±0.04 in psi-TRAP2, 1.980±0.01 in psi-control. Assayed by TRAP-ELISA, the activity oftelomerase was inhibited, and knocked down 36.3 % in psi-TRAP 1,7.1%in psi-TRAP2. MTT cell proliferation curve showed that theproliferation of SGC-7901 transfected with psi-TRAPlor psi-TRAP2was inhibited.The results showed that psi-TRAP could significantlydecreased the expression of TRAP mRNA, hTERT expression, and thetelomerase activity, decrease cellular proliferation capacity, and increasecell apoptosis.4 Effects of psi-hTERT1combined with psi-hTERT2 orpsi-TRAP1 on proliferation of SGC-7901 cellpsi-hTERT1 were respectively combined with psi-hTERT2 (name as psi-h1+h2) or psi-TRAP1(name as psi-h1+t1), then,were introducedit into SGC-7901 cells, the level of hTERT mRNA and protein weredeteced, the activity of telomerase and the anti-proliferation effects weredetected. The results showed that psi-h1+h2 and psi-h1+t1 couldsignificantly decrease the expression of hTERT, decrease cellularproliferation capacity, and increase cell apoptosis, but psi-h1+t1 couldnot significantly decreased the telomerase activity.5 Effects of psi-hTERT combined with chemotherapy drugson proliferation of SGC-7901 Cellpsi-hTERT and chemotherapy drugs (DDP,5-Fu)were used to treatSGC-7901 Cell, drug sensitivity test was performed with MTTmethod, the result showed that psi-hTERT increased susceptibility toDDP or 5-FU in SGC-7901 Cell.In summary, These data indicate that hTERT is ideal target forcancer therapy and psi-hTERT and psi-TRAP have long-termanti-proliferation effects on SGC-7901 cells,and increased susceptibilityof chemotherapy drugs. These findings suggest that RNA interferencehTERT gene or/and TRAP gene may provide a new pathway for cancerbiotherapy, the methods of related gene combination therapy orcombined with chemotherapy drugs is developing trend of cancertherapy.