| Telomeres, the physical ends of linear chromosomes consist of tandem repeat DNA sequences and associated proteins. The telomere play a key role in replication of chromosomes, protects them from recombination, end to end fusion, and recognition of chromosomes, etc. Telomerase is a ribonucleoprotein(RPN) complex,a unique reverse transcriptase. It consists of three essential parts: an RNA component (hTR in humans), telomerase associate protein (TP1), and the catalytic subunit-human telomerase reverse transcriptase (hTERT). hTERT is the only limiting factor for telomerase activity. Telomerase synthesizes telomeric DNA sequences and prevents the telomere shortening. It plays an important role in cellular aging and tumorigenesis. Because the majority of human cancers show high levels of telomerase activity, it is considered that telomerase as a promising diagnostic and therapeutic target for neoplasia. Many approaches targeting telomerase developed in cancer arena, but they are blocked in pre-clinic. Since the RNA interference (RNAi) first described in 1998, it has largely been exploited as a powerful tool of cancer therapy targeting telomerase. In our research, we want to utilize RNAi to inhibit hTERT gene expression in vitro and in vivo, and to observe the biological behaviors of HeLacells after silencing of hTERT, and to investigate the potential mechanisms. Thus we can explore the feasibility of cancer therapy targeting hTERT. The results were as follows:The first part: screening of a valid shRNA targeting hTERT and the stable cell lines with telomerase activity inhibition by RNAi.According to the rules of RNAi, we designed four oligonucleotides targeting hTERT gene. Four shRNAs (A,B,C,D) were produced by transcription in vitro. We assessed the expression of hTERT by immunofluorescence under LCSM after transient transfection of shRNAs by lipid formulation and found that the shRNA (B) down-regulated hTERT gene efficiently. Thereby, we constructed the plasmid, pGCsilencerTMH1/Neo/GFP/shRNA, to produce the shRNA (B) in vivo. Accordingly, we choose pGCsilencerTMH1/Neo/GFP/non as control. Both of the two plasmids were transfected to HeLacells. After selection by G418(1000μg/ml), we got two stable cell lines with silenced hTERT gene (T1,T2) and the other two sable cell lines with normal hTERT gene expression(N1,N2). Then, we examined the expression of hTERT gene by Western Blot, and the telomerase activity by TRAP-ELISA, and the telomere length by Southern Blot to certify the validity of RNAi. Thus we can make more advance experiments both in vitro and in vivo.The second part: observation on the biological behaviors of HeLacells after hTERT gene silencing by RNAi.We assessed the biological behaviors of experimental HeLacells and control cells, that is①cells transfected transiently by shRNA(B) vs. non-silencing RNAi;②T1 and T2 vs. parental HeLacells and N1 cell clone. We investigated cell proliferation by cell growth curve and Brdu incorporation assay, examined the cell adhesion by CCK-8 assay, and observed cell spreading and migration by LCSM, and assessed cell invasion by boyden chamber assay, and at last observed the tumor growth of xenografts in nude mice. We found that the biological behaviors of HeLacells changed notably after the telomerase activity inhibited by RNAi. The cell proliferation was impaired, and the number of Brdu incorporation positively decreased. The ability of cell spreading and adhesion on fibronectin declined significantly. At the same time, the ability of cell migration and invasion were also down-regulated. At last the volumes of xenografts in nude mice diminished obviously.The third part: to explore the potential mechanisms.We assessed the cell cycles by FCM, and utilized Hoechst33258 and TUNEL staining to investigate the apoptosis cells. We examined the activation of caspase-3 and caspase-8 by absorption spectrometry method. We observed the ultra-microstructures of HeLacells under transmission electron microscope. And under laser scanning confocal microscope (LSCM), the change of calcium ion (Ca2+) channel was dynamically observed by Fluo-3/AM staining. We observed the expression of cell skeleton proteins and FN assembly under LSCM. At last, the p-AKT was examined by Western Blot assay. The results as followed: after inhibition telomerase activity by RNAi, the cell cycle of HeLacells change markedly. The number of S-phase cells decreased, and most cells were inhibited in G0/G1 phase, and the level of SPF and PI declined. The number of apoptosis cells increased notably, caspase-3 and caspase-8 activated in some degree. Under the transmission electron microscope, we observed the changes of ultra-microstructure, which is the endoplasmic reticulum (ER.) broadened strongly. The result of Fluo-3/AM staining showed that the store of intracellular Ca2+ elevated. The results of IF staining show that the expression of filamine reduced, and the ability of FN assembly decreased significantly. At last, the level of p-AKT declined remarkably. All of these results called the attention to that there would be some signals produced in the process of RNAi, and the signals might transduce by the AKT signal transduction.To sum up, we utilized the transcription methods both in vitro and in vivo to screen a valid shRNA targeting hTERT gene and get stable cell lines with silenced telomerase activity. The biological behaviors of HeLacells after RNAi appeared significantly changes, which were impair cell proliferation, increased of apoptosis cells, and decreased ability of cell spreading, cell adhesion, cell migration and cell invasion. Telomerase inhibition by stable RNAi impaired the tumor growth of xenografts in nude mice. The ER broadened strongly and the intracellular store of Ca2+ elevated. Besides of these, there were down-regulation expression of filamin, FN assembly and the p-AKT. All of these results called the attention to that there would be some signals produced in the process of RNAi, and the signals might convey by the AKT signal transduction between intra and extra cellular. |
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