Adenovirus-Delivered HTR-siRNA Inhibits Telomerase Activity In Cervical Cancer And Its Anti-tumor Activity

Background:Telomerase is a specialized reverse transcriptase responsible for synthesizing telomeric DNA at the ends of chromosomes. Telomerase has been found to be up-regulated in 85-90% of tumor cells, but only 4.2% in mormal somatic cells or benign tumor cells. Thus, telomerase is an attractive target for anti-tumor therapy. Six subunits comprising the telomerase complex have been identified. Of them, both human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) are very important. The template region of hTR encompasses 11 nucleotides (5'-CUAACCCUAAC-3'), which is complementary to the human telomere sequence (TrAGGG)_n. The hTR serves as a template, and together with hTERT, synthesizes and adds new TTAGGG repeats to the ends of telomeres, thereby elongating it. The RNA component of telomerase is absolutely required for the function of telomerase complex and is therefore a natural target for anti-telomerase agents. M ny methods, such as short interfering RNA (siRNA), hammerhead ribozymes and antisense oligonucleotides, have been designed to target hTR, especially the template region of the 11 nucleotides. Of these methods, siRNA has been shown to induce strong and efficient RNAi in mammalian cells. Thus it has been routinely used in gene silencing by transfection of synthesized 21-23 nucleotide siRNA or plasmid derived shRNA. Nevertheless, transient siRNA expression, low and variable transfection efficiency remained problems for chemically synthesized and vector derived siRNA. Some scientists prefer to use viral vectors to RNA interference. Recently, several retrovirus, lentivirus and adenovirus plasmid systems have been developed for efficient delivery of shRNA into mammalian cells, where the shRNA might be cut into siRNA by Dicer. These viral vectors have been designed to produce shRNA driven by either the U6 or the H1-RNA promoter for efficient, uniform delivery and immediate knockdown of target gene(s). Now, the shRNA-encoding viruses have opened numerous novel opportunities for tumor, viral infection and nervous system deseases research across the field of bilolgy. Because adenoviruses have many advantages in terms of anti-tumor therapy, such as high efficiency, transient duration, targeting both proliferative and non-proliferative tumor cells, no risk of insertional mutagenesis, and liver tropism, they are popular for gene therapy trials, and extensive to be used to transfect cells with expressing hairpin-RNA. The two traditional methods of adenovirus construction are based on homologous recombination either in Escherichia coli BJ5183 or in human embryonic kidney 293 cells. But for difficult to manipulate or low efficient to recombinate, adenoviruses generation has poven difficult by using the two methods.Objective:In this study, we will utilize a commercially available adenovirus system (BDTM Knockout Adenoviral RNAi System 1) to construct an adenovirus vector against hTR template region by a ligation method in vitro, which depended on pSIREN and pAdeno-X. A series of experiments will be then conducted to evaluate the effect of the hTR-siRNA adenovirus on hTR mRNA gene silence, telomerase activity inhibition and anti-tumor in vitro and in vivo. All these results will offer reliable data and powerful way to tumor gene therapy. Furthermore, there are not similar reports about RNAi adenovirus which was constructed by ligation and used to silence hTR gene.Methods:1 Construction of RNAi adenovirus(1) construction the shuttle plasmidBased on BD siRNA designing system, principals and BLAST assay, the 67-bp oligonucleotides and complementary oligonucleotides encoding hTR-specific siRNA were synthesized. The target sequence corresponds to bases 136-154 of hTR (GenBank accession number U86046). The oligonucleotides were annealed and ligated to the BamH I and EcoR I sites of RNAi-Ready pSIREN-Shuttle to produce pSIREN-hTR plasmid. The inserted sequences were confirmed by Pst I digestion and sequencing. The negative control siRNA annealed oligonucleotides were also introduced into pSIREN-NT as described above for hTR.(2) construction the skeleton plasmidThe U6-RNA promoter and the siRNA coding insert were cut from pSIREN-hTR with PI-Sce I and I-Ceu I, and ligated with pAdeno-X(32.6kb) digested by the same restriction enzymes. The products of ligation were transformed into E. coli (DH5a) to get the pAd-hTR. The pAd-hTR was confirmed by restriction enzyme mapping, PCR screening and sequencing.(3) package of adenovirusRecombinant adenoviral pAd-hTR was linearized with Pac I and purified using the PCR purified kit. HEK-293 cells with 90% density were used to pack this adenovirus and transfected with 4μg Pac I linearized pAd-hTR in the presence of LipofectamineTM 2000 in serum-free and non-antibiotic medium. Ad-hTR-siRNA was generated. Then this virus was harvested, concentrated, titrated, amplified and purified. Ad-NT-siRNA, which derived from pSIREN-NT as a negative virus control, was constructed and packed in a similar way.2 RNAi adenovirus silenced hTR gene in tumor cellsDifferent tumor cells and liver cell line, HL-7702, were infected with 100 MOl of the recombinant adenoviruses Ad-hTR-siRNA and Ad-NT-siRNA, respectly. End-Point Dilution Assay, TRAP-ELISA, Real-time PCR and FCM were used to analyze virus sensitivity, telomerase activity, hTR mRNA, apoptosis rate and hTERT protein expression.3 anti-tumor assay In vivo with RNAi adenovirusThis assay was performed using BALB/c nu/nu mice. HeLa cells were suspended as single cell suspension in serum-free DMEM shortly before subcutaneous inoculation into the right armpit. Each mouse received a 0.2 ml of 0.8×10⁶ HeLa cells. After successful implantation, the mice were randomized into four groups. The mice were intratumorally injected with 0.1ml of serum-free DMEM alone, or Ad-hTR-siRNA (10⁹ pfu/mouse), or Ad-NT-siRNA (10⁹ pfu/mouse), or 120μg of cisplatin as positive control. These different treatments were given once every 3 days for 15 consecutive days. After the last injection, mice were observed for 7 days continuously and sacrificed at the end. Tumors were excised, weighed, sectioned and assessed for morphology by HE and for apoptosis by the TUNEL assayResults:1 Restriction enzyme mapping, PCR screening and sequencing demonstrated that pSIREN-hTR, pAd-hTR and Ad-hTR-siRNA were constructed successfully. At the same time, the negative control virus Ad-NT-siRNA was obtained. The titers of the fourth passage viruses were 1.0×10¹⁰ pfu/ml and 1.5×10¹⁰pfu/ml, separately. 2 The arrangement of virus sensitivity in turn was HeLa≥HepG2＞A549＞HL-7702. As compared with Ad-NT-siRNA, Ad-hTR-siRNA reduced both hTR mRNA levels (70.21%) and telomerase activity (58.87%) of HeLa cells significantly, increased apoptosis rate (29.7%). But the telomerase activity of HL-7702 and hTERT protein didn't show the tendency of decrease.3 Tumors-implanted were established successfully by 100% in the right armpit with HeLa cells. As compared with DMEM- and Ad-NT-siRNA-treated mice, Ad-hTR-siRNA could slow down tumor growth, decrease tumor volume (45.48%) and tumor weight (34.68%) and push forward the apoptosis and necrosis of tumor cells. The TUNEL positive cells were about 11.8%. But the anti-tumor activity of Ad-hTR-siRNA didn't catch on cisplatin's.Conclusions:Taken together, our results demonstrated that adenovirus-delivered siRNA could efficiently and specifically knock-down hTR, decrease telomerase activity, and inhibit tumor cells growth in vitro and in vivo. All of these indicated the prospect of applying this siRNA expressing recombinant adenovirus system in cancer gene therapy.