Construction Of SiRNA Expression Vector And Its Inhibition Of HTERT Gene Expression In MCF-7 Cells

Objectives: To construct the RNAi expression vector of hTERTgene and explore the specific inhibitory effect of this vector on hTERT mRNAexpression in MCF-7 cells, and to lay the experimental foundation for theadvanced research on the protein and cell levels. This research can provide notonly new and effective target of RNA interference on hTERT gene, but also thenew experiment evidence of the usage of expression vector in vivo mediatedRNA interference acting as an efficient gene silencing technology. Thisresearch may present a new approach to the gene therapy of breast cancer,which aim at inhibiting telomerase activity, and is also a helpful exploration ofthe possibility that RNAi applies to tumor research and gene therapy.Methods: We designed an interference target sequence TGT TCA GCG TGCTCA ACT A, which aimed at hTERT gene, and then synthesize two strands ofoligonucleotides which including a hairpin structure according to the sequenceabove. The sequence of the oligonucleotides that formed a double-strand DNAthrough annealing contained seven parts: the BamHⅠsite, 19nt positive-sensesequence, 9nt loop sequence, 19nt antisense sequence, the terminator of RNApolymerase Ⅲ (TTTTTT), the SalI site and the HindⅢ site. The pGenesil-1vector was digested with BamHI and HindIII, and then purified and retrievedthe 4800bp fragment by agarose electrophoresis separation,then the linearvector was generated. It was the double-strand DNA that was inserteddownstream from the U6 promoter of the linear pGenesil-1 vector by T4 ligase,so the siRNA expression plasmid pGenesil-hTERT was constructed throughrecombination. The next step was transfecting the recombinant into thesusceptible cell E.coli DH5α which was prepared by CaCl2 method, andchoosing the positive colony which was screened by kanamycin resistance toamplify by shaking, then extracting the plasmid. To make sure the interferencefragment was accurate, that is to say, the siRNA expression vector targetinghTERT had been constructed successfully, the extracted recombinant wasdigested by PstI and SalI respectively,and to analyze the result by agaroseelectrophoresis and DNA sequencing. At the same time, we constructed thenegative control recombinant that did not aim at any gene by the same method.MCF-7 cells that were in good condition were plated in 6-well cell cultureplates 24 hours prior to transfection, and set up four experimental groups, theywere blank control group, liposome group, negative control group andpGenesil-hTERT group. We added 7.5μl LipofectamineTM2000 + 3μgplasmid/well, and then transfected the compound to the MCF-7 cells (theplasmid was replaced by sterile water in liposome group and do not add anyreagent to blank control group). At 48 hours post transfection, the GFP (greenfluorescent protein) gene of recombinant could express, so transfection ratecould be judged by observing the ratio of green fluorescence cells under thefluorescence microscope. The total RNA from each well of cells was isolatedby TRIzol reagent at the same time. And the concentration, purity andintegrality of total RNA were detected by ultraviolet spectrophotometer andagarose gel electrophoresis. The semi-quantitative two-step RT-PCR whichtook the total RNA as template and took β-actin as the control was used todetermine the interference efficiency. Agarose gel electrophoresis andsemi-quantitative gray scale scan were used to detect the relative expression ofhTERT mRNA in every group. Results: ① After the pGenesil-1 plasmid hadbeen digested with BamHI and HindIII, a linear vector strip which was about4800bp could been seen through electrophoresis analysis. ② The recombinantswere digested by PstI and SalI respectively and agarose gel electrophoresisshowed that the recombinants could not be digested by PstI because the PstIsite was replaced by the insert, but the recombinants could be digested by SalIbecause a SalI site was designed in the target gene fragment which inserted intothe plasmid, so yielded a nearly 400bp fragment. Besides, the sequencingidentification also confirmed that the target sequence had inserted into thepredicted site precisely and the recombining plasmids were successfullyconstructed. ③ Observe the MCF-7 cells under the fluorescence microscope48h after transfection, and judge the transfection rate that LipofectamineTM2000 to MCF-7 cells was above 80% according to the ratio of the cells shinninggreen fluorescence. ④ Total RNA sample in each group was determined by UVspectrophotometer 48h after transfection. The results indicate that the density ofRNA was 0.7~3.0 μg/μl, and the purity (OD260/OD280) of it was 1.7~1.9, whichcould satisfy the request of reverse transcription. 2% agarose gelelectrophoresis indicated that three rRNA strips were visible, including 28S,18S and 5S,and among them, the strips of 28S and 18S were seen clearly, andthe luminosity of the former was about twice as high as the latter, whichshowed the total RNA samples were enough integral to acting as amplificationtemplate of RT-PCR. ⑤ The result of electrophoretic analysis of theamplification product of RT-PCR showed that the lightness of the strips ofβ-actin (546bp) in the four channels were similar, while the luminosity ofhTERT strip (111bp) were obviously weaker in the pGenesil-hTERT groupwhen compared with the other three groups. And the result of thesemi-quantitative gray scale scan showed that the relative expression quantitiesof hTERT mRNA in pGenesil-hTERT group, negative control group,lipofectamine group and blank control group were 0.188 ± 0.020, 0.737 ± 0.009,0.733 ± 0.015 and 0.753 ± 0.014. These data were analyzed by q test ofANOVA that the relative expression of hTERT mRNA of pGenesil-hTERTgroup was 75% lower than the others groups, and the difference had statisticalsignificance (P<0.01). But the comparation among the other three groups hadno significant difference (P>0.05). Conclusions: ① The shRNA interferencesequence that had designed and synthesized had cloned to pGenesil-1expression vector accurately, and the siRNA expression vector that aimed athTERT constructed successfully, which can provide new method to the deepresearch of the effect of telomere and telomerase in tumor cell. ② Therecombinant could transfect to MCF-7 cells high efficiently throughlipofectamineTM 2000, and then ensured the highly potent expression of therecombinant in cells. ③ The target sequence of RNAi which aimed at hTERTgene designed efficiently. And the RNAi expression vector in vivo that aimed atthis target sequence efficiently and specifically inhibited the expression ofhTERT mRNA in MCF-7 cells. Compared to the antisensenucleic acidstechnology, the inhibitory effect elevated obviously, so it is an effectual andfeasible approach of using expression vector in vivo mediated RNAi tosuppress the expression of the tumor related gene. ④ Because of the highlydependability between the expression of hTERT mRNA and the telomeraseactivity, when the siRNA expression vector that constructed in this experimentexerted a distinct inhibitory effect on the expression of hTERT mRNA, it mustbe able to down-regulate the telomerase activity, and then depress the cellgrowth and induce apopotosis. Hence, this is a feasible strategy of gene therapyof tumor.