The Effect Of DNAzymes Targeting Against HTERT On Telemerase Activity In Human Liver Cancer SMMC-7721 Cell Line

Primary carcinoma of the liver is one of common malignant tumors in China. Although the etiology and pathogenesis is still not entirely sure, may be associated with a variety of factors. Activiation of telomerase and the stable of telomere length are essentially required for cellular immortalization and genetic alteratens. Telomerase , a ribonucleoprotein enzyme , synthesizes DNA-protein complexes which is called telomere at the end of eukaryotic chromosomes that protects chromosome ends from fusion and degradation,ensures the complete replication of telomeric DNA. Telomerase is thought to be necessary for cellular immortality and carcinogenesis consists of three subunits:human telomerase RNA(hTR),human telomerase reversetranscriptase(hTERT),and telomerase- associated protein 1 (TP1). hTERT is a catalytic subunit of telomerase and its expression is the rate-limiting factor of the enzymatic activity of human telomerase. According to studies,the most of examined tumors showed telomerase activity correlating with the expression of the activity-limiting component hTERT. hTERT was found in nearly all types of cancer but not in most normal somatic cells. So it is thought the target of anticancer strategy. Primary caicinoma of the liver has been reported to have a high frequency of detectable telomerase activity and hTERT gene expression was consistent with detectable telomerase activity in 89.5% of hepatoma tissues. This suggested a strong relationship between the expression of hTERT mRNA and telomerase activity in hepatoma tissues. DNAzyme is a kind of DNA molecule with enzyme activities. It can pairs with specific target mRNA and inactivate target mRNA by cutting. At present, most researches focus on 10~23DNAzyme.The structure of 10~23DNAzyme is like hammer ribozyme, containing a highly conserved catalyzing domain and two variable side arm domains. It was shown that transfection with 10~23DNAzyme specifically reduces the expression of target genes. By use of antisense strategy,10~23DNAzyme complementary to hTERT mRNA could be used to inhibit hTERT gene expression and telomerase activity and induce tumor cell apoptosis.Objectives: In this study, we designed and synthesized 10~23DNAzyme aimed directly to hTERT mRNA , then observed the cleavation of hTERT mRNA by 10~23DNAzyme in vitro and in a human liver cancer SMMC-7721 cell line, and observed its inhibition on cell proliferation and the effect on telomerase activity in SMMC-7721 cells. Thus to explore the possibility of DNAzymes become a new gene medicine treated to primary liver cancer in cellular level.Methods: 10~23 DNAzymes DzTi targets against hTERT mRNA and its analogues (DzTiscr ) were synthesized [Oligonucleotides were synthesized with an inverted thymidine Ti at the 3'position to provide resistance to nucleolytic degradation. DzTiscr is the size- matched counterpart of DzTi, containing an active catalytic (15 nt) domain but scrambled arms. The scrambled DNAzyme (DzTiscr) served as a control for sequence specificity]; The cleavage ability of DNAzymes was tested in vitro with mRNA from SMMC-7721 cells; The in vivo hTERT fragment from the SMMC-7721 cells transfected with the DNAzymes were tested by semiquantitative RT-PCR; and the cell proliferation and the telomerase activity from the SMMC-7721 cells transfected with DNAzymes were measured by MTT colormetric assay and TRAP-PCR sliver staining assay. The data was analyzed by q test of ANOVA.Results: 1. The result of electrophoretic analysis of the amplification product of RT-PCR fragments of human telomerase transcriptase(hTERT) mRNA after in vitro cleavage with DNAzymes showed that the lightness of the strips ofβ-actin(592bp) in the three channels were similar, while the luminosity of hTERT strip(222bp)were obviously weaker in the DzTi group when compared with the other two groups. And the result of the semi-quantitative gray scale scan showed that the relative expression quantities of hTERT mRNA in blank control group, DzTi group and DzTiscr control group were 0.718±0.014, 0.283±0.012,0.716±0.019. These data were analyzed by q test of ANOVA that the relative expression of hTERT mRNA of DzTi group was 61% lower than the others groups, and the difference had statistical significance(P<0.01). But the comparation among the other two groups had no significant difference(P>0.05).2. The result of electrophoretic analysis of the amplification product of RT-PCR after transfected with DNAzymes for 48 hours showed that the lightness of the strips ofβ-actin(592bp) in the four channels were similar, while the luminosity of hTERT strip(222bp) were obviously weaker in the DzTi group when compared with the other three groups. And the result of the semi-quantitative gray scale scan showed that the relative expression quantities of hTERT mRNA in blank control group, lipofectamine group, DzTi group and DzTiscr negative control group were 0.726±0.018,0.725±0.011,0.170±0.029,0.710±0.013. These data were analyzed by q test of ANOVA that the relative expression of hTERT mRNA of DzTi group was 76% lower than the others groups, and the difference had statistical significance(P<0.01). But the comparation among the other three groups had no significant difference(P>0.05).3. The result of the rate of the cell proliferation inhibition measured by MTT colormetric assay showed that routine concentration of Lipo-DzTi can significantly inhibited proliferation of SMMC-7721 cells(P<0.05) in a concentration-dependent and time-dependent manner. But the comparation among the other two groups had no significant difference(P>0.05).4. The result of the telomerase activity from the SMMC-7721 cells transfected with DNAzymes for 48 hours detected by TRAP-PCR sliver staining assay showed that the TRAP produets ladders in blank control group, lipofectamine group and DzTiscr negative control group were apparente, but the TRAP produets ladders in DzTi group was decreased significantly, and the lightness of the strips in in DzTi group were obviously weaker than the other three groups.Conclusion: The above results suggested that the designed and synthesized 10~23 DNAzymes targeting against hTERT mRNA could effectively and specifically intertfere with hTERT expression at gene level and decrease the telomerase activity , and then depress the cell growth in human liver cancer SMMC-7721 cells. As a newly found catalytic nucleic acid, DNAzymes will play an important role in the antitumor field.