Abstract Objective To investigate the liposome-mediated c myc gene antisense oligonucleotide (Antisense phosphorothioate oligodeoxynucleotide, ASODN) MCF 7 cell growth and human telomerase reverse transcriptase (human of telomerase reverse transcriptase, hTERT) the impact of gene expression. C myc positive antisense oligonucleotide (ODN) were introduced into each group MCF 7 cells, MTT method, reverse transcription polymerase chain method and flow cytometry to detect the expression of growth, hTERT mRNA levels and the rate of apoptosis. C myc ASODN results transfected MCF 7 cells 24h, cell growth was inhibited (P <0.05), and hTERT mRNA expression was significantly reduced; gradually increased with the prolonged duration of action of antisense nucleic acid, the number of apoptotic cells, cell growth and hTERT expression decreased gradually apparent over time. Control group, LR SODN group, LR ASODN 24h group LR ASODN 48h, 72h group compared to significant differences (P <0.05). The conclusion c myc antisense oligonucleotide significantly lowered cell hTERT gene expression activity, induced MCF 7 cell apoptosis and inhibit MCF 7 cell growth; certain extent, its effect was positively correlated with the time.
Key words breast tumors c myc human telomerase reverse transcriptase antisense nucleic acid MCF 7 cells
Telomerase to maintain chromosome structure and function of a ribose nucleoprotein complexes are highly expressed in almost all human malignant cells . The most important component of human telomerase catalytic subunit, also known as human telomerase reverse transcriptase (human of telomerase reverse transcriptase, hTERT), is considered to be the rate-limiting factor of telomerase activation. With the hTERT gene cloning  success, the focus of its study of the regulation of telomerase research.
c myc proto-oncogene involved in cell proliferation and differentiation by gene amplification and expression of reverse transcriptase insertion and chromosomal translocation disorders have a high level of expression in the majority of tumors [3,4 and c myc is a transcription factor regulating hTERT gene expression . Many studies have reported that c myc and hTERT expression is closely related to the malignant cells. Oh et al  reported that, compared with normal cells, myc protein expression in tumor cells was significantly higher; activate or inhibit the expression of myc protein can change the hTERT promoter activity that myc protein regulation of hTERT promoter may promote tumor with formation. At present, studies have shown , c myc and hTERT expression regulation was no significant correlation. Thus, researchers have c myc expression and regulation of hTERT relations still hold different views.
The selection of c myc and hTERT were highly expressed in breast cancer MCF 7 cells, mediated antisense technique Lipfect AMINETM c myc antisense oligonucleotide was transfected into MCF-7 cells, the observed inhibition of cell growth, cell apoptosis and hTERT gene expression changes, provide a theoretical basis for telomerase inhibition in breast cancer gene therapy, and to explore further from the reverse angle c myc role in regulation of hTERT gene expression.
1 Materials and methods
1.1 Cell culture
Using DMEM medium containing 5% FBS (60 ℃ the inactivated 30min), 100U/ml penicillin, 100μg/ml streptomycin, human breast cancer MCF 7 cell lines cultured under 37 ℃, 5% CO2 saturated humidity conditions. The experiment used the exponential growth phase cells.
1.2c myc antisense oligonucleotide transfection
c myc is an antisense oligonucleotide by Shanghai Sangon synthesis parallel full-thio phosphorylation NCBI blast confirmed no homology to other known human gene. Sequences: antisense sequence (ASODN): 5 ‘ AAC GTT GAG GGG CAT 3’; sense sequence (SODN): 5 ‘-ATG CCC CTC AAC GTT 3’. The cells were divided into a control group, LR SODN group LR ASODN group. Transfected in accordance with the literature  above, and some changes: ASODN and SODN, final concentration of 2.5 μmol / L, the liposome is relatively concentration of 10%; cells of the control group added only the same amount of medium.
1.3MTT assay cell growth
To remove c myc SODN and ASODN respectively role 24h, 48h, 72h after each group of cells, each well was added at a final concentration of 5mg/ml MTT solution 200 μl of 5h after the DMSO dissolved oscillation 10min microplate reader measured 570nm wavelength OD value. Draw the cell survival curve. The inhibitory rate (%) = (1 – treated OD value / OD value of the control group) × 100%. The experiment was repeated five times.
The 1.4 RT PCR was used to detect mRNA expression levels of hTERT gene
the hTERT gene PCR amplification cited material reference Takakura such as the approach to design, and by the GenBank retrieved confirmed of hTERT upstream primer sequence: 5 ‘ CGG AAG AGT GTC TGG the AGC AA 3’; of hTERT downstream primer sequence: 5 ‘ GGA TGA the AGC GGA GTC TGG A 3 ‘, amplified fragment length 145bp. An internal control β actin (β actin) gene amplified fragment length of 540bp. The primers were synthesized by Shanghai Sangon.
Were collected groups MCF-7 cells, total RNA was extracted using the UV spectrophotometer RNA concentration and purity, 1.2% formaldehyde denaturing agarose gel electrophoresis detection of RNA extracted sample integrity. Accordance with MMLV one-step RT PCR amplification reaction, β muscle actin (β actin) as an internal control, set 50μl reaction system: template RNA 1μl, 2 × reaction mixture of 25 μl MMLV / Taq Mix 1 μl of of hTERT upstream and downstream primers 1μl, internal control β actin gene downstream primer 0.3μl, added to 50 μl of sterile double distilled water. 37 ℃ 30min, 94 ℃ 2min; 94 ℃ 15sec, 57 ℃ 30sec, 68 ℃ 90sec, 35 Ge circulation final 68 ° C for 5min. Amplification products on a 1.7% agarose gel electrophoresis, the electrophoresis results of RT PCR products were analyzed by gel image analysis, hTERT mRNA relative expression levels of hTERT β actin grayscale scanning ratio, the experiment was repeated 5 times.
1.5Annexin Ⅴ FITC / PI double staining flow cytometry analysis of transfected cells count detected apoptosis
Annexin Ⅴ FITC / PI apoptosis detection kit was purchased from the the Biovision company. Collection of cells in each group with 0.25% trypsin digestion, cold PBS and washed twice with 1 × Annexin Ⅴ buffer suspension cells, adjusting the cell concentration of 1 × 10 6 / ml, take 100 cell suspension (1 × 105 cells) added 5μl Annexin Ⅴ the FITC and 5μl PI, mix well and incubated for 15min at room temperature in the dark. 400μl 1 × Annexin Ⅴ buffer per tube, flow cytometry within 1h after filtration. Annexin Ⅴ positive / PI negative judgment for early apoptosis. The experiment was repeated five times.
1.6 statistical analysis
Data ± s single-factor analysis of variance by SPSS11.0 software statistical package complete. Inspection standards: α = 0.05.
2.1 different treatment groups MCF 7 cell growth
LR mediated by antisense c-myc role after 24h, cell growth appears inhibited; After 48h, cell growth inhibition rate was significantly higher. Each group OD values were compared: LR ASODN 24h, 48h, 72h group and the control group, compare LR SODN group, the difference was statistically significant (P <0.05). 0.05)，见表1。">Blank control group compared with the LR SODN group, no significant difference (P> 0.05), are shown in Table 1. Table 1 LR c myc antisense oligonucleotide mediated growth of MCF 7 cells with the control group, the LR SODN group, P <0.05; * with the control group, LR SODN each group and LR ASODN 24h group, P <0.012.2c myc ASODN MCF 7 cells hTERT mRNA expression levels
The MMLV one-step RT PCR reaction amplification, gel electrophoresis under UV light directly observed each group to see the light and shade of different hTERT 100bp DNA ladder Marker 145bp amplification product was visible a consistent shading within the control β 540bp at actin amplification product, as shown in Figure 1. UV gel imaging grayscale scanning, one-way ANOVA analysis, results showed that: in ASODN role hTERT mRNA relative expression in MCF 7 cells 24,48,72 h respectively with the control group, compared to LR SODN group, the difference 0.05）。">statistically significant (P <0.01); the while SODN acting on MCF 7 cells 24,48,72 h hTERT mRNA relative expression, respectively, compared with the control group, the difference was not statistically significant (P> 0.05). And within a certain range, LR ASODN hTERT mRNA relative expression level with time increments significantly reduced, as shown in Table 2.
M: 100bp DNA ladder was Marker;: LR ASODN for 72h; 2: LR ASODN for 48h;: LR ASODN for 24h; 4: LR SODN for 72h; 5: LR SODN handle 48h ; 6: LR SODN for 24h; 7: control group Table 2 LR c myc antisense oligonucleotide mediated MCF 7 cell apoptosis and expression of hTERT gene # with the control group and LR SODN, each compared with the control group and the LR SODN each group, P <0.01; group, P <0.05; * Δ with the control group, the LR SOND each group and LR ASODN 24h group, P <0.01; ※ with control group, LR SODN group and LR ASODN compared to other groups, P <0.012.3 flow cytometry detection analysis of different time segment transfected cells to apoptosis
MCF 7 cells by the LR-mediated c myc antisense treatment, flow cytometry within a certain range, with time, the rate of apoptosis was significantly higher, as shown in Table 2. Statistical analysis showed that: the control group, no significant difference (P> 0.05) between the LR ASODN 24h group and LR SODN groups; LR ASODN 48h, 72h group and the control, LR ASODN 24h and LR SODN each for The difference was statistically significant (P <0.05) compared.
The upregulation of hTERT gene expression in telomerase activation and c myc is an important factor involved in hTERT regulation, it can lead to tumorigenesis by activating the expression of hTERT. Sequence analysis of hTERT found [8,9], multiple potential transcription factor binding sites in the intron of the hTERT gene structure and promoter sequence, c myc and hTERT gene promoter E-box binding and activation of target genes transcription, thus affecting the expression of telomerase activity and cell proliferation and differentiation. Lin Yong et al  using transient transfection assays using liposomes LipfectAMINETM (LR) mediated containing c mycDNA plasmids were transfected into the liver cancer HepG2 monkey kidney COS 7 cells and NIH3T3 cells, results found that c myc can directly activate the expression of hTERT promoter activation of c myc was associated with dose was positively correlated. Oh et al  also applied c myc antisense nucleic role in Hela cells, c myc and hTERT gene expression were decreased and both were parallel.
In this study, using relative quantitative RT PCR detection the antisense c myc role MCF 7 cells, hTERT mRNA expression level: c myc SODN role MCF 7 cells 24h, 48h and 72h, hTERT mRNA expression had no obvious change (P> 0.05); c myc ASODN role in MCF 7 cells after 24h of hTERT mRNA in the relative expression of the amount that decreased inhibition rate of 25%, 48h inhibition rate rises further after 72 hours, respectively. 62% and 80%, compared with the control group, there was significant difference (P <0.01). The suppressing effect can be seen to gradually increase with time. This result is further proof, c myc hTERT gene expression regulatory factors. c myc upregulation of hTERT gene expression levels, and further up the telomerase activity, which is an important mechanism for the regulation of telomerase activity.
In addition, in this experiment, c myc antisense oligo nucleotide role 72h MCF 7 cells, hTERT mRNA expression levels can be attained 0.143 ± 0.011, inhibition rate of 80%, that is not completely hTERT gene expression inhibition, consistent with the Superficial et al  conclusions. That c myc antisense nucleic acid can only cut hTERT gene expression, the gene can not be completely closed. This result is further proof of c myc hTERT regulation is not the only factor.
Although the hTERT promoter region E box c myc interaction, and many research results have confirmed that, but the study by RT PCR method to detect telomerase in breast tissue and c myc mRNA  and hTERT mRNA c myc mRNA expression levels , found no significant relationship between c myc mRNA expression of telomerase, and no significant relationship between hTERT mRNA and c myc mRNA expression. And there was no apparent relationship between hTERT protein and c myc protein expression in breast cancer . Therefore, the authors suggest that hTERT regulation is undoubtedly very complex, in conjunction with other cell regulatory pathways comprehensive evaluation. For example, the researchers pointed out , Mad1 can be a factor, as the regulatory pathways of myc inhibit hTERT transcription levels. Therefore, to understand what is the regulation of hTERT and c myc relations, should be combined with the two levels of the cells and tissues further confirmed.
In recent years, many studies have shown that telomerase catalytic subunit-hTERT activity is down, the telomere sequence incomplete copy, as cell division, the telomere repeat fragment loss, thus tumor cells chromosomal instability, induction of DNA damage response cell cycle is blocked, The ultimate tumor cells to apoptosis or death. This experiment, c-myc antisense oligonucleotide breast cancer MCF 7 cells transfected cells at 24 h, 48 h, 72 h inhibition rates were 5.85%, 32.18%, 62.47%; flow cytometry to detect antisense c myc oligonucleotide role 24,48,72 h, apoptosis was 4.64%, 13.50%, 28.76%, and the apoptotic index was significantly higher than the normal control group cells. C myc antisense liposome-mediated down hTERT gene expression, while inhibition of MCF-7 cell growth and induction of apoptosis, to some extent, its effect was positively correlated with the time. Found that the proportion of early necrotic cells and not elevated by flow cytometry, ASODN cell inhibition is not a direct cytotoxic effect, but the antisense nucleic acid antisense effect. Yamaguchi et al  the hTERT antisense expression vector transfected into glioma cells, telomerase activity is inhibited, but two months after transfection cell death have not yet seen. These results suggest that affect telomere length, telomerase also possible through other means (such as cysteine protein kinase family members waterfall signaling pathways) involved in apoptosis, leading to apoptosis occurred at various speeds not identical.
Telomerase is a most broad spectrum of molecular markers currently found in malignant cells, and is activated in a majority of tumor cells, while expression in normal somatic cells is generally negative Telomerase is therefore a tumor gene therapy The ideal target. Cancer gene therapy research, combined with the results of this study, the authors suggest that targeting c myc for telomerase inhibition of tumor gene therapy is an effective strategy for gene therapy. But regulation of telomerase activity in tumor cells, multi-factor, multi-level, multi-stage, in different types of cells, telomerase activation mechanism is different, therefore telomerase targeted gene therapy is still The need for multi-target gene, multiple ways to further explore.