Effects of DNMT1 silencing on malignant phenotype and methylated gene expression in cervical cancer cells
- Yi Zhang†1, 2,
- Fu-qiang Chen†1,
- Ye-hong Sun†1,
- Shu-yan Zhou†1,
- Ti-yuan Li1Email author and
- Rui Chen†1, 2
© Zhang et al; licensee BioMed Central Ltd. 2011
Received: 17 July 2011
Accepted: 17 October 2011
Published: 17 October 2011
DNA methylation has been widely used in classification, early diagnosis, therapy and prediction of metastasis as well as recurrence of cervical cancer. DNMT methyltransferase 1 (DNMT1), which plays a significant role in maintaining DNA methylation status and regulating the expression of tumor suppressor genes. The aim of this research was to investigate the relationship between DNMT1 and abnormal methylation of tumor suppressor genes and malignant phenotype in cervical cancer.
Levels of DNMT1 mRNA and protein were detected using qPCR and Western blot, respectively. Cell proliferation was analyzed by MTT and apoptosis was performed by Annexin V-FITC/PI double staining flow cytometry, respectively. MeDIP-qPCR and qPCR were performed to measure demethylation status and mRNA re-expression level of 7 tumor-suppressor genes (CCNA1, CHFR, FHIT, PAX1, PTEN, SFRP4, TSLC1) in Hela and Siha cells after silencing DNMT1.
The average expression levels of DNMT1 mRNA and protein in Hela and Siha cells were decreased significantly compared with control group. The flow cytometry and MTT results showed that Hela and Siha cells apoptosis rates and cell viabilities were 19.4 ± 2.90%, 25.7 ± 3.92% as well as 86.7 ± 3.12%, 84.16 ± 2.67% respectively 48 h after transfection (P < 0.01). Furthermore, the promoter methylation of five tumor suppressor genes was decreased with the increased mRNA expression after silencing DNMT1, whereas there were no significant changes in PTEN and FHIT genes in Hela cells, and CHFR and FHIT genes in Siha cells.
Our experimental results demonstrate that methylation status of DNMT1 can influence several important tumor suppressor genes activity in cervical tumorigenesis and may have the potential to become an effective target for treatment of cervical cancer.
Cervical cancer is the second most common cancer in women worldwide and the leading cause of cancer deaths in women in developing countries. It is obviously that many genetic and epigenetic alternations occur during cervical tumorigenesis. Among those changes, aberrant promoter methylation of tumor-suppressor genes gives rise to its silencing functions and results in the significant carcinogenesis of cervical cancer.
Currently, the known repressor genes are related to cervical cancer including CCNA1, CHFR, FHIT, PAX1, PTEN, SFRP4, TSLC1 and etc . All these genes mentioned above have performed a wide variety of functions to regulate the transcription and expression, any of which down-regulation as well as promoter hypermethylation will lead to the precursor lesions in cervical development and malignant transformation. DNA methylation is catalyzed by several DNA methyltransferases, including DNMT1, DNMT3a, DNMT3b and etc. DNMT1 is responsible for precise duplicating and maintaining the pre-existing DNA methylation patterns after replication. As reported by Szyf , DNMT1 inhibited the transcription of tumor suppressor genes and facilitated the formation of tumorigenesis, which linked to the development of cervical cancer. Meanwhile, Inhibition of DNMT1 activity could reduce hypermethylation of repressive genes and promote its re-expression, and reverse phenotype of malignant tumor. Thus, specific inhibition of DNMT1 could be one strategy for cervical therapy.
In our study, we detected the demethylation and re-expression levels of seven cervical cancer suppressor genes with DNMT1 silencing in Hela and Siha cells. The aim was to elucidate the relations between DNMT1 and abnormal methylation of these genes' promoter as well as the malignant phenotype of tumor cells, which might contribute to the investigations of functions and regulation roles of DNMT1 in cervical cancer.
Materials and methods
Cell culture and transfection
The Hela and Siha human cervical cancer cells lines were obtained from American Type Culture Collection (Manassas, VA, USA). Lipofectamine TM2000 was purchased from Invitrogen Co. These cells grown in Dulbeco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and incubated at 37°C in a humidified chamber with 5% CO2. The siRNA primer sequences for DNMT1 were 5'-UUAUGUUGCUCACAAACUUCUUGUC-3' (forward) and 5'-GACAAGAAG UUUGUGAGCAACAUAA-3' (reverse), which were custom synthesized by Shanghai Sangon (Shanghai, China). After transfection, the inhibition efficiency was examined using quantitative polymerase chain reaction (qPCR). Transfections were performed with Lipfectamine TM2000 according to the protocol (Invitrogen Co.).
Real-time qPCR assay
Primers used in RNA expression
Primers used in MeDIP-qPCR assay
Western blot analysis
Cells were harvested and rinsed twice in ice-cold PBS, and kept on ice for 30 min in cell lysis buffer containing 1 mM PMSF while agitating constantly, and insoluble cell debris was discarded by centrifugation for 10 min at 12,000 rpm at 4°C. The protein samples were separated with 12% SDS-PAGE and subsequently transferred to PVDF membranes (Millipore). Membranes were blocked with 5% nonfat dry milk solution either at room temperature for 2 h, and incubated with Rabbit anti-DNMT1 and secondary antibody at 37°C for 2 h respectively. The Membranes were stained with an enhanced chemiluminescence solution. Band intensities are normalized to β-actin as a loading control.
Annexin V-FITC/PI staining and flow cytometry
Cell cycle analysis: Cells were digested by typsin (0.25%) and fixed with cold 70% ethanol at 48 h after transfection. After washed in phosphate-buffered saline, samples were incubated with 100 μl RNase A at 37°C for 30 min and stained with 400 μl propidium iodide (Sigma). Flow cytometric analysis was performed at 488 nm to determine the DNA contents.
Apoptosis analysis: Cells were harvested as described above. After adding of 10 μl Binding reagent and 1.25 μl Annexin V-FITC, samples were suspended in 0.5 ml cold 1 × Binding Buffer and stained with 10 μl PI. The samples were then analyzed for apoptosis by flow cytometry.
Cellular proliferation was measured using MTT assay. 104 cells were seeded in 96-well plates and cultured with siRNA-DNMT1 at 37°C in a humid chamber with 5% CO2 for 24 h. 50 μl 1 × MTT was then added to each well and incubated with cells at 37°C for 4 h. After removal of supernatant, 150 μl DMSO were added to each well. The optical density (OD) was measured at 550 nm. The percentage of viability was calculated according to the following formula: viability% = T/C×100%, where T and C refer to the absorbance of transfection group and cell control, respectively.
Transfections were performed as described above. MeDIP assay combined with qPCR were used to quantitatively assess the status of demethylation. Hela and Siha cells were transfected with siRNA and treated with 1.0 μM 5-az-dC (Sigma) respectively, and harvested at 72 h after incubation. Genomic DNA was extracted and randomly sheared to an average length of 0.2-1.0 kb by sonication. Dilution buffer and 60 μl Protein G Magnetic Bead suspension were added into the fragmented DNA and allowed for more than 10 min of incubation. DNA was then incubated overnight at 4°C with 8 μg antibody (Epigentek) against 5-methylcytosine, followed by 2 h incubation with Mouse-IgG magnetic beads at 4°C. The methylated DNA/antibody complexes were then washed with 1 ml cold WB1, WB2 and WB3 buffer. Purified DNA was analyzed by qPCR on an Applied Biosystems 7500 Real-Time PCR System. Real-time PCR was performed in a total 8 μl volume containing 1 μl of DNA template, 5 μl of 2 × Master Mix, 1 μl ddH2O and 1 μl of each primer. The relative changes in the extent of promoter methylation were determined by measuring the amount of promoter in immunoprecipitated DNA after normalization to the input DNA: %(MeDNA-IP/Input) = 2^[(Ct(input)-Ct(MeDNA-IP)×100.
Statistical analyses were performed with SPSS version 13.0(SPSS, Chicago, USA). Quantitative results were given as mean ± SD and statistical analysis was carried out by t-test. P values less than 0.05 were considered as statistically significant.
Effects of siRNA on DNMT1 mRNA and protein level
Effects of DNMT1 silencing on cell cycle and apoptosis
Effects of DNMT1 silencing on cell growth and proliferation
Effects of DNMT1 silencing on gene demethylation and mRNA expression level in Hela cell
Effects of DNMT1 silencing on gene demethylation and mRNA expression level in Siha cell
DNMT1 silencing in cervical cancer cells could induce re-expression of most tumor suppressor genes by demethylating its promoter region, and co-silencing of DNMT1 and DNMT3b might perform a greater inhibitory effect on tumorigenesis . Sowinska  demonstrated that combined DNMT1 and DNMT3b siRNAs could enhance promoter demethylation and re-expression of CXCL12 in MCF-7 breast cancer as well as AsPC1 in pancreatic carcinoma cell lines, and suggested that they acted synergistically in inhibiting CpG island hypermethylation of tumor suppressor genes. Rhee et al  reported that DNMT3b deletion in a colorectal cancer cell line reduced global DNA methylation by less than 3%, but co-silencing of both DNMT1 and DNMT3b nearly eliminated methyltransferase activity, and reduced genomic DNA methylation by greater than 95%. Thus, DNMT1 and DNMT3b play the significant role in promoter methylation of tumor suppressor genes and tumorigenesis in its early status. Currently, functions and mechanisms of DNMTs in cervical cancer cells remained unclear, and whether DNMT1 and DNMT3b act synergistically or through other ways exploration efforts were still required study.
In human bladder cancer cells, selective depletion of DNMT1 with siRNA induced demethylation and reactivation of the silenced tumor-suppressor gene CDKN2A . RNAi-mediated knockdown of DNMT1 resulted in significant reduction of promoter methylation and re-expression of RASSF1A, p16, and HPP1 in HCC1954 breast cancer cells . In ovarian cancer cell line CP70, DNMT1 siRNA treatment led to a partial removal of DNA methylation from three inactive promoter CpG islands, TWIST, RASSF1A, and HIN-1, and restored the expression of these genes . Thus, RNAi-mediated DNMT1 depletion in different tumor cells could induce demethylation of various tumor suppressor genes and enhance re-expression. However, contradictory results were reported even in the same cell line. Ting et al  found that hypermethylation of CDKN2A, SFPR1, GATA4 and GATA5 were still maintained in HCT116 colorectal cancer cells after transiently or stably depleted of DNMT1, and suggested that DNMT1 might not play the dominant effect which caused hypermethylation of CpG islands in tumor suppressor genes. Knockout of DNMT1 in HCT116 cells by homologous recombination only reduced global DNA methylation by 20% and p16 maintained completely methylated status. Besides, methylations of HMLH1, p16 and CDH1 in gastric-cancer tissue samples at different progress periods do not correlate with the expression of DNMT1 directly . Therefore, whether over-expression of DNMT1 accounts for the only or key causes of hypermethylation of tumor suppressor genes remains to be confirmed.
Currently, correlation between methlylation and mRNA expression still remains unclear. In our study, methylation status of five suppressor genes (such as PAX1) in transfection group was significantly lower than that in control group or blank control, and the mRNA expression levels were higher as compared to the two types of control, suggesting that lower level of methylation facilitates mRNA expression. This trend was confirmed when CCNA1, SFRP4, TSLC1 and CHFR in Hela cells and CCNA1, PTEN, SFRP4 and TSLC1 in Siha cells were analyzed.
Surprisingly, transfection did not affect the methylation status and mRNA expression of FHIT and PTEN in Hela cells and FHIT and CHFR in Siha cells in our study, even though both of these two genes might achieve high mRNA expression through low methylation. It was previously reported that there was no PTEN mutation in 63 cases of squamous cervical carcinomas, but 58% of the cases showed high methylation of PTEN promoter [11, 12]. Wu et al  reported that FHIT was highly methylated in Hela, C33A and Siha cervical cancer cells, and that aberrant methylation of the FHIT gene might be a key mechanism for cervical tumorigenesis, which could be reactivated and whose tumor suppressing function could be restored by treatment of demethylating agent. Banno et al  reported that cervical smears showed aberrant methylation of CHFR in 12.3% of adenocarcinoma specimens, while aberrant DNA methylation was not detected in normal cervical cells. These researches demonstrated us that FHIT and PTEN in Hela cells and FHIT and CHFR in Siha cells might have the other regulation pathways for carcinogenesis or transcription control, and which needs more tests of cervical cancer cells and clinical specimens.
Apart from DNMT1 silencing, we treated Hela and Siha cells with 5-aza-dC, which revealed the similar results with transfection group. Five repressor genes were demethylated to various degrees and the mRNA expressions were also increased. These results are in accordance with the findings of other reports [15–19], which could be important in the development of new and effective strategy in cervical treatment.
In conclusion, our study demonstrates that DNMT1 silencing could suppress proliferation and induce apoptosis of Hela and Siha cells. DNMT1-siRNA induces demethylation of five tumor suppressor genes, including CCNA1, CHFR, PAX1, SFRP4 and TSLC1 in Hela cells and CCNA1, PTEN, PAX1, SFRP4 and TSLC1 in Siha cells, and enhances their mRNA expression. In a word, DNMT1 represents an important potential diagnostic and therapeutic target for cervical cancer.
This study was supported by the Shenzhen major research projects of healthy department.
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