| Background and ObjectiveGastric cancer is one of the most common tumors in human beings. The mechanism of gastric cancer development is yet not fully understood. Cancer formation is a multi-step process. Generally speaking, genetics and epigenetics are closely related within the occurrence of cancer. DNA methylation is an epigenetic modification. During cancer development, there is a shift in methylation patterns and some promoter region CpG islands become methylated leading to silencing of the adjacent genes and this process is considered to be a critical step in cancer development.70%–80% of CpG dinucleotides are heavily methylated in human cells. While CpG islands should be protected from methylation and approximately 60% of human genes are associated with a CpG island. Aberrant methylation of normally unmethylated CpG islands, is associated with transcriptional inactivation that is as effective as inactivation by gene mutation or deletion.Previously reports on gastric cancers showed varying degree of promoter methylation of many tumor suppressor genes. Unmethylated CpG islands may become methylated in cancer cells with resultant loss of gene function. RUNX3 was originally cloned as AML2 and is localized on chromosome 1p36.1. RUNX3 protein combines with Smads and acts synergistically to regulate various target genes. The human runt-related transcription factors (RUNXs) are an important target of transforming growth factor (TGF-β) superfamily signaling. In mammals, the Runt domain transcription factor family includes three members, RUNX1, RUNX2, and RUNX3, they share a common NH2-terminal domain. RUNX1 is associated with human acute leukemia. RUNX2 is essential for osteogenesis and is associated with the human bone disease cleidocranial dysplasia.A tumor suppressor function has been attributed to RUNX3, a member of the RUNX family of transcription factors. RUNX3 is a novel tumor suppressor gene that is frequently silenced by promoter hypermethylation in gastric cancer. It was recently reported that RUNX3 gene expression is significantly downregulated in human gastric cancer cells due to hypermethylation of its promoter region or hemizygous deletion. 5-Aza-2'-deoxycytidine (5-Aza-dC) is a specific inhibitor of DNA methyl- transferases, it can reverse the function of gene silenced by hypermethylation. In this study, to explore whether 5-Aza-dC could reverse hypermethylation of RUNX3 CpG island and restore function of RUNX3 gene in gastric cancer cell SGC-7901, methylation specific PCR (MSP) technique was used to detect the effect of 5-Aza-dC on methylation pattern of RUNX3 CpG island, Real-time-RT-PCR technique was used to detect effect of it on RUNX3 mRNA level, and flow-cytometry (FCM) technique was used to detect effect of it on cell cycle.Materials and methods1. Cell culture Gastric cancer cell line SGC-7901 was cultured in RPMI-1640 supplemented with 10% FBS, in a 37℃, 50ml/L CO2 incubator. The culture medium was replaced one time each day, and the cell was subcultured one time three day.2. Effect of 5-Aza-dC on methylation pattern of RUNX3 gene promoter CpG island in gastric cancer cell SGC-7901 Gastric cancer cell SGC-7901 was cultured with medium containing 5-Aza-dC at final concentration of 12μmol/L for 72h. Genomic DNA was extracted and methylation specific PCR (MSP) technique was used to detect effect of 5-Aza-dC on methylation pattern of RUNX3 gene promoter CpG island.3. Effect of 5-Aza-dC on RUNX3 mRNA level in gastric cancer cell SGC-7901 Gastric cancer cell SGC-7901 was cultured with medium containing 5-Aza-dC at final concentration of 1.5μmol/L, 3μmol/L, 6μmol/L and 12μmol/L for 72h, respectively. Total RNA of each group was extracted, and Real-time-RT-PC technique was performed to investigate effect of 5-Aza-dC on RUNX3 mRNA level in gastric cancer cell SGC-7901.4. Effect of 5-Aza-dC on cell cycle of gastric cancer cell SGC-7901 Gastric cancer cell SGC-7901 was cultured with medium containing 5-Aza-dC at final concentration of 1.5μmol/L, 3μmol/L, 6μmol/L and 12μmol/L for 72h, respectively. The cells were collected and FCM technique was used to detect effect of 5-Aza-dC on cell cycle of gastric cancer cell SGC-7901.5. Statistical analysis Experimental data were showed as mean±SD ( x±s) and analyzed with SPSS18.0 statistical software. P<0.05 was selected as test level.Results1. Effect of 5-Aza-dC on methylation pattern of RUNX3 gene promoter CpG island in gastric cancer cell SGC-7901 Results of MSP showed that RUNX3 gene promoter CpG island of gastric cancer cell SGC-7901 was methylation status; after treatment of 12μmol/L 5-Aza-dC for 72h, it was converted from methylation to unmethylation status.2. Effect of 5-Aza-dC on RUNX3 mRNA level in gastric cancer cell SGC-7901 Results Real-time- RT-PCR showed that RUNX3 mRNA level in gastric cancer cell SGC-7901 was low. After treatment with 5-Aza-dC at final concentration of 1.5μmol/L, 3μmol/L, 6μmol/L and 12μmol/L for 72h, RUNX3 mRNA level in group was 0.32±0.05, 0.95±0.07, 1.22±0.052 and 1.85±0.03, respectively; RUNX3 mRNA level was increased in a concentration-dependent manner. There was a significantly positive correlation between RUNX3 mRNA level and 5-Aza-dC concentration (r>0.95,P<0.05).3. Effect of 5-Aza-dC on cell cycle of gastric cancer cell SGC-7901 Results of FCM showed that, after treatment of 5-Aza-dC for 72h, gastric cancer cell SGC-7901 was arrested at G0/G1 phase, and the ratio of G0/G1 phase cells was increased in a concentration-dependent manner. A significantly positive correlation occurred between the ratio of G0/G1 phase cells and 5-Aza-dC concentration (r>0.95,P<0.01).Conclusions5-Aza-dC can reverse methylation status of RUNX3 gene promoter CpG island in gastric cancer cell SGC-7901, upregulate expression of RUNX3 gene, and induce gastric cancer cell G0/G1 phase arrest.
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