The Study Of The Interaction Between SMG1and MTOR Signaling Pathway In Acute Myeloid Leukeia

Background:Acute myeloid leukemia (AML) is a clonal disorder of hematopoiesis characterized by the uncontrolled proliferation and accumulation of immature and dysfunctional hematopoietic progenitors. Cytotoxic chemotherapy has been widely used as the main approach for AML treatment.Recent studies have proved that, not only the successive accumulation of genetic alterations in oncogenes and tumor suppressor genes, but also the epigenetic alterations contribute to carcinogenesis. The most extensively studied epigenetic mechanisms is the methylation of the fifth carbon of a cytosine nucleotide. Alterations of the methylation status in oncogenes and tumor suppressor genes, which could affect the mRNAs and proteins expression levels, result in uncontrolled cell growth finally. Because epigenetic alterations are thought to be reversible, epigenetic drugs offer great promise for treatment of cancer.5-aza-2’-deoxycytidine (decitabine, DAC), as a kind of DNA methyltransferase inhibitor, is the most widely used epigenetic modulator to date. Recently, many studies have come to a conclusion that DAC offers a promising alternative therapeutic option for AML patients who are not candidates for standard remission induction chemotherapy and DAC has activity in all phases of AML treatment. Currently, the potential roles of DAC in the treatment of AML are being explored in numerous clinical trials. In our study, we found that SMG1(suppressor with morphogenetic effect on genitalia family member) was hypermethylated in the promoter region in AML. SMG1is a well known member of phosphoinositide3-kinase-related kinases (PIKK) family. It mainly involves in nonsense-mediated Mrna decay (NMD), which is the process of eliminating mRNAs that contain premature termination codons to prevent the accumulation of truncated proteins. However, up to date SMG1functional mutations, deletions, or reduced expression in human cancer is rarely studied. Recently, SMG1was suggested to be a potential tumor suppressor and could be downregulated due to promoter hypermethylation in human papillomavirus (HPV)-positive head and neck squamous cell carcinoma. However, there is no data showing the relation between SMG1promoter methylation status and its expression level in AML.Cristina et al. reported that SMG1and mammalian target of rapamycin complex1(Mtorc1) act antagonistically to regulate response to injury and growth in planarians and their study indicated that SMG1is likely to be a potential human tumor suppressor gene product. Mammalian target of rapamycin (Mtor) signaling pathway regulates cell growth and proliferation and is essential for the process of protein synthesis, which is consistent with that many human genetic defects and tumors associating with Mtor upregulation manifest as uncontrolled cell growth. As a result Mtor signaling is currently the most targeted signaling pathway in drug development for the treatment of cancers. Many human tumor suppressors negatively regulate Mtor signaling. Although it has been proved that Mtor signaling pathway is overactivated in AML, the relation between SMG1and Mtor remains unknown in AML so far.Objective:To detect the expression level of SMG1and Mtor in AML patients bone marrow samples; to study the effect of epigenetic modified methylation status of SMG1on the expression of SMG1and Mtor; to investigate the roles of SMG1and Mtor signaling pathway in the effect of demethylation and their mechanisms, so as to provide the basis for molecular targeted therapy.Materials and methods: 1. Patient samples:BM mononuclear cells from50AML patients and32healthy donors were taken after patients’ consent and approval from the Local Ethics Committee (Qilu Hospital, China). BM specimens obtained from14healthy donors with no history of cancer were included as controls. AML cells were isolated by density gradient separation from the BM samples of patients.2. Quantitative real-time RT-PCR:Total RNA was extracted from cells using Trizol reagent and reverse transcription was performed with M-MLV reverse transcriptase. The expression of SMG1、Mtor was detected.3. DNA extraction and MSP:Genomic DNA from patient samples was isolated by DNA Extraction Kit and chemically modified by using an EZ DNA Methylation-Gold Kit according to the manufacturer’s instructions. The methylation status of SMG1promoter region was determined by methylation-specific polymerase chain reaction (MSP).4. Demethylating agent DAC treatment:AML cells were seeded at a density of1×106cells per ml in10-cm dishes. After24h of culture, cells were treated with DAC for2days. Control cells were treated in parallel with DMSO. Cells were harvested and Mrna expression of SMG1and Mtor were analyzed by quantitative RT-PCR.5. SMG1knockdown and transfection:Transfection of siRNA was performed with Lipofectamine2000reagent in accordance with the manufacturer’s protocol. AML cell lines with SMG1expression were transfected with SMG1-siRNA or control-siRNAs. Knockdown efficiency was evaluated at48h after transfection by quantitative RT-PCR.6. Apoptosis assay:after treatment with DAC or transfection with SMG1-siRNA for48h,2X105cells were harvested and apoptosis was detected using an Annexin V/FITC and PI Apoptosis Detection Kit by Galios flow cytometer.7. Western blot analysis:Cells were solubilized in radio immunoprecipitation assay (RIPA) lysis buffer and cell lysates were separated by10%SDS-polyacrylamide gel electrophoresis. The protein levels of SMG1、Mtor、p-Mtor were determined in AML cells and BM samples by western blot methods. 8. Statistical analysis:Data was collected from at least three experiments in duplicate. Statistical analysis was carried out using SPSS software (version17.0). Differences with p-values of less than0.05were considered statistically significant. The statistical comparisons between the controls and AML patients were made with the Mann-Whitney U nonparametric tests.Results:1. SMG1was downregulated in AML patient samples: SMG1was expressed at a lower level in50AML patients, but was readily expressed in32controls confirmed by quantitative RT-PCR2. SMG1was hypermethylated in AML cell lines and patient samples:①SMG1was methylated by varying degrees in four AML cell lines (Kasumi-1, HL60,THP-1,HEL).(2) MSP confirmed SMG1hypermethylation in33out of50(66%) AML samples and none SMG1hypermethylation (0/14) in the controls.3. DAC treatment affect SMG1expression:①HEL cells treated with DAC shown decreased SMG1Vmethylation degree compared with the control group.②HEL cells treated with DAC were induced to express SMG1in dose-dependent manner.4. DAC treatment induced AML cells apoptosis:DAC treatment caused a significant increase in early apoptotic cells and late apoptotic cells compared with that of the control group.5. Knockdown of SMG1inhibited AML cells apoptosis:flow cytometry detection found that both early apoptotic cells and late apoptotic cells among SMG1-siRNA-transfected HEL cells decreased compared with those among control-siRNA-transfected HEL cells.6. Mtor expression level was negatively correlated with SMG1in AML:①Mtor was overexpressed in AML samples compared with that in normal controls confirmed by quantitative RT-PCR. ②Mtor was expressed at lower levels in the DAC treated group compared with the control group confirmed by western blotting analysis④The Mrna and protein expression levels of Mtor increased in SMG1-siRNA-transfected group compared to that in the control-siRNA-transfected group confirmed by quantitative RT-PCR analysis and western blotting analysis.Conclusion:1. SMG1was downregulated in AML cell lines and patients due to the hypermethylation of its promoter region.2. The DAC demethylating treatment of AML cells induced apoptosis by reversing SMG1methylation status and restoring SMG1expression while knockdown of SMG1inhibited apoptosis.3. SMG1acts as a potential tumor suppressor with epigenetic regulation in AML by acting antagonistically with Mtor to regulate AML cell growth..