The Role Of H3K9Methyltransferase G9a In Regulation Of Cell Growth And Proliferation In Neuroblastoma Cells

Insect stem cell biology was defined as one of the hot and difficult frontier in the entomology research. But except Drosophila, the research of insect stem cells is still not sufficient. As the typical representative of Lepidoptera, Bombyx mori experience repeatedly alternations of old and new organizations and structural changes in morphology. Therefore, the development of silkworm stem cell research, especially the research of regulation mechanism of stem cell in development and metamorphosis process of silkwrom, will lay the foundation for elucidating the mechanisms of insect development, and provide a theoretical basis for the new management strategy for lepidoptera pests. Compared with the relatively perfect stem cell research system in Drosophila and mammals, it is urgent to establish the technical system of silkworm stem cell research. In view of the current key technical problem that lacking of Bombyx mori specific stem cell molecular markers, we choose neuroblastoma cells as an alternative to study, which originated from embryonic development period and with differentiation potentials. To seek the key enzyme gene for regulation of neuroblastoma cell proliferation and differentiation, to clarify the mechanism of target gene on neuroblastoma cell proliferation and differentiation, to explore the occurrence mechanism of neuroblastoma, referring to the research result, will lay the foundation for carrying out the identification of silkwrom stem cell, and provide a theoretical reference for the regulation and control of the maintenance and differentiation of silkwrom stem cell.Neuroblastoma is a common childhood malignant tumor of neural crest origin, arising in the sympathetic nervous system, accounting for approximately7%of pediatric cancers and15%of cancer-related deaths in children. It has long been known that neuroblastoma is a heterogeneous group of tumors, and the differentiation state of neuroblastoma is of prognostic significance. Histologically, neuroblastoma is ranging from tumors with poorly differentiated neuroblasts to those fully differentiated sympathetic neurons. Patients with poorly differentiated neuroblastomas have significantly poorer survival than those with neuroblastomas of differentiating histology. Tumorigenesis is considered to be a multi-step progress from normal histologic features to carcinoma features. Epigenetic mechanism was recently defined as descendible changes in gene expression which was not due to any alteration in the DNA sequence. Histone methylation is the fundamental epigenetic mechanism to regulate gene expression in cancers, which linked to the silence of a number of critical tumor suppressor genes in tumorigenesis. Recently, G9a was reported as a major H3K9mel and H3K9me2histone methyltransferase in vivo, and some studies have identified the critical role that G9a plays in various biological processes, including embryo development, immune response, drug response and tumor cell growth. Moreover, current evidences suggest that G9a promotes invasion and metastasis in lung cancer, and highly expressed G9a was observed in hepatocellular carcinomas. Therefore, G9a might be a key regulator that serves as a potential therapeutic target in tumor formation. In this study, we investigated the role of G9a in neuroblastoma tumor growth together with BIX01294(BIX), which was the specific inhibitor of G9a. We provide experimental evidence supporting the role of G9a in the transcriptional regulation of cell proliferation and autophagy in neuroblastoma cells. Pharmacological inhibition or RNA interference (RNAi) of G9a led to increased LC3B expression and autophagosome formation. Collectively, we identify G9a as a prognostic marker for survival in patients with neuroblastoma, and a regulator of neuroblastoma cell growth, proliferation and autophagy. Our results provide a novel potential role of G9a in regulation of autophagy signaling pathway in neuroblastoma, and suggest that G9a might be served as a potential therapeutic target for neuroblastoma treatment, and provide a theoretical support for the regulation of maintenance and differentiation of silkworm stem cells.The main results are as follows:1. G9a expression in neuroblastoma is associated with poor prognosis In this study, we investigate the possibility of G9a as a prognostic marker in neuroblastoma. We had undertaken a microarray-based search using the Tumor Neuroblastoma public-Versteeg database, which is available from the online R2: microarray analysis and visualization platform. The Versteeg database contains a cohort of88patients with neuroblastoma representative of various tumor stages and genetic alterations. Kaplan-Meier analysis of progression-free survival for the Versteeg database showed that high G9a expression was associated strongly with poor outcome, whereas low G9a expression was correlated with good overall survival. We confirmed that high expression of G9a is prognostic for unfavorable outcome with the Neuroblastoma Prognosis Database available from the online Oncogenomics database, which includes a cohort of56neuroblastoma patients. Moreover, G9a expression was different in various tumor stages, and G9a expression increased significantly in stage4tumors compared with stage3and stage4S. Then we examined the correlation of G9a expression levels with patient death cause, and the result revealed that G9a expression level was significantly higher in tumor caused death than normal death. As younger patient at diagnosis is associated with good prognosis, we examined the correlation of G9a expression levels with patient age at diagnosis by using information available from the Versteeg database. We chose the cutoff at18months of age because it gave the most significant P value for prognostic evaluation. The younger age group expressed significantly lower levels of G9a than the older age group, showing a correlation of low G9a expression with younger age at diagnosis. Together, our analyses of two independent microarray databases indicated that G9a is a prognostic marker in neuroblastoma.2. Inhibition of G9a represses neuroblastoma cell growth and proliferationWe next checked G9a expressions in five neuroblastoma cells SK-N-AS, BE(2)-C, SK-N-DZ, SK-N-F1, and SHEP1. The result showed that G9a is commonly expressed in five neuroblastoma cells SK-N-AS, BE(2)-C, SK-N-DZ, SK-N-F1, and SHEP1. Therefore we examined the functional consequence of G9a high expression in neuroblastoma cells. Five neuroblastoma cells SK-N-AS, BE(2)-C, SK-N-DZ, SK-N-F1, and SHEP1were treated with BIX. We found that all cells were very sensitive to BIX-treatment, which dramatically repressed cell proliferation after4days treatment, which was confirmed by cell counting kit-8(CCK8) assay, and the cell number was highly decreased after5μM BIX-treatment. We further examined the cell cycle of neuroblastoma cells. After drug treatment, G1phase was increased and S phase was decreased in five cells, which mean that G9a inhibition caused by BIX-treatment induced cell arrested in G1phase. Western blot analysis showed that G9a inhibition led to a marked down-regulation of CyclinDl, CDK4and CDK6, which are collectively required for the cell cycle progression through G1to S phases. Moreover, G9a inhibition led to an obvious down-regulation of CDK2, no big difference on CyclinE, which are required for cell cycle progressions through S to G2phases. These results showed that G9a inhibition in neuroblastoma cells completely blocked cell proliferation, with cell arrested in the Gl phase. Except inhibition G9a by inhibitor BIX, we knocked down G9a in five neuroblastoma cells. Biochemically, we noted that G9a knockdown in neuroblastoma cells induced cell growth arrest, which was determined by cell growth and proliferation assays. Western blot analysis showed that G9a knockdown also led to a marked down-regulation of CyclinD1, CDK4and CDK6in three neuroblastoma cells. These results showed that down-regulation of G9a repress neuroblastoma cells growth and proliferation.3. Inhibition of G9a induces neuroblastoma cell autophagyTo elucidate the contribution of G9a in neuroblastoma cell proliferation inhibition, we carried out morphologic examination and immunofluorescence staining. After2days treatment, the autophagosome was observed in neuroblastoma cells, and was so obvious in time-dependent manner after4days treatment. Next, we checked LC3B expression, which was a marker of autophagy. We found abundant LC3B expression in cells with G9a inhibition which was induced by BIX-treatment. To gain insight into the molecular mechanism underlying G9a inhibition-induced LC3B expression, we analyzed the protein expression of all five neuroblastoma cells. BIX-treatment had no significant effect on G9a expression in neuroblastoma cells at all, but led to a marked down-regulation of H3K9me2which was reported as a major product in G9a-mediated H3K9methylation. These findings indicated that the inhibitor BIX had no effect on G9a protein expression, but suppressed the methyltransferase function of G9a. Moreover, we investigated whether BIX-treatment can induce the expression of genes known to participate in autophagosome formation. Western blot assay revealed that expressions of ATGs and LC3B were markedly up-regulated after treatment. Furthermore, we noted that G9a knockdown in three neuroblastoma cells also induced autophagosome formation and LC3B expression, as determined by morphology and immunofluorescence assays. In addition, knockdown of G9a in neuroblastoma cells markedly inhibited H3K9me2level and up-regulated ATGs and LC3B expressions. These findings demonstrated that loss of G9a function or down-regulation of G9a induces autophagy and formation of autophagosome in neuroblastoma cells.4. Inhibition of G9a decreases tumorigenicity of neuroblastoma cellsTo evaluate the role of G9a for the self-renewal of neuroblastoma cells, we examined the possibility in soft agar clonogenic assays. BE(2)-C cells were plated at1×103cells per well (six-well culture plates). After plating, immediately examined under a microscope would reveal mostly individual cells, and colonies which defined as a collection of more than50cells appeared after14to21days. BE(2)-C cells treated with5μM BIX01294were observed to give rise to small and scanty colonies in soft agar compared with cells treated with water. Similar results were also obtained with several other neuroblastoma cells, including SK-N-AS, SK-N-DZ, and SK-N-F1. Moreover, we examined the self-renewal of G9a knockdown cells. The result showed that neuroblastoma cells with G9a knockdown failed to form large colonies in soft agar Together, these results indicated that inhibition of G9a prevents the clonogenic forming activity of neuroblastoma cells. Next, we examined the effect of G9a on tumorigenicity of neuroblastoma cells in immunodeficient mice. Neuroblastoma cells BE(2)-C and SK-N-AS were injected subcutaneously into the flanks of NOD/SCID mice. After1week tumor growth, BIX01294were injected intraperitoneally at4mg/kg. NOD/SCID mice were injected with water as control. The mice without BIX01294treatment developed large tumor masses after three weeks, whereas BIX01294injection significantly diminished the tumorigenic activity of neuroblastoma cells during the same time period. All above results demonstrated that inhibition of G9a decreases tumorigenicity of neuroblastoma cells.