The Study On The Molecular Mechanism Of A Candidate Tumor Suppressor Gene-HoxD10in The Pathogenesis Of Gastric Cancer

Gastric cancer is a common malignant tumor worldwide. Both genetic and epigenetic alterations contribute to the progression of gastric cancer. The transcriptional silencing of tumor suppressor genes (TSGs) by promoter methylation is a major epigenetic event in the origin of many cancers, including gastric cancer. Thus, identification of novel TSGs will be of great importance in the understanding of the mechanisms of gastric cancer, and which could be utilized as biomarkers for the intervention of gastric carcinogenesis.The homeobox (Hox) superfamily genes, including HoxD10. encode transcription factors that control cell differentiation and morphogenesis during development. We have previously found that HoxD10maybe a candidate tumor suppressor in gastric cancer through CpG island screening and cDNA microarray analysis. The role of HoxD10in the progression of gastric cancer needs further investigation.In this study, we firstly determined whether ectopic expression of HoxD10had tumor suppressing effect in gastric cancer in in vivo and in vitro. In addition, the relationship between HoxD10expression and promoter methylation was evaluated in primary gastric cancer tissues as well as a penal of gastric cancer cell lines. We also testify the potential downstream targets of HoxD10by cDNA microarray assay and qPCR validation. The regulation of IGFBP3and caspases signaling pathways by HoxD10was further investigated.Material and Methods1. The expression and its fuctional role of HoxD10in gastric cancerâ‘ The expression of HoxD10mRNA in33paired endoscopic biopsy samples from gastric cancer and normal stomach tissues were determined by qPCR. and HoxD10expression in a panel of gastric cancer cell lines (n=8) were examined by PCR and Western blot analysis.â‘¡The number of surviving colonies formed on the plates were observed. and cell proliferation by ectopic expression of HoxD10was confirmed by MTS assay.â‘¢Cell apoptosis and cell-cycle were assessed by flow cytometry. Cell migration and invasion were investigated in the24-transwell cell migration and collagen-based cell invasion system.â‘£xenografted gastric tumor model was used to validate the tumor suppressing effect by ectopic expression of HoxD10in vivo.2. The relationship between promoter hypermethylation and the expression of HoxD10in gastric cancer and its potential ulitility in monitering of gastric carcinogenesisâ‘ The expression of HoxD10before and after de-methylated treatment with5-aza-2*-deoxycytidine (5-Aza) were examined with RT-PCR and Western blot analysis.â‘¡The methylation status of HoxD10in gastric cancer cells was determined by methylation specific PCR (MSP).â‘¢HoxD10methylation status in different endoscopic biopsy specimens among primary gastric cancers, intestinal metaplasia and normal stomach tissues were investigated by MSP. 3. Screening and validation of the downstream targets of HoxD10, and the influence of apoptotic related caspases by reintroduction of HoxD10in gastric cancer cellsâ‘ cDNA microarray and qPCR validation analysis were performed in MNK28cells transfected with HoxD10or empty vector.â‘¡We further determined whether HoxD10modulates IGFBP3and related apoptotic caspases in gastric cancer cells which tranfected with HoxD10or empty vector.Results1. HoxD10significantly inhibited proliferation, impaired migration and invasion of gastric cancer cellsâ‘ HoxD10expression was significantly downregulated in tumor tissues relative to normal gastric tissues (p<0.001). HoxD10expression was absent or reduced in all gastric cancer cell lines when compared to normal gastric tissues.â‘¡We observed that the number of surviving colonies formed on the plates was significantly reduced in HoxD10transfected cells when compared to the control vector transfectants (p<0.01), The cell proliferation inhibition of HoxD10in AGS cells was confirmed by MTS assay which showed significant reduction in growth in HoxD10-transfected cells (p<0.05).â‘¢Ectopic expression of HoxD10significantly induced early apoptois of gastric cancer cells (12.29%and11.43%respectively in AGS and MKN28) when compared to empty vector transfected cells (7.51%and8.82%. respectively). Additionally, we observed that HoxD10-transfected cells (AGS and MKN28) showed lower S phase populations in comparison to the empty vector transfectants. Furthermore, ectopic expression of HoxD10significantly suppressed AGS cell migration (p<0.01) and cell invasion (p<0.01).â‘£Ectopic expression of HoxD10suppresses tumor growth in a mouse xenograft model.2. HoxD10is downregulated mainly through promoter hypermethylationâ‘ The expression of HoxD10mRNA and protein was dramatically re-activated after demethylated treatment with5-Aza in gastric cancer cell lines.â‘¡All tested gastric cancer cell lines showed full (MKN28, MKN45and NCI-N87) or partial (AGS) methylation of HoxD10promoter DNA. The MSP results showed that the DNA promoter of HoxDIO in primary gastric cancer biopsy tissues exhibited frequent methylation (85.7%,24/28). Intestinal metaplasia tissues showed a relative high rate of HoxD10methylation (60%,18/30), whereas no methylation was observed in normal stomach tissues (0/10).3. HoxDI0modulates multiple downstream genes, and apoptotic related caspasesâ‘ Our results showed that HCLS1. ANP32A, PDGFRL. IGFBP3. and CXCL9were up-regulated, while RAC2. NTS. KRT5. and TUSC3were down-regulated by overexpression of HoxD10in MKN28cells.(2)The expression of IGFBP3protein was significantly upregulated by ectopic expression of HoxD10. Ectopic expression of HoxD10leads to activating caspase8and caspase3.Conclusions1. Ectopic expression of HoxD10significantly inhibited proliferation, impaired migration and invasion of gastric cancer cells. In particular, re-expression of HoxD10suppresses tumor growth in a mouse xenograft model. HoxD10functions as a candidate tumor suppressor in gastric cancer.2. DNA methylation may be associated with the transcriptional silencing of HoxD10in gastric cancer cells. HoxD10potentially serves as a biomarker for the monilering the progression from precancerous lesions to gastric cancer. HoxD10could be a useful therapeutic target of gastric cancer.3. The induction of cell apoptosis by HoxD10may be mediated through IGFBP3. and subsequent activation of apoptotic caspases.