Differentially Expressed Genes Profiling In ESCC And Effect Of HPGD On The Carcinogenesis Of ESCC

Background:China is the high incidence area for esophageal cancer, more than90%of which isesophageal squamous cell carcinoma (ESCC). The etiology and molecular mechanism ofESCC is still not well clarified. Lots of differentially expressed genes play a key role inthe transformation from normal to malignancies. Study on deep understanding themechanism underlying ESCC at molecular biological level, is of great significance todiagnosis, prevention and treatment for ESCC. Combination of microarray andbioinformatics can be used to detect key genes in the progression of ESCC and providenovel tagets for diagnosis and treatment.Hydroxyprostaglandin dehydrogenase (HPGD) is a key enzyme of prostaglandindegradation and is speculated as a physiological antagonist of cyclooxygenase (COX-2).Down-regulation or loss of15-PGDH has been observed in several human cancers,including colon cancer, gastric cancer, lung cancer, breast cancer, thyroid cancer andhepatocellular cancer.Objective:1.To find key differentially expressed genes (DEGs) and pathways in ESCC for abetter understanding of molecular mechanism by microarray incorporated withbioinformatics.2.To elucidate the role of key candidate gene HPGD in ESCC,including the effecton the proliferation and invasion of ESCC cells and possible molecular mechasim.Material and Mehods:1. For the fist part, Microarray was carried out on8-paired fresh resected ESCCspecimens with adjacent normal tissue. Next, bioinformatics was used to analyze theDEGs, significant enrichment of GO terms and pathways, and construction of geneco-operative net compared ESCC with normal tissues.2.For the second part, a candidate gene-HPGD was further validated. qRT-PCR andwestern blot were applied to detect the expression of HPGD in both ESCC tissues atmRNA and protein level, respectively. Immunohistochemcial assays were used toinvestigate the HPGD and COX-2expresison in sixty ESCC and adjacent normal tissues,which obtained from Beijing Friendship Hosptial,capital University Medicine.Spearon analysis was used to investigate the relationship between HPGD and COX-2.Western Blot was used to detect expression of HPGD in ESCC cell lines. Plasmids forover-expression and knockdown-expression of HPGD was constructed and transienttransfected into relatively up-regulated and down regulated cells for functional study. Theeffects on proliferation and invasion of HPGD in vitro were evaluated by MTT assay andMatrigel chambers.Methylation status in ESCC cell lines and tissues were evaluated bymethylation-specific polymerase chain reaction (MSP) and bisulfite sequencing (BSP).After treated with demethylating agent5-aza-deoxycytydine (5-aza-dC), the changes ofHPGD expression were detected by RT-PCR.Results:1. A total of1208DEGs including529up-regualted genes and679down-regulatedgenes were screened. GO analysis showed that the significant functional process forup-regulated DEGs included extracellular matrix organization, extracellular matrixdisassembly, cell cycle, cell adhesion,et al. while the significant down-regulated DEGsinvolved in small molecule metabolic process, keratinocyte differentiation, arachidonicacid metabolic process,et al. In pathway analysis, some classical pathways, such asECM-receptor interaction?focal adhesion and PI3K-Akt pathway were detect forup-regulated DEGs involving in the tumorigenesis of ESCC. Metabolic pathways,arachidonic acid metabolism, drug metabolism-cytochrome P450were the mostsignificant pathways for down-regulated DEGs. Based on above findings, HPGD werefound as a candidate gene and may play a crucial role in ESCC.2. HPGD was down-regulated in ESCC tissues in consistence with Microarray.Immunohistochemical assays revealed that HPGD was negative in41ESCC tissues andCOX-2was positive in46ESCC tissues. There is an inverse correlation between HPGDexpression and COX-2expression. Besides, negative expression of HPGD and positiveexpression of COX-2were correlated with lymph node metastasis and outer membraneinvolvement. HPGD protein expressed higher in Het1A than that in ESCC cell lines. TE1was the relatively higher expressed and TE2was less expressed. MTT showed thatover-expression of HPGD inhibited the proliferation of TE2cells, while knock-down expression of HPGD promoted the proliferation of TE1cells (P?0.01). Ttranswellinvasion array revealed that this capability of TE2was elevated after transfected withover-expression plasmid. Cell invasion of TE1was suppressed after knockdownexpression of HPGD. Non-methylation status was observed in Het1A, methylation statusin TE2and partial methylation in other three cell lines by MSP and BSP. HPGD mRNAexpression in TE2and EC9706was restored or increased by after treatment of5-aza-dC.Conclusions:1. Differentially expressed genes in ESCC can be rapidly detected by microarray andbioinformatics. HPGD is a key down-regulated DEGs in ESCC, with multiple significanttargeted function as well as pathways.2. HPGD may act as a putative tumor suppressor; methylation of HPGD may becontributed to loss or down expression of HPGD in ESCC. Silencing of HPGD promotescell proliferation and invasion. Down-regulated expression of HPGD appears to beinversely related to the expression of COX-2.