Characterization Of The Adenosine Deaminase Acting On RNA-1in Esophageal Squamous Cell Carcinoma

Background and PurposeEsophageal cancer is one of the most aggressive cancers worldwide and it is considered as the sixth cause of cancer leading deaths.Esophageal squamous cell carcinoma(ESCC) is the major histological form of esophageal cancer,the occurrence of ESCC has obvious geographic variation, especially in the northern China with the highest incidence rate.Epidemiologic studies have indicated that smoking, micronutrient deficiency and HPV could cause esophageal cancer. However,these risk factors play very weak role in high-incidengce areas such as LinZhou city of Henan province. Some research shows that there is more significant familial aggregation in high-risk areas of northern China and genetic susceptibility may play a very important role in esophageal carcinogenesis. In the past few decades,though the great advance in diagnosis and chemoradiotherapy has achieved,the prognosis for patients is very poor. Like other kinds of solid tumors,ESCC is thought to arise from the interaction of various factors.The activation of oncogenes and inactivation of tumor supperssor genes maybe the key molecular changes. But so far,we know little about the molecular mechanisms in the development and progression of ESCC,such as dysregulation of cell growth control. Therefore,it is essential for systematically analysis of the mechanism in the development and progression of ESCC, to raise the early diagnostic rate and therapeutic efficacy.In order to investigate the factors,which is closely related to the pathogenesis of tumors. We collected180pairs surgically resected EC specimens,with tumors and their adjacent non-tumors,then we constructed a set of ESCC tissue microarray including clinical pathologic data and prognosis. At the same time,we collected89pairs fresh specimens of esophageal cancer tissue and their adjacent non-tumor tissue from Linzhou of Henan Province,which has the highest incidence of esophageal carcinoma.we extracted the tissue’s RNA and DNA.Then,we applied Q-PCR technology to detect the ADAR1different expression between tumors and non-tumors.The result revealed that ADAR1expression was obviously higher in the tumors than their adjacent non-tumor tissue.Materials and Methods1Patients and clinical specimensThe89pairs surgically resected ESCC tumor tissue and their matched nontumorous tissue obtained from Linzhou city(Henan,China),all specimens were fixed in10%formaldehyde,embedelinged in paraffin and stained by HE.All pathologic diagnosis results are esophageal squamous cell carcinoma,the nontumorous tissue didn’t contain carcinoma cells.None of patients received chemoradiotherapy and immunotherapy.2MethodsWe applied Q-PCR technology to detect the mRNA level of ADAR1in tumor tissue,compared with the matched non-tumors.At the same time,we applied immunohistochemistry to detect the protein level of ADAR1in ESCC TMA.In order to investigate the function of ADARl,we transfected lenti-ADAR1into two low expressing ESCC cell lines EC109、 KYSE180.Stable expressing ADAR1cell clones were selected by blasticidin and confirmed by Q-PCR and western blot.At the same time, we transfected siADAR1into high expressing ESCC cell lines KYSE510, stable expressing siADAR1cell clones were selected by puromycin and confirmed by Q-PCR and western blot.To analysis the function of ADARl,we carried out cell proliferation assay,foci formation,soft agarose assay,cell invasion and migration assay to confirmation. Meanwhile flow cytometry was used to study siADARl effect on ESCC cell cycle.ResultADAR1is upregulated in ESCC tissue compared with the matched nontumor tissue in protein and mRNA levels.At the same time,we find that the expression of ADAR1is closely related to the prognosis of ESCC pateins.Our study identifys that ADAR1contributes to the development of tumor by cell proliferation assay,foci formation,soft agarose assay,cell invasion and migration assay.The overexpression of ADAR1in EC109、KYSE180cell lines can enhance cell proliferation,colony formation,cell invasion and migration.Knockout ADAR1expression of KYSE510cell line can inhibit the corresponding function.We also discover that ADAR1is closely related to cell cycle of tumor cells by flow cytometry.Knockdown expression of ADAR1can inhibit proliferation of tumor cells by inhibiting the Gl/S transition.That is,ADAR1can help the cancer cells to grow.ConclusionIn conclusion,the expression of ADAR1is obviously upregulated in ESCC tissue comparisoning with the matched nontumor tissue,ADAR1can promote cancer cell to grow、 invasion and migration.ADAR1is the independent factor that affects the ESCC patients’prognosis.