| Background and ObjectionsEsophageal cancer(EC),which keeps threatening human health,has a high morbidity and mortality globally and in China.Esophageal cancer develops rapidly,at time of detection,the patients are usually at advanced stages or suffered with lymph node metastasis.Recently,although the diagnosis and treatment for esophageal cancer have been highly improved,Esophageal cancer remains low the overall survival rate.Therefore,it is particularly important to study the mechanism of tumorigenesis and progression of esophageal cancer for assisting its diagnosis and treatment.Micro RNAs(mi RNAs),which are a class of conservative non-coding regulatory RNAs with 18-24 nucleotides in length,can exert posttranscriptional regulatory function by binding complementarily to the 3' untranslated region(3'UTR)of target gene m RNA.Emerging evidences demonstrated that mi RNAs play important roles in the biological processes of cell,such as differentiation,apoptosis,proliferation and metabolism.Moreover,mi RNAs are usually actively involved in the pathogenesis of multiple cancers as oncogenes or tumor suppressors.A large number of studies have found many functional mi RNAs with dysregulation in esophageal cancer tissues.However,the whole landscape of mi RNAs in esophageal cancer remains incomplete and further exploring is awaited.DNA polymerase ?(pol ?)is the smallest eukaryotic DNA polymerase and participates in the key enzymes of base excision repair(BER)process.As a repair enzyme in mammalian cell nucleus,pol ? also plays an important role in base excision repair,DNA replication and DNA synthesis.Furthermore,more and more evidences showed that pol ? acts as a key factor in tumorigenesis and progression and a potential therapeutic target in multiple cancers.Previous gene chip analysis of esophageal cancer and their matched para-cancerous tissues in our laboratory showed that there was differential expression of micro RNA-149.Bioinformatics analysis showed that there were binding sites between micro RNA-149 and DNA pol ?.In this study,we examined the direct interaction between micro RNA-149 and DNA Pol ? and studied the effects of this relationship on phenotype of esophageal cancer cell.Our findings provide better understanding of occurrence and development mechanisms of esophageal cancer and give a potential target to facilitate the diagnosis and treatment of this disease.Methods1.From 2014 to 2015,64 samples of esophageal cancer tissues and their matched para-cancerous tissues were collected from hospitalized patients in the First Affiliated Hospital of Zhengzhou University and Linzhou Cancer Hospital,and the samples were stored in liquid nitrogen for subsequent use.2.The expression levels of mi RNA-149 and pol ? in 64 cases of esophageal cancer and corresponding adjacent tissues were detected by q RT-PCR.3.Pearson correlation analysis was used to analyze the correlation between the expression levels of mi RNA-149 and pol ? in esophageal cancer tissues.4.Bioinformatics analysis was performed to predict the putative target genes of mi RNA-149.5.Mi RNA-149 mimics and mi RNA-149 NC were synthesized and transfected into EC-9706 esophageal cancer cells by liposome.The experiment was conducted in EC-9706 cells which were divided into three groups including mi RNA-149 group(transfected with mi RNA-149 mimics),NC group(transfected with mi RNA-149 NC)and Blank group(only added with liposome).6.Western-blot assay was conducted to detect the expression level of pol ? protein in EC-9706 cells transfected with mi RNA-149 mimic or scramble.the target gene of mi RNA-149 was verified.7.Dual-luciferase reporter assay was performed to validate direct interaction of mi RNA-149 and pol ? by using recombinant reporter plasmids containing wild type or mutant type sequence of mi RNA binding site in 3'UTR of pol ? m RNA. The experimental cells were divided into four groups:co-transfection of micro RNA-149 mimics and pmir GLO-Wt-pol ?,co-transfection of micro RNA-149 mimics and pmir GLO-Mu-pol ?,co-transfection of micro RNA-149 NC and pmir GLO-Wt-pol ?,co-transfection of micro RNA-149 NC and pmir GLO-Mu-pol ?.8.SPSS 21.0 software was used for statistical analysis of the experimental data, ?=0.05 was served as the significance test standard.Results1.The results of q RT-PCR showed that compared with adjacent normal tissue,theexpression of pol ? in esophageal cancer tissues was significantly upregulated(P< 0.05).Moreover,the expression of mi RNA-149 in esophageal cancer tissues was markedly downregulated(P < 0.05).2.Pearson correlation analysis showed that there was a negative correlation between the expression of pol ? and mi RNA-149 in esophageal cancer tissues (r=-0.671,P < 0.001).3.Bioinformatics analysis revealed that there were complementary binding sites in the 3'UTR region of pol ? m RNA for mi RNA-149.4.Western blot results showed that the expression level of pol ? protein in mi RNA-149 group was significantly lower than that in Blank group and NC group.5.The results of dual-luciferase reporter assay showed that in EC9706 cells,the luciferase activity of cells co-transfected with mi RNA-149 mimics and pmir GLO-Wt was significantly lower than that of the other three groups(P <0.05).It was confirmed that pol ? was the target gene of mi RNA-149. Western-blot results suggest that mi RNA-149 negatively regulates the expression of pol ? protein by binding to the 3'UTR region of pol ? m RNA.ConclusionsIn esophageal cancer tissues,the expression of micro RNA-149 was abnormal and low,and was negatively correlated with the expression of pol ?.Pol ? is a target gene of mi R-149 in esophageal cancer EC9706 cells and mi R-149 can negatively regulate pol? expression by binding to its 3'UTR of its m RNA. |
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