MiR-429Up-regulation Induces Cell Apoptosis And Suppresses Invasion By Targeting Bcl-2and SP-1in Esophageal Carcinoma

Background and PurposeEsophageal carcinoma (EC) is one of the six most common malignancy worldwid. in China, EC ranks fourth in cancer mortality (17.38/105). Early EC may be asymptomatic or only mild symptoms. The clinical diagnosis is established only after late development that appears to be progressive dysphagia, but the best time for treatment is lost. Nowdays, human have conducted research on post-genome. Therefore, it’s particularly important to screen susceptible populations of EC at high risk and explore its causes and treatment at genetic level.MicroRNAs (miRNAs), typically18-25nucleotides long, constitute a class of small non-coding single-stranded RNAs (ssRNAs). miRNAs have been linked to the development and progression of several cancers by regulating gene expression targeting, and thus play the role of oncogenes or tumor suppressor genes. To explore miRNA expression level in EC, our labor have previously conducted a miRNA chip-based expression analysis of primary EC tissues and found that the expression of miR-429in these tissues was lower than that in adjacent paired non-tumor tissues. The purpose of this study is to find out the expression levels of miR-429in a cohort of EC tissues and adjacent tissues, and investigate the relationship between miR-429and pathological features of EC patients. We also want to reveal the effect of miR-429up-regulation on apoptosis and invasion in EC cells, explore the targets of miR-429and its regulatory mechanism. Methods1. EC specimens (n=32) were obtained from eligible patients between June2011and May2012, from the First Affiliated Hospital of Zhengzhou University, and preservated at-80℃. qRT-PCR assays were used to quantify the expression levels of miR-429, Bcl-2and SP1in32paired EC samples and adjacent non-neoplastic tissues. We determined the expression levels of Bcl-2and SP1by Western blot assay to analyze the relationship between the expression levels and clinical pathological feature of patients.2. Using liposomes to transfect synthetic miR-429agomir or miR-429negative control into EC9706and KYSE30. The cells were divided into three groups:miR-429, cells transfected with miR-429agomir; NC, cells transfected with miR-429negative control; Blank, cells transfected with only liposomes. Transfected cells were placed in carbon dioxide incubator and cultured for subsequent experiments.3. CCK-8growth experiment was used to evaluate the cell growth effect of miR-429expression on EC cells.4. Flow cytometry and Caspase activity assays were used to evaluate the cell apoptosis effect of miR-429expression on EC cells.s for, apoptosis, caspase activity and by observed effects of miR-429on apoptosis.5. Transwell assay was used to evaluate the cell invasion effect of miR-429expression on EC cells.6. Bioinformatic analyses using TargetScan and miRanda predicted the targets of miR-429. Constructed wild type and mutant type recombinant vectors that contained Bcl-2/SP13’UTR region. Respectively, co-transfected them with miR-429agomir or miR-429negative control into EC9706and KYSE30. Luciferase reporter and Western blotting assays were used to confirm the major targets of miR-429.7. We constructed expression vectors containing Bcl-2/SP1lacking the3’UTR sequence. Transfected expression vectors alone or co-transfected with miR-429agomir into EC9706and KYSE30. Restore assay was used to analyse mechanism that miR-429regulated the expression of Bcl-2and SP1.Results 1. The expression levels of miR-429in EC tissues were found to be lower than those in adjacent non-neoplastic tissues (P<0.05), expression levels of Bcl-2and SP1were significantly higher than in adjacent tissues (P<0.05). The expression levels of miR-429and Bcl-2, SP1were negatively correlated. miR-429, Bcl-2and SP1gene expression were found to be significantly associated with the occurrence of lymph node metastases, degree of differentiation and TNM stage (P<0.05), and not with gender, age or tumor location (P>0.05).2. miR-429expression levels of miR-429group was significantly higher than NC group and Blank group (P<0.05). After transfection miR-429mimics, miR-429expression levels of esophageal cancer cells increases.3. CCK-8growth experiments showed that, compared to the Blank and NC groups, the OD490values for the miR-429group on days2,3and4were significantly decreased in both EC9706and KYSE30(P<0.05).4. Our flow cytometry and Caspase activity assays showed that, compared to the Blank and NC groups, the apoptosis levels of cells and caspase-3/7activity of miR-429group were both significantly enhanced(P>0.05).5. Using a trans-well assay we found that, compared with the NC group and Blank group, the mean numbers of cells penetrating the membrane for miR-429group were significantly decreased (P<0.05).6. Western blot and luciferase reporter assays showed that Bcl-2and SP1act as targets of miR-429.7. Restore assay showed that miR-429can bind to putative binding sites within the Bcl-2or SP1mRNA3’untranslated regions (UTR) to negative regulation of its expression, thus to participate in apoptosis and invasion of EC cells.Conclusions1. In EC tissues miR-429was expressed at abnormally low levels. The expression was associated with the occurrence of lymph node metastases, degree of differentiation and TNM stage.2. Up-regulation of miR-429can effectively inhibit growth and invasion and promote apoptosis in EC cells.3. miR-429negatively regulated the expression levels of Bcl-2and SP1by targeting their3’UTR region, and thus play a biological role.