| Esophageal cancer has become the sixth leading cause of death and the eighth most frequently diagnosed cancer worldwide. Esophageal squamous cell carcinoma (ESCC) is predominant in most parts of the world, especially in high risk regions such as China where it accounts for over 90% of the total cases of esophageal cancer. Despite significant advances in the diagnosis, staging and treatment of ESCC in recent decades, few significant improvements in overall survival have been achieved:the 5-year overall survival rate remains between 15% and 25%. Local recurrences and distant organ metastases are the leading causes of treatment failure and poor prognosis. Therefore, it is imperative to explore the underlying mechanism and identify additional biomarkers and therapeutic targets for ESCC.AJUBA belongs to the Zyxin/Ajuba family which are characterized by the conservation of three tandem C-terminal LIM domains and a unique N-terminal preLIM region. AJUBA has been reported to interact with a variety of proteins or DNA and affect cell proliferation and the control of tissue size and be involved in cell-cell adhesion, proliferation, migration and cell fate decision by acting as a scaffold or adaptor protein. However, the expression level, function and underlining mechanisms of AJUBA in ESCC remain largely unknown. In the present study, we analyzed the mRNA levels of AJUBA in 179 pairs of ESCC tumor tissues and corresponding non-tumor tissues and detected the protein levels of AJUBA in 81 ESCC tumor tissues and 60 corresponding non-tumor tissues by immunohistochemistry (IHC). The results showed that the AJUBA levels were significantly higher in ESCC tissues compared with corresponding non-tumor tissues, overexpression of AJUBA was correlated with the differentiation and depth of invasion (T stage) of ESCC tissues. We showed that knockdown of AJUBA with shRNAs inhibited the proliferation, migration and invasion ability of KYSE450, KYSE510 and KYSE180. On the contrary, overexpression of AJUBA promoted the migration and invasion ability of KYSE450 and KYSE510 in vitro and in vivio.RNA-Sequencing is a kind of technology which can detect the transcriptome sequencing and RNA levels with high sensitivity and high accuracy. In this study, RNA-Sequencing was used to identify the altered genes following AJUBA knockdown in KYSE450, KYSE510 and KYSE180 cells. Genes that were significantly upregulated or downregulated by AJUBA knockdown in three cell lines were selected for Gene Ontology (GO) analysis. The GO analysis revealed that a number of genes involved in cell motility, cell adhesion and cell junctions were significantly dysregulated following AJUBA knockdown. Among these genes, the mRNA levels of MMP10 and MMP13 were downregulated by 5.6-folds and 5.5-folds, respectively, in AJUBA depleted cells compared with the control cells. In addition, mRNA levels of AJUBA were significantly associated with elevated MMP10 and MMP13 expression in 179 ESCC tumor tissues (r= 0.441, P< 0.001 and r= 0.404, p< 0.001 respectively). Furthermore, AJUBA promoted migratory and invasive potential of ESCC cells as well as the expression of MMP10 and MMP13 through activating the ERK1/2.Moreover, AJUBA could suppressed cisplatin-induced cell apoptosis, promoted the survival of ESCC cells.In conclusion, our study revealed that AJUBA was frequently upregulated in ESCC tumor tissues. The overexpression of AJUBA promoted the tumorigenicity and motility of ESCC. The overexpression of AJUBA increased the resistance of ESCC cells to cisplatin, which could serve as a new strategy for treatment.DNA cytosine methylation is one of the most important epigenetic modifications and is involved in various biological processes, such as genomic imprinting, X chromosome inactivation, and gene expression regulation. In the mammalian genome, almost all DNA methylation occurs at the C-5 atom of cytosine in CpG dinucleotides. 5-Methylcytosine (5-mC) is initially generated by the DNA methyltransferases Dnmt3a and Dnmt3b and is maintained by Dnmtl during DNA replication.Ten-eleven translocation (TET) enzymes, a family of Fe(2+) and a-oxoglutarate-dependent dioxygenases, catalyze the oxidation of 5-mC into 5-hydroxymethylcytosine (5-hmC),5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC) and play a key role in DNA demethylation. There are three TET genes in mammalian cells, TET1, TET2 and TET3, and these genes are responsible for the tissue-dependent conversion of 5-mC to 5-hmC.It was reported that 5-hmC levels were decreased in a variety of cancers and could be regarded as an epigenetic hallmark of cancer. In the present study,5-hmC levels were detected by immunohistochemistry (IHC) in 173 esophageal squamous cell carcinoma (ESCC) tissues and 91 corresponding adjacent non-tumor tissues; DNA dot blot assays were used to detect the 5-hmC level in another 50 pairs of ESCC tissues and adjacent non-tumor tissues. In addition, the mRNA level of TET1, TET2 and TET3 in these 50 pairs of ESCC tissues was detected by real-time PCR. The IHC results showed that 5-hmC levels were significantly downregulated in ESCC tissues compared with adjacent non-tumor tissues. Overall,62% (56/91) of non-tumor tissues showed positive 5-hmC expression, whereas 47% (82/173) of tumor tissues were positive for 5-hmC expression, this difference was statistically significant (P= 0.029). DNA dot blot results showed 5-hmC levels were significantly decreased in tumor tissues compared with paired normal tissues (P< 0.001). TET2 and TET3 expression was also significantly decreased in tumor tissues compared with paired non-tumor tissues (TET2, P< 0.0001; TET3, P= 0.009), and the decrease in 5-hmC was significantly associated with the downregulation of TET2 expression (r= 0.405, P= 0.004). Moreover, the loss of 5-hmC in ESCC tissues was significantly associated with poor overall survival among patients with ESCC (P= 0.043); multivariate Cox regression analysis showed that the loss of 5-hmC in ESCC tissues was an independent unfavorable prognostic indicator for patients with ESCC (HR= 1.569, P = 0.029).In addition to the downregulated expression of TET genes, it was reported that decreased 5-hmC expression in malignant tumors could be caused by mutations in the TET, IDH1 or IDH2 genes. Whole exome sequencing data for 105 of the 173 cases with IHC staining in the present study were obtained from our previous study, and 12 of the 105 cases had mutations in the TET 1/2/3 or IDH1/2 genes. We analyzed potential correlations between TET or IDH1 gene mutations and 5-hmC levels in these 105 cases. The result showed lower 5-hmC expression in patients with TET or IDH1 mutations than in those with wild type versions of these genes, although the P value was not significant (P= 0.051).In conclusion,5-hmC levels were decreased in ESCC tissues, and the loss of 5-hmC in tumor tissues was an independent unfavorable prognostic factor for patients with ESCC. |
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