| BackgroundHuman esophageal cancer is one of the leading causes of cancer-related mortality worldwide, with occurrence rates that vary greatly by geographic location. According to the histological classification, esophageal cancer can be divided into two main forms: esophageal squamous cell carcinoma(ESCC) and esophageal adenocarcinoma(EAC). ESCC occurs with high prevalence in many areas of the world especially in China. Despite advances in diagnosis and treatment, the overall 5-year survival rate for ESCC patients is still very low. The reason for this may be a combination of a late stage at diagnosis and limited access to timely and standard treatment. Therefore, it is imperative to seek more effective biomarkers and therapeutic targets for ESCC.Non-coding RNAs(nc RNAs) are emerging as new regulators in the cancer paradigm. They are classified into small non-coding RNAs and long non-coding RNAs(IncRNAs) based on size. IncRNAs, a new research hotspot among cancer-associated nc RNAs, is an important compoent of epigenetic, transcriptional and post-transcriptional regulation. Multiple lines of evidences have highlighted Inc RNAs as drivers of tumor progression and as oncogenic suppressors in various cancer types. Since first clue indicative of such relationship was found in the biological functions lnc RNA implicated in HOTAIR(HOX transcript antisense RNA) and breast cancer, intense attentions were drawn on this area leading to an increasing number of discovers demonstrating that dysfunction of lnc RNAs is closely associated with tumor formation, proliferation, invasion, and metastasis. Thus, identification of cancer-associated lncRNAs and the interplay between lnc RNAs and protein-coding genes are of profound importance in the field of cancer biology, in which lncRNAs may provide a missing piece of the oncogene and tumor suppressor network puzzle.Inc RNA H19 is produced from imprinted genes H19, whose expression is depending on the parental origin of the chromosome. Recent studies disclosed that H19 is up-regulated in various human malignancies such as bladder cancer, breast cancer, hepatocellular cancer, suggesting that it has oncogenic properties. While other studies revealed that H19 may suppress malignant proliferation, invasion and metastasis, and tumor angiogenesis. More striking is the predictive value of H19 for tumor recurrence, and its prognostic significance.ObjectiveAlthough much investigated, the exact biological functions of H19 and its mechanism in ESCC currently remain to be elucidated. The purpose of this study was to elucidate the role of H19 in development and progression of ESCC and its corresponding molecular mechanism.Materials and Methods1.A total of 64 ESCC patients who underwent surgery resection at Southwest Hospital(Third Military Medical University, Chongqing, China) between 2007 and 2012 were enrolled in this study. The expression levels of H19 were first evaluated in 64 paired of ESCC samples and adjacent, nontumorous tissues by q RT-PCR. The relationship between the clinicopathologic features and the expression of H19 was analyzed.2.Expressions of H19 in human normal esophageal epithelial cell line(Het-1A) and human esophageal squamous cell carcinoma cell lines(EC109, EC9706, KYSE150, and KYSE450) were detected respectively by qRT-PCR.The ESCC cell lines of high level of Inc RNA H19 were used in the following tests.3.Then we knocked down H19 in ESCC cell lines EC109 and EC9706 by small interfering RNAs(siRNAs). Experiments were divided into two groups, experimental group(si-H19) and negative control group(si-NC). Cell proliferation were evaluated by CCK-8 cell viability analysis,flow cytometry cell cycle, and Xenograft assays in nude mice,Cell migration and invasive capability were evaluated by in vitro cell migration and invasion assay.4.the effects of H19 knockdown on the regulation of the epithelial-mesenchymal transition(EMT) markers and metastasis-associated protein such as MMP-9 was detected by Western blot analysis.Results1.H19 levels were significantly up-regulated in ESCC tissues, which may play an important role in the carcinogenesis and progression of ESCC. Furthermore, correlation of H19 expression with pathological features of ESCC patients, revealed a significant association between H19 up-regulation and tumor invasion depth(P=0.01) and lymph node metastasis(P=0.007). Other clinical factors, such as age, gender and tumor differentiation degree, were not found to be significantly associated with H19 expression in this study.2.To further investigate the functional effects of H19 in ESCC cells, qRT-PCR was performed to investigate H19 expression in four human ESCC cell lines(EC109, EC9706, KYSE150, and KYSE450) and a normal esophageal epithelial cell line(Het-1A). compared with Het-1A, all ESCC cell lines expressed higher H19 levels. The relatively higher H19-expressing cell lines(EC109 and EC9706) were selected for further studies.3.Then, knocked down of H19 expression was performed in EC109 and EC9706 cells lines by si RNA. The results of the CCK-8 assay showed that the proliferation of EC109 and EC9706 cells were significantly inhibited by transfection of si RNA that target H19 compared with negative control.Using cell-cycle analysis, The data showed that the cell cycle progression of H19 transfected cells was significantly stalled at G0/G1 phase in both cell lines compared with that of control groups.To validate the influence of H19 on growth of ESCC cells in vivo, EC9706 cells transfected with si-H19 were subcutaneously injected into a nude mice xenograft model.The results showed that tumors grew slower in si-H19 transfected mice than that in the NC group four weeks after injection. The detection of migration by transwell suggested that cell migration was remarkably reduced by H19 knockdown. In agreement with this finding, the invasiveness of these two cell lines were also reduced separately following knockdown of H19.4.Western blot analysis showed that knockdown of H19 substantially increased the expression of epithelial marker(E-cadherin) with that in the negative control group, while causing a significant decrease in the expression of mesenchymal marker(vimentin) and metastasis-associated protein(MMP-9).ConclusionIn summary, these findings provided evidence to support that H19, a lncRNA up-regulated in ESCC, is an oncogene that promotes tumor growth and metastasis, probably through its role in an EMT-like process. H19 seems to be an important regulator of the metastasis of ESCC and the potential target for treatment of this cancer. |
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