miR-32 promotes esophageal squamous cell carcinoma metastasis by targeting CXXC5

MicroRNA-32 (miR-32) functioned as a tumor oncogene in some cancer, which control genes involved in important biological and pathological functions and facilitate the tumor growth and metastasis. However, the role of miR-32 modulates esophageal squamous cell carcinoma (ESCC) malignant transformation has not been clarified. Here, we focused on the function and the underlying molecular mechanism of miR-32 in ESCC. Results discovered a significant increased expression of miR-32 in ESCC tissues and cells. Downregulation of miR-32 inhibited the migration, invasion, adhesion of ESCC cell lines (EC9706 and KYSE450), and the levels of EMT protein in vitro. In vivo, miR-32 inhibitors decrease tumor size, tumor weight, and the number of metastatic nodules. Hematoxylin and eosin (H&E) results revealed that inhibition of miR-32 attenuate lung metastasis. Immunohistochemistry and immunofluorescence assay showed increased level of E-cadherin and decreased level of N-cadherin and Vimentin with treatment of miR-32 inhibitors. Furthermore, miR-32 targeted the 3′-untranslated region (3′-UTR) of CXXC5, and inhibited the level of mRNA and protein of CXXC5. There is a negative correlation between the expressions of CXXC5 and miR-32. Then, after EC9706 and KYSE450 cells cotransfected with si-CXXC5 and miR-32 inhibitors, the ability of cell migration, invasion, and adhesion was significantly reduced. In addition, the protein expression of EMT and TGF-β signaling was also depressed. Collectively, these data supply an insight into the positive role of miR-32 in ESCC progression and metastasis, and its biological effects may attribute the inhibition of TGF-β signaling mediated by CXXC5.     

Tumor-suppressive microRNA-10a inhibits cell proliferation and metastasis by targeting Tiam1 in esophageal squamous cell carcinoma

Aberrant microRNAs (miRNAs) expressions could contribute to the progression of numerous cancers, including esophageal squamous cell carcinoma, while miR-10a participates in multiple biological processes on cancers. However, the molecular mechanism of miR-10a in esophageal squamous cell carcinoma (ESCC) has not been investigated. Herein, miR-10a was significantly reduced in ESCC clinical tissues and ESCC cell lines (EC109 and TE-3). In addition, immunohistochemistry indicated that the expressions of α-SMA, Ki-67, and PCNA in tumor tissues were higher than that of controls. In vitro, overexpression of miR-10a dramatically suppressed cell proliferation and enhanced cell apoptosis, while the decrease of miR-10a expressed the opposite outcome. Specially, overexpression of miR-10a caused a G0/G1 peak accumulation. Moreover, miR-10a also negatively regulated ESCC cell migration and invasion. Furthermore, targetscan bioinformatics predictions and the dual-luciferase assay confirmed that Tiam1 was a direct target gene of miR-10a. The statistical analysis showed Tiam1 was negatively in correlation with miR-10a in ESCC patient samples. And silencing Tiam1 could lead to a decline on cell growth, invasion, and migration in ESCC cell lines, while it could enhance cell apoptosis and cause a G0/G1 peak accumulation. In vivo, it revealed that miR-10a notably decreased the tumor growth and metastasis in xenograft model and pulmonary metastasis model. And it showed a lower expressions of Tiam1 in the miR-10a mimics group by immunohistochemistry. Taken together the results, they indicated that miR-10a might function as a novel tumor suppressor in vitro and in vivo via targeting Tiam1, suggesting miR-10a to be a candidate biomarker for the ESCC therapy.     

Over-expression of Oct4 in human esophageal squamous cell carcinoma

The Octamer 4 gene (Oct4) is a master pluripotency controller that has been detected in several types of tumors. Here, we examine the expression of Oct4 in human esophageal squamous cell carcinoma (ESCC). We found that punctate Oct4 protein was expressed in most (93.7%) ESCC samples but it was not observed in esophageal mucosa. Some ESCC cells had the capacity to form tumorospheres; those with an Oct4⁺-rich cell phenotype had increased proliferation and Oct4 mRNA levels compared to those of differentiated cells in culture or xenograft tumors. The over-expression of Oct4 in ESCCs suggests that it is a potential target for ESCC therapy. Oct4 could be a useful tumor marker in an immunohistochemical panel designed to differentiate between ESCC and esophageal mucosa. Expression of Oct4 in tumorospheres might indicate the presence of a population of ECSCs and its expression in xenograft tumors suggests that Oct4 is also associated with tumor metastasis.     

Silencing HMGN5 suppresses cell growth and promotes chemosensitivity in esophageal squamous cell carcinoma

Previous study has demonstrated that high mobility group nucleosome‐binding domain 5 (HMGN5) is involved in tumorigenesis and the development of multidrug resistance in several human cancers. However, the role of HMGN5 in esophageal squamous cell carcinoma (ESCC) remains unclear. Here, we showed that HMGN5 was significantly upregulated in ESCC cells. Knockdown of HMGN5 significantly inhibited cell growth and induced cell apoptosis of ESCC cells. Moreover, knockdown of HMGN5 increased the sensitivity of ESCC cells towards cisplatin. By contrast, overexpression of HMGN5 showed the opposite effects. Further experiments demonstrated that HMGN5 regulated the expression of multidrug resistance 1, cyclin B1, and Bcl‐2. Overall, our results reveal that HMGN5 promotes tumor progression of ESCC and is also an important regulator of chemoresistance. Our study suggests that inhibition of HMGN5 may be a potential strategy for improving effectiveness of ESCC treatment.     

Upregulation of long noncoding RNA SNHG20 promotes cell growth and metastasis in esophageal squamous cell carcinoma via modulating ATM-JAK-PD-L1 pathway

Increasing evidence have proved that long noncoding RNAs (lncRNAs) play significant roles in tumorigenesis and development of various cancers. However, the effect of small nucleolar RNA host gene 20 (SNHG20) on the progression of esophageal squamous cell carcinoma (ESCC) remains to be discovered. Herein, we aim to find out the function and the possible mechanism of SNHG20 in ESCC progression. In our study, we demonstrate that SNHG20 is markedly upregulated in ESCC tissues and cell lines. Besides, the level of SNHG20 is closely associated with tumor size, lymph node metastasis, TNM stage, and tumor grade. In addition, SNHG20 level is an independent predictor for clinical outcomes of ESCC patients. Then the gain- and loss-of-function assays reveal that SNHG20 overexpression promotes cell proliferation, migration, invasion, and epithelial-mesenchymal transition as well as represses apoptosis, whereas depletion of SNHG20 exhibits opposite effects. Moreover, we uncover that SNHG20 modulates the expression of ataxia telangiectasia–mutated kinase (p-ATM), p-JAK1/2, and programmed cell death 1 ligand 1 (PD-L1) in ESCC cells and ATM upregulation restores the suppressive effect of SNHG20 inhibition on ESCC progression. Therefore, we conclude that SNHG20 serves as a carcinogen in ESCC by promoting growth and metastasis via ATM-JAK-PD-L1 pathway, supplying a possibly effective therapeutic target for ESCC.     

PAX5 gene as a novel methylation marker that predicts both clinical outcome and cisplatin sensitivity in esophageal squamous cell carcinoma

Therapeutic strategies for esophageal cancer largely depend on histopathological assessment. To select appropriate treatments of individual patients, we examined the background molecular characteristics of tumor malignancy and sensitivity to multidisciplinary therapy. Seventy-eight surgically-resected esophageal squamous cell carcinoma (ESCC) cases during 2001–2013 were examined. PAX5, a novel gene methylation marker in ESCC, was evaluated in the specimens, as methylation of this gene was identified as an extremely tumor-specific event in squamous cell carcinogenesis of head and neck. PAX5 methylation status was evaluated by quantitative MSP (QMSP) assays. Mean QMSP value was 15.7 (0–136.3) in ESCCs and 0.3 (0–8.6) in adjacent normal tissues (P < 0.001). The 78 cases were divided into high QMSP value (high QMSP, n = 26) and low QMSP value (low QMSP, n = 52). High QMSP cases were significantly associated with downregulated PAX5 expression (P = 0.040), and showed significantly poor recurrence-free survival [Hazard Ratio (HR) = 2.84; P = 0.005; 95% Confidence Interval (CI): 1.39–5.81] and overall survival (HR = 3.23; P = 0.002; 95%CI: 1.52–7.01) in multivariable analyses with histopathological factors. PAX5-knockdown cells exhibited significantly increased cell proliferation and cisplatin resistance. PAX5 gene methylation can predict poor survival outcomes and cisplatin sensitivity in ESCCs and could be a useful diagnostic tool for cancer therapy selection.     

Circular RNA circ-Foxo3 inhibits esophageal squamous cell cancer progression via the miR-23a/PTEN axis

Circ-Foxo3 is a circRNA encoded by the human FOXO3 gene and works as a sponge for potential microRNAs (miRNAs) to regulate cancer progression. However, the role of circ-Foxo3 in esophageal squamous cell cancer (ESCC) is not clear. In this study, circ-Foxo3 was lowly expressed in cell lines and ESCC tissues. Meanwhile, overexpression of circ-Foxo3 inhibited cell growth, migration, and invasion, whether in vivo or in vitro. Mechanically, we found a potential miRNA target, miR-23a, which negatively correlated with circ-Foxo3 in ESCC. Then, a luciferase assay confirmed the relationship between the circ-Foxo3 and miRNA. Moreover, circ-Foxo3 upregulation of PTEN occurred through “sponging” miR-23a. Taken together, these results indicated that the circ-Foxo3/miR-23a/PTEN pathway was critical for inhibiting the ESCC progression. This may provide a promising target for treat ESCC.     

LINC00473/miR-374a-5p regulates esophageal squamous cell carcinoma via targeting SPIN1 to weaken the effect of radiotherapy

Esophageal squamous cell carcinoma (ESCC) is the most prevalent type in esophageal cancers. Despite accumulating achievements in treatments of ESCC, patients still suffer from recurrence because of the treatment failures, one of the reasons for which is radioresistance. Therefore, it is a necessity to explore the molecular mechanism underlying ESCC radioresistance. Long intergenic noncoding RNA 473 (LINC00473) has been reported to be aberrantly expressed in several human malignancies. However, its biological function in radiosensitivity of ESCC remains to be fully understood. This study explored the role of LINC00473 in radiosensitivity of ESCC cells and whether LINC00473 acted as a competing endogenous RNA to realize its modulation on radioresistance. We found that LINC00473 was markedly upregulated in ESCC tissues and cell lines, and its expression was remarkably related to cellular response to irradiation. In addition, knockdown of LINC00473 could sensitize ESCC cells to radiation in vitro. As for the underlying mechanism, we uncovered that there was a mutual inhibition between LINC00473 and miR-374a-5p. Spindlin1 (SPIN1) was verified as a downstream target of miR-374a-5p, and LINC00473 upregulated SPIN1 expression through negatively modulating miR-374a-5p expression. Furthermore, we revealed that SPIN1 could aggravate the radioresistance of ESCC cells. Finally, overexpression of SPIN1 reversed the LINC00473 silencing-enhanced radiosensitivity in ESCC cells. To sum up, we demonstrated that LINC00473 facilitated radioresistance by regulating the miR-374a-5p/SPIN1 axis in ESCC.     

miR-204-5p regulates cell proliferation,invasion, and apoptosis by targeting IL-11 in esophageal squamous cell carcinoma

Esophageal cancer (EC) is the world's eighth most common malignant neoplasm and is ranked as the sixth leading cause of death related to cancer. Aberrant microRNA (miRNA) expression has been reported to be associated with esophageal squamous cell carcinoma. However, the molecular mechanism of miR-204-5p in esophageal squamous cell carcinoma (ESCC) is not clear. Therefore, the aim of this study was to investigate the potential role of miR-204-5p in ESCC. In the present study, we found that miR-204-5p could affect ESCC proliferation, invasion, apoptosis, and cell cycle in cell and mouse models. A dual-luciferase reporter assay showed that miR-204-5p expression was negatively correlated with interleukin-11 (IL-11) expression. IL-11 overexpression reversed the suppressive effects of miR-204-5p in the cell lines. These results indicated that miR-204-5p functions as a tumor suppressor by directly targeting IL-11 in ESCC.     

MicroRNA-143 functions as a tumor suppressor in human esophageal squamous cell carcinoma

Esophageal cancer is one of the most common cancers worldwide with a poor prognosis. MicroRNAs(miRNAs) are a class of naturally occurring small noncoding RNAs and play an important role in cancer initiation and development. In this study, we demonstrate that the expression levels of miR-143 and miR-145 were significantly decreased in ESCC tissues in comparison with adjacent normal esophageal squamous tissues(NESTs). Furthermore, an inverse correlation between miR-143 and tumor invasion depth and lymph node metastasis was observed. The enforced expression of miR-143 induced growth suppression and apoptosis of ESCC cells. Rescue of miR-143 significantly suppressed the ESCC cells migration and invasion capabilities. Moreover, we show that functions of miR-143 in ESCC are mediated at least in part by the inhibition of extracellular signal regulated kinase-5(ERK-5) activity. These results prove that miR-143 may act as a tumor suppressor in ESCC.     

Linc-ROR regulates apoptosis in esophageal squamous cell carcinoma via modulation of p53 ubiquitination by targeting miR-204-5p/MDM2

The long intergenic noncoding RNA, regulator of reprogramming (linc-ROR) has been reported to participate in tumorigenesis, while its functions and fundamental mechanisms in esophageal squamous cell carcinoma (ESCC) remain unclear. In this study, gain-of-function assays showed that linc-ROR upregulation enhanced cell viability, promoted cell proliferation, and inhibited apoptosis. Mechanistically, the regulatory network of linc-ROR/miR-204-5p/MDM2 was established with bioinformatics analysis and online databases, then validated via dual-luciferase reporter assays, RNA immunoprecipitation assays in ESCC cells. Linc-ROR positively regulates the expression of MDM2 as a molecular sponge of miR-204-5p. Moreover, results of western blot and coimmunoprecipitation indicated that linc-ROR overexpression enhanced the ubiquitination level of p53, and its downstream apoptosis-related genes have showed higher bcl-2 expression, lower bax, and cleaved caspase-3 expressions, while miR-204-5p could counteract with this effect. Finally, small interfering RNAs tailored to linc-ROR were established to further evaluate its effects on ESCC comprehensively. In summary, this study revealed that linc-ROR modulated cell apoptosis and regulated p53 ubiquitination via targeting miR-204-5p/MDM2 axis, which provides a novel therapeutic insight into treatments for ESCC.     

Serum miR-1297: a promising diagnostic biomarker in esophageal squamous cell carcinoma

We aimed to value the diagnostic potential of serum miR-1297 in esophageal squamous cell cancer (ESCC). Its expression level was detected in 156 pairs of patients with ESCC and healthy volunteers using quantitative real-time polymerase chain reaction (qRT-PCR) method. It was statistically decreased in ESCC patients compared with healthy controls. AUC based on serum miR-1297 was 0.840?±?0.035 in discovery group and 0.837?±?0.034 in validation group. Further analysis on early-stage patients revealed that the AUC was 0.819?±?0.053 in discovery group and 0.814?±?0.044 in validation group. Its sensitivity and specificity were promising. In conclusion, serum miR-1297 can serve as an ideal indicator for the diagnosis of ESCC.     

Apoptosis inhibitor 5 increases metastasis via Erk-mediated MMP expression

Apoptosis inhibitor 5 (API5) has recently been identified as a tumor metastasis-regulating gene in cervical cancer cells. However, the precise mechanism of action for API5 is poorly understood. Here, we show that API5 increases the metastatic capacity of cervical cancer cells in vitro and in vivo via up-regulation of MMP-9. Interestingly, API5-mediated metastasis was strongly dependent on the Erk signaling pathway. Conversely, knock-down of API5 via siRNA technology decreased the level of phospho-Erk, the activity of the MMPs, in vitro invasion, and in vivo pulmonary metastasis. Moreover, the Erk-mediated metastatic action was abolished by the mutation of leucine into arginine within the heptad leucine repeat region, which affects protein-protein interactions. Thus, API5 increases the metastatic capacity of tumor cells by up-regulating MMP levels via activation of the Erk signaling pathway. [BMB Reports 2015; 48(6): 330-335]     

Overexpression of Aurora-A kinase promotes tumor cell proliferation and inhibits apoptosis in esophageal squamous cell carcinoma cell line

Attrora-A kinase, a serine/threonine protein kinase, is a potential oncogene. Amplification and overexpression of Aurora-A have been found in several types of human tumors, including esophageal squamous cell carcinoma （ESCC）. It has been demonstrated that cells overexpressing Attrora-A are more resistant to cisplatin-induced apoptosis. However, the molecular mechanisms mediating these effects remain largely unknown. In this report, we showed that overexpression of Attrora-A through stable transfection of pEGFP-Aurora-A in human ESCC KYSE150 cells significantly promoted cell proliferation and inhibited cisplatin- or UV irradiation-induced apoptosis. Cleavages of caspase-3 and poly （ADPribose） polymerase （PARP） in Attrora-A overexpressing cells were substantially reduced after cisplatin or UV treatment. Furthermore, we found that silencing of endogenous Aurora-A kinase with siRNA substantially enhanced sensitivity to cisplatin- or UV-induced apoptosis in human ESCC EC9706 cells. In parallel, overexpression of Aurora-A potently upregulated the expression of Bcl-2. Moreover, the knockdown of Bcl-2 by siRNA abrogated the Aurora-A＇s effect on inhibiting apoptosis. Taken together, these data provide evidence that Aurora-A overexpression promoting cell proliferation and inhibiting apoptosis, suggesting a novel mechanism that is closely related to malignant phenotype and anti-cancer drugs resistance of ESCC cells.     

Upregulation of lncRNA GAS5 inhibits the growth and metastasis of cervical cancer cells

This study aims to figure out the methylation of long non-coding RNA GAS5 promoter in cervical cancer and the mechanism of GAS5 on the progression of cervical cancer cells. The expression of GAS5 and methylation state of GAS5 in cervical cancer tissues and cells were determined. With the aim to to explore the ability of GAS5 in the proliferation, cell cycle progression, apoptosis, invasion, migration as well as the tumor growth, and metastasis in nude mice were determined. The expression of GAS5 was decreased and methylation state of GAS5 was elevated in cervical cancer. Overexpression of GAS5 inhibited proliferation, cell cycle progression, invasion, migration while inducing apoptosis of cervical cancer cells as well as suppressed tumor growth and metastasis in nude mice. Our study demonstrates that abnormal methylation of GAS5 contributes to poor expression of GAS5 in cervical cancer. In addition, upregulation of GAS5 inhibits the cervical cancer development.