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1.  MicroRNA analysis of microdissected normal squamous esophageal epithelium and tumor cells 
Previous studies have identified several dysregulated microRNAs in esophageal squamous cell carcinoma (ESCC); however, to date there are no ex vivo analyses comparing expression levels of these regulatory molecules in esophageal squamous cell tumors versus patient-matched normal epithelium. We describe here a technical strategy to evaluate microRNAs in normal esophageal basal cells (NB), normal esophageal differentiated cells (ND), and tumor cells (T). Laser capture microdissection was used to procure target populations from five cases and 18 ESCC-associated microRNAs were measured by RT-qPCR. Five microRNAs (miR-25, miR-106b, miR-21, miR-203, and miR-145) demonstrated consistent differential expression in at least one of the three comparisons: T vs. NB, T vs. ND, or NB vs. ND. The potential regulatory role of the microRNAs in ESCC was further evaluated by correlating their expression with a matched mRNA dataset, which included the same five cases and cell populations. In conclusion, the present work demonstrates the feasibility of studying microRNA levels in precisely dissected cell populations from clinical samples, and sheds light on the molecular mechanisms associated with ESCC.
PMCID: PMC3142940  PMID: 21796275
Esophageal squamous cell carcinoma; laser capture microdissection; microRNA; basal layer; differentiated layer; miR-25; miR-106b; miR-21; miR-203; miR-145
2.  The Cluster of miR-143 and miR-145 Affects the Risk for Esophageal Squamous Cell Carcinoma through Co-Regulating Fascin Homolog 1 
PLoS ONE  2012;7(3):e33987.
MicroRNAs (miRNAs), 18–24 nt non-coding RNAs, are thought to play important roles in cell proliferation, differentiation, apoptosis, and development. Recent studies suggest that some of the known microRNAs map to a single genomic locale within a single polycistronic transcript. But the roles of the cluster remain to be known. In order to understand the role and mechanism of a cluster of miR-143 and miR-145 in esophageal squamous cell carcinoma (ESCC), the association of mature miR-143 and miR-145 expression with the risk for esophageal cancer was evaluated in ESCC patients with a case-control study, and target protein regulated by mature miRNA was analyzed in ESCC cell lines with 3′UTR luciferase reporter assay. The expression levels of miR-143 and miR-145 were determined in 110 pairs of esophageal cancer tissues and adjacent normal tissues using real-time reverse transcription PCR. The relative expression of miR-143 and miR-145 were statistically different between cancer tissues and matched controls. The combined expression of miR-143 and miR-145 was significantly associated with the risk for esophageal cancer. Meanwhile, the reduced expression of two miRNAs in tumor patient was supposed to have a trend of lymph node metastases. The co-expression pattern of miR-143 and miR-145 was analyzed with Pearson correlation. It showed a significant correlation between these two miRNAs expression both in tissues and tumor cell lines. 3′UTR luciferase reporter assay indicated that Fascin Homolog 1 (FSCN1) could be co-regulated by miR-143 and miR-145. The protein level of FSCN1 showed no significant linear correlation with miR-143 and miR-145 expression in ESCC cell lines with Western blotting analysis. In conclusion, since miR-143 and miR-145 could regulate oncogenic FSCN1 and take part in the modulation of metastases, the result suggested the combination variable of miR-143 and miR-145 as a potential biomarker for earlier diagnosis and prognosis of esophageal cancer.
PMCID: PMC3311581  PMID: 22457808
3.  Down-regulation of microRNA 10a expression in esophageal squamous cell carcinoma cells 
Oncology Letters  2010;1(3):527-531.
This study identified significantly down-regulated microRNAs (miRs) specific for esophageal squamous cell carcinoma (ESCC) cells. Total RNA was extracted from ESCC cell lines (OE21 and TE10) and a non-malignant human esophageal squamous cell line (Het1A), and subjected to microarray analysis. Expression levels of miRs that showed significant down-regulation in ESCC cells compared to Het1A cells based on the comprehensive analysis were analyzed by quantitative reverse transcription polymerase chain reaction. Among the significantly down-regulated miRs, miR-10a expression levels in the five ESCC cell lines examined were significantly lower than in Het1A and the esophageal adenocarcinoma cells. Since miR-10a is a specific miR in ESCC, its clinical relevance was examined. Using ESCC tumor samples and non-cancerous tissue obtained endoscopically, the involvement of miR-10a in the clinicopathological findings was examined. MiR-10a expression was comparably down-regulated in the tumors of high-grade intraepithelial neoplasm and non-invasive ESCC, while the expression levels were elevated in the invasive ESCC tumors. Treatment with a demethylating agent, 5-aza-2′-deoxycytidine, restored miR-10a expression in OE21 cells. Only a modest additive or synergistic effect was observed in the presence of a histone deacetylase inhibitor, trichostatin A. These results imply that miR-10a may be differentially expressed in ESCC cells and may be involved in ESCC development and progression. The unique epigenetic regulation of miR-10a expression can be mediated via hypermethylation of the CpG islands proximal to its gene locus, at least in certain ESCC cells.
PMCID: PMC3436400  PMID: 22966337
microRNA; microRNA 10a; esophageal squamous cell carcinoma; DNA methylation
4.  MiRNA-205 modulates cellular invasion and migration via regulating zinc finger E-box binding homeobox 2 expression in esophageal squamous cell carcinoma cells 
Esophageal squamous cell carcinoma (ESCC) is often diagnosed at later stages until they are incurable. MicroRNA (miR) is a small, non-coding RNA that negatively regulates gene expression mainly via translational repression. Accumulating evidence indicates that deregulation of miR is associated with human malignancies including ESCC. The aim of this study was to identify miR that could be specifically expressed and exert distinct biological actions in ESCC.
Total RNA was extracted from ESCC cell lines, OE21 and TE10, and a non-malignant human esophageal squamous cell line, Het-1A, and subjected to microarray analysis. Expression levels of miR that showed significant differences between the 2 ESCC and Het-1A cells based on the comprehensive analysis were analyzed by the quantitative reverse transcriptase (RT)-PCR method. Then, functional analyses, including cellular proliferation, apoptosis and Matrigel invasion and the wound healing assay, for the specific miR were conducted. Using ESCC tumor samples and paired surrounding non-cancerous tissue obtained endoscopically, the association with histopathological differentiation was examined with quantitative RT-PCR.
Based on the miR microarray analysis, there were 14 miRs that showed significant differences (more than 2-fold) in expression between the 2 ESCC cells and non-malignant Het-1A. Among the significantly altered miRs, miR-205 expression levels were exclusively higher in 5 ESCC cell lines examined than any other types of malignant cell lines and Het-1A. Thus, miR-205 could be a specific miR in ESCC. Modulation of miR-205 expression by transfection with its precursor or anti-miR-205 inhibitor did not affect ESCC cell proliferation and apoptosis, but miR-205 was found to be involved in cell invasion and migration. Western blot revealed that knockdown of miR-205 expression in ESCC cells substantially enhanced expression of zinc finger E-box binding homeobox 2, accompanied by reduction of E-cadherin, a regulator of epithelial mesenchymal transition. The miR-205 expression levels were not associated with histological differentiation of human ESCC.
These results imply that miR-205 is an ESCC-specific miR that exerts tumor-suppressive activities with EMT inhibition by targeting ZEB2.
PMCID: PMC3076245  PMID: 21426561
5.  miR-22 is down-regulated in esophageal squamous cell carcinoma and inhibits cell migration and invasion 
Esophageal squamous cell carcinoma (ESCC) is one of the most common and deadly forms of cancer. Despite advances in the diagnosis and treatment of this cancer, the survival rate at five years is poor. Lately, miR-22 is identified as a tumor-suppressing microRNA in many human cancers. However, the specific function of miR-22 in ESCC is unclear at this point.
We first measured miR-22 expression level in 30 paired of ESCC and matched normal tissues, ESCC cell lines by real-time quantitative RT-PCR. Invasion assay, MTT proliferation assay and wound-healing assay were performed to test the invasion and proliferation of ESCC cell after overexpression of miR-22.
We found that the expression of miR-22 in ESCC tissues and cell lines were much lower than that in normal control, respectively. The expression of miR-22 was inversely correlated with ESCC metastatic ability. Furthermore, transfection of miR-22 expression plasmid could significantly inhibit the cell proliferation, migration and invasion in Eca109 and Kyse410 ESCC cell lines.
Our findings suggest that miR-22 act as tumor suppressor and inhibiting ESCC cell migration and invasion. The findings of this study contribute to the current understanding of the functions of miR-22 in ESCC.
PMCID: PMC4267162  PMID: 25516722
miR-22; Esophageal squamous cell carcinoma; Invasion; Migration
6.  SOX4 interacts with EZH2 and HDAC3 to suppress microRNA-31 in invasive esophageal cancer cells 
Molecular Cancer  2015;14:24.
Tumor metastasis is responsible for 90% of cancer-related deaths. Recently, a strong link between microRNA dysregulation and human cancers has been established. However, the molecular mechanisms through which microRNAs regulate metastasis and cancer progression remain unclear.
We analyzed the reciprocal expression regulation of miR-31 and SOX4 in esophageal squamous and adenocarcinoma cell lines by qRT-PCR and Western blotting using overexpression and shRNA knock-down approaches. Furthermore, methylation studies were used to assess epigenetic regulation of expression. Functionally, we determined the cellular consequences using migration and invasion assays, as well as proliferation assays. Immunoprecipitation and ChIP were used to identify complex formation of SOX4 and co-repressor components.
Here, we report that SOX4 promotes esophageal tumor cell proliferation and invasion by silencing miR-31 via activation and stabilization of a co-repressor complex with EZH2 and HDAC3. We demonstrate that miR-31 is significantly decreased in invasive esophageal cancer cells, while upregulation of miR-31 inhibits growth, migration and invasion of esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) cell lines. miR-31, in turn, targets SOX4 for degradation by directly binding to its 3′-UTR. Additionally, miR-31 regulates EZH2 and HDAC3 indirectly. SOX4, EZH2 and HDAC3 levels inversely correlate with miR-31 expression in ESCC cell lines. Ectopic expression of miR-31 in ESCC and EAC cell lines leads to down regulation of SOX4, EZH2 and HDAC3. Conversely, pharmacologic and genetic inhibition of SOX4 and EZH2 restore miR-31 expression. We show that SOX4, EZH2 and HDAC3 form a co-repressor complex that binds to the miR-31 promoter, repressing miR-31 through an epigenetic mark by H3K27me3 and by histone acetylation. Clinically, when compared to normal adjacent tissues, esophageal tumor samples show upregulation of SOX4, EZH2, and HDAC3, and EZH2 expression is significantly increased in metastatic ESCC tissues.
Thus, we identified a novel molecular mechanism by which the SOX4, EZH2 and miR-31 circuit promotes tumor progression and potential therapeutic targets for invasive esophageal carcinomas.
PMCID: PMC4374188  PMID: 25644061
Esophageal cancer; Oncogene; MicroRNA; miR-31; EZH2; SOX4; HDAC3; Epigenetics
7.  Dysregulation of miR-31 and miR-21 induced by zinc deficiency promotes esophageal cancer 
Carcinogenesis  2012;33(9):1736-1744.
Zinc deficiency (ZD) increases the risk of esophageal squamous cell carcinoma (ESCC). In a rat model, chronic ZD induces an inflammatory gene signature that fuels ESCC development. microRNAs regulate gene expression and are aberrantly expressed in cancers. Here we investigated whether chronic ZD (23 weeks) also induces a protumorigenic microRNA signature. Using the nanoString technology, we evaluated microRNA profiles in ZD esophagus and six additional tissues (skin, lung, pancreas, liver, prostate and peripheral blood mononuclear cells [PBMC]). ZD caused overexpression of inflammation genes and altered microRNA expression across all tissues analyzed, predictive of disease development. Importantly, the inflammatory ZD esophagus had a distinct microRNA signature resembling human ESCC or tongue SCC miRNAomes with miR-31 and miR-21 as the top-up-regulated species. Circulating miR-31 was also the top-up-regulated species in PBMCs. In ZD esophagus and tongue, oncogenic miR-31 and miR-21 overexpression was accompanied by down-regulation of their respective tumor-suppressor targets PPP2R2A and PDCD4. Importantly, esophageal miR-31 and miR-21 levels were directly associated with the appearance of ESCC in ZD rats, as compared with their cancer-free Zn-sufficient or Zn-replenished counterparts. In situ hybridization analysis in rat and human tongue SCCs localized miR-31 to tumor cells and miR-21 to stromal cells. In regressing tongue SCCs from Zn-supplemented rats, miR-31 and miR-21 expression was concomitantly reduced, establishing their responsiveness to Zn therapy. A search for putative microRNA targets revealed a bias toward genes in inflammatory pathways. Our finding that ZD causes miR-31 and miR-21 dysregulation associated with inflammation provides insight into mechanisms whereby ZD promotes ESCC.
PMCID: PMC3514898  PMID: 22689922
8.  Up-regulation of miR-335 predicts a favorable prognosis in esophageal squamous cell carcinoma 
Introduction: MicroRNAs (miRNAs) are noncoding RNAs that regulate multiple cellular processes during cancer progression. MiR-335 has recently been identified to be involved in tumorigenesis of several cancers such as ovarian cancer and gastric cancer. However, the regulation of miR-335 in esophageal squamous cell carcinoma (ESCC) has not been reported yet. Methods: Expression of miR-335 in tumor and their normal matched tissues was determined by quantitative real-time PCR in 67 ESCC patients and its association with overall survival of patients was analyzed by statistical analysis. Results: The expression level of miR-335 was reduced in malignant tissue samples in comparison to normal matched tissue (P < 0.05). It was also proved that miR-335 expression was associated with ESCC histological grade, lymph node metastasis, tumor stage and clinical stage (P < 0.05). In addition, the Kaplan-Meier survival curves revealed that low miR-335 expression was associated with poor prognosis in ESCC patients. Multivariate analysis showed that miR-335 expression was an independent prognostic marker of overall survival of ESCC patients. Conclusions: The study proves for the first time that miR-335 is down regulated in a majority of ESCC patients. Our results indicate that miR-335 expression is an independent prognostic factor for patients with esophageal cancer, which might be a potential valuable biomarker for ESCC.
PMCID: PMC4203243  PMID: 25337272
miR-335; esophageal squamous cell carcinoma; quantitative real-time PCR; prognosis
9.  Tumor suppression by miR-31 in esophageal carcinoma is p21-dependent 
Genes & Cancer  2014;5(11-12):436-444.
microRNA regulation network is important for the cancer genetic heterogeneity. Relative to the increasing numbers of microRNA's targets identified, upstream regulatory mechanisms that control functional microRNAs are less well-documented. Here, we investigated the function of miR-31, a pleiotropically-acting microRNA, in esophageal squamous cell cancer (ESCC). We demonstrated that miR-31 only exerted tumor-suppressive effects in TE-7 ESCC cells, but not in TE-1 ESCC cells, although both of these cell lines harbor inactive p53. Interestingly, TE-1 cells highly expressed p21, while p21 levels were virtually undetectable in TE-7 cells, suggesting a p21-dependent mechanism of miR-31-mediated tumor suppression. Accordingly, knockdown of p21 in TE-1 cells reversed the tumor suppressive actions of miR-31. In patient ESCC specimens, real-time RT-PCR analysis revealed that expression of E2F2 and STK40, two known miR-31 target oncogenes, was negatively correlated with the expression of miR-31 in a p21-dependent manner, supporting the conclusion that miR-31 only downregulates its target oncogenes when p21 levels are low. Collectively, these data suggest a novel mechanism through which the tumor-suppressive effect of miR-31 is p21-dependent. In addition, we speculate that delivery of miR-31 could provide therapeutic benefit in the personalized management of a subgroup of ESCC patients with p21-deficient tumors.
PMCID: PMC4279440  PMID: 25568668
microRNA; miR-31; p21; esophageal squamous cell cancer; personalized medicine
10.  MicroRNA-126 is down-regulated in human esophageal squamous cell carcinoma and inhibits the proliferation and migration in EC109 cell via PI3K/AKT signaling pathway 
MicroRNA-126 (miR-126) was found down-regulated in different types of cancer including esophageal squamous cell carcinoma (ESCC). However, the onco-genetic role of miR-126 in ESCC still remains unknown. In the present study, we found the relative expression of miR-126 in ESCC was significant decreased in ESCC tissues compared to adjacent normal tissues. Overexpression of miR-126 in EC109 cells resulted in significant decrease in cell proliferation, colon formation and migration. PI3K regulatory subunit p85 beta (PIK3R2), a member of PI3K/AKT signaling pathway was found upregulated in ESCC tissues and there is a negative relation between expression of PIK3R2 and miR-126. Restoration of miR-126 in EC109 cells induced a reduction in PIK3R2 protein levels, accompanied with a substantial reduction in phosphorylated AKT levels in EC109 cells, suggesting impairment in PI3K/AKT signaling pathway. The luciferase reporter assay confirmed that PIK3R2 was a direct target of miR-126. Furthermore, we also indicated overexpression of miR-126 suppresses G2/M transition in EC109 cells. Taken together, our study suggests that miR-126 functions as a potential tumor suppressor in ESCC progression via regulating PI3K/AKT signaling pathway partly by targeting PIK3R2, and targeting of miR-126 may provide a novel strategy for the diagnosis and treatment of ESCC.
PMCID: PMC4503036  PMID: 26191164
miR-126; ESCC; PIK3R2; AKT; cell cycle arrest
11.  MicroRNA Expression Profiling in the Histological Subtypes of Barrett's Metaplasia 
The histological definition of Barrett's esophagus (BE) is debated, particularly regarding the phenotype of its metaplastic columnar epithelium. Histologically proven intestinal metaplasia (IM) was the sine qua non condition for a diagnosis of BE but, more recently, non-intestinalized (i.e., cardiac gastric-type; GM) columnar metaplasia has been re-included in the spectrum of Barrett's histology. MicroRNAs modulate cell commitment, and are also reportedly dysregulated in Barrett's carcinogenesis. This study investigates miRNA expression in the histological spectrum of esophageal columnar metaplastic changes, specifically addressing the biological profile of GM vs. IM.
A study was performed to discover microRNA microarray in 30 matching mucosa samples obtained from 10 consecutive BE patients; for each patient, biopsy tissue samples were obtained from squamous, GM and intestinalized epithelium. Microarray findings were further validated by qRT-PCR analysis in another bioptic series of 75 mucosa samples.
MicroRNA profiling consistently disclosed metaplasia-specific microRNA signatures. Six microRNAs were significantly dysregulated across the histological phenotypes considered; five of them (two overexpressed (hsa-miR-192; -miR-215) and three under-expressed (hsa-miR-18a* -miR-203, and -miR-205)) were progressively dysregulated in the phenotypic sequence from squamous to gastric-type, to intestinal-type mucosa samples.
A consistent microRNA expression signature underlies both gastric- and intestinal-type esophageal metaplasia. The pattern of microRNA dysregulation suggests that GM may further progress to IM. The clinico-pathological implications of these molecular profiles prompt further study on the “personalized” cancer risk associated with each of these metaplastic transformations.
PMCID: PMC3671360  PMID: 23677165
12.  Pri-miR-124 rs531564 and pri-miR-34b/c rs4938723 Polymorphisms Are Associated with Decreased Risk of Esophageal Squamous Cell Carcinoma in Chinese Populations 
PLoS ONE  2014;9(6):e100055.
MicroRNAs are a new class of small non-protein-coding RNAs that sometimes function as tumor suppressors or oncogenes. Aberrant expression and structural alteration of microRNAs have been reported to be involved in tumorigenesis and cancer development. Recently, rs531564/pri-miR-124-1, rs4938723/pri-miR-34b/c, rs7372209/pri-miR-26a-1, rs895819/pre-miR-27a, and rs11134527/pri-miR-218 were reported to be associated with risks of various cancers. In order to evaluate the relationship of these SNPs and esophageal squamous cell carcinoma (ESCC) risk, we conducted a case-control study with 1109 ESCC patients and 1275 control subjects to examine the potential association of these pri/pre-miRNA polymorphisms with ESCC susceptibility. As a result, two SNPs were associated with a significant risk of ESCC. We found that the GG genotype of pri-miR-124-1 rs531564 was associated to a significantly decreased risk of ESCC comparing with the CC/CG genotypes (p = 0.005; OR = 0.61, 95% CI = 0.43–0.86). In addition, the CC genotype of pri-miR-34b/c rs4938723 was associated with a significant decreased risk of ESCC (CC VS. TT/TC: p = 0.007, OR = 0.82, 95% CI = 0.71–0.95) in Chinese population. The present study provides the first evidence that pri-miR-124-1 rs531564 and pri-miR-34 rs4938723 were associated with the risk of ESCC in Chinese population.
PMCID: PMC4063769  PMID: 24945256
13.  MiR-130b plays an oncogenic role by repressing PTEN expression in esophageal squamous cell carcinoma cells 
BMC Cancer  2015;15:29.
Esophageal carcinoma is one of the most common malignancies with high cancer-related morbidity and mortality worldwide. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate a wide variety of cellular processes, and also play an important role in the development and progression of cancers. In a previous microarray study, we demonstrated that miR-130b was upregulated in esophageal squamous cell carcinoma (ESCC) tissues. However, the biologic functions and the molecular mechanism of miR-130b in ESCC remain to be elucidated.
qRT-PCR assays were used to quantify miR-130b expression levels in ESCC samples. Novel targets of miR-130b were identified via a bioinformatics search and confirmed using a dual-luciferase reporter system. Western blotting and qRT-PCR assays were used to quantify the expression of the target gene PTEN (phosphatase and tensin homolog) and the downstream effector, Akt. ESCC cells over- or underexpressing miR-130b were analyzed for in vitro biologic functions.
High levels of miR-130b were identified in 20 ESCC samples following comparison with adjacent non-neoplastic tissues. We confirmed that miR-130b interacted with the 3′-untranslated region of PTEN, and that an increase in the expression level of miR-130b negatively affected the protein level of PTEN. However, the dysregulation of miR-130b had no obvious impact on PTEN mRNA. As Akt is a downstream effector of PTEN, we explored if miR-130b affected Akt expression, and found that miR-130b indirectly regulated the level of phosphorylated Akt, while total Akt protein remained unchanged. Overexpression of miR-130b increased the proliferation of ESCC cells and enhanced their ability to migrate and invade. In contrast, the proliferation, migration, and invasion of ESCC cells were weakened when miR-130b expression was suppressed, which was reversed by PTEN-targeted siRNA.
The results indicate that miR-130b plays an oncogenic role in ESCC cells by repressing PTEN expression and Akt phosphorylation, which would be helpful in developing miRNA-based treatments for ESCC.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1031-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4318221  PMID: 25637514
Esophageal squamous cell carcinoma; miR-130b; Oncogenic; PTEN
14.  Diagnostic and prognostic potential of miR-21, miR-29c, miR-148 and miR-203 in adenocarcinoma and squamous cell carcinoma of esophagus 
Diagnostic Pathology  2015;10:42.
Esophageal cancer is the malignant tumor with very poor prognosis and increasing incidence often diagnosed at very late stage, so the prognosis of affected patients is unsatisfactory, despite the development of therapeutic option such as surgery, chemotherapy and radiotherapy. Consequently, there is a great need for biomarkers to allow a tailored multimodality approach with increased efficiency. Altered expression of microRNAs has been reported in wide range of malignancies, including esophageal cancer. The aim of this study was to examine the expression levels of candidate microRNAs in esophageal cancer and evaluate their diagnostic and prognostic potential.
Using quantitative real-time PCR, expression levels of 9 candidate microRNAs were examined in 62 tissue samples, 23 esophageal adenocarcinomas, 22 esophageal squamous cell carcinomas and 17 adjacent esophageal mucosa samples. MicroRNA expression levels were further analyzed in regards to clinico-pathological features of esophageal cancer patients. We observed significantly decreased levels of miR-203 and increased levels of miR-21 in adenocarcinoma tissues when compared to normal mucosa. MiR-29c and miR-148 indicated good ability to distinguish between histological subtypes of esophageal cancer. MiR-203 and miR-148 were linked to disease-free survival and overall survival in esophageal adenocarcinoma patients, and miR-148 also in esophageal squamous cell carcinoma patients.
Our data suggest that altered expression of miR-21, miR-29c, miR-148 and miR-203 are related to neoplastic transformation and progression of the disease and these microRNAs could serve as a potential diagnostic and prognostic biomarkers in esophageal cancer.
Virtual slides
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PMCID: PMC4411933  PMID: 25928282
15.  MicroRNA-218 inhibits the proliferation and metastasis of esophageal squamous cell carcinoma cells by targeting BMI1 
MicroRNAs (miRNAs or miRs) play a pivotal role in esophageal carcinogenesis either as oncogenes or as tumor suppressor genes. In the present study, we found that the expression level of miR-218 was significantly reduced in esophageal squamous cell carcinoma (ESCC) tissues and ESCC cell lines. Moreover, its expression was found to correlate with the clinicopathological stage of ESCC; miR-218 expression was lower in the stage III tissue samples than in the stage I and II tissue samples. Furthermore, the decreased expression of miR-218 was found to be associated with an enhanced ESCC cell proliferation and metastasis. Western blot analysis and luciferase reporter assay revealed that miR-218 decreased BMI1 expression by binding to the putative binding sites in its 3′-untranslated region (3′-UTR). The BMI1 mRNA expression levels were markedly increased and negatively correlated with the miR-218 expression level in the ESCC tissues. Functional analyses revealed that the restoration of miR-218 expression inhibited ESCC cell proliferation, migration and invasion and promoted apoptosis. The knockdown of BMI1 by siRNA showed the same phenocopy as the effect of miR-218 on ESCC cells, indicating that BMI1 was a major target of miR-218. In the present study, our findings confirm miR-218 as a tumor suppressor and identify BMI1 as a novel target of miR-218 in ESCC. Therefore, miR-218 may prove to be a useful biomarker for monitoring the initiation and development of ESCC, and may thus be an effective therapeutic target in ESCC.
PMCID: PMC4494586  PMID: 25999024
microRNA-218; esophageal squamous cell carcinoma; BMI1; proliferation; metastasis
16.  Identifying microRNA/mRNA dysregulations in ovarian cancer 
BMC Research Notes  2012;5:164.
MicroRNAs are a class of noncoding RNA molecules that co-regulate the expression of multiple genes via mRNA transcript degradation or translation inhibition. Since they often target entire pathways, they may be better drug targets than genes or proteins. MicroRNAs are known to be dysregulated in many tumours and associated with aggressive or poor prognosis phenotypes. Since they regulate mRNA in a tissue specific manner, their functional mRNA targets are poorly understood. In previous work, we developed a method to identify direct mRNA targets of microRNA using patient matched microRNA/mRNA expression data using an anti-correlation signature. This method, applied to clear cell Renal Cell Carcinoma (ccRCC), revealed many new regulatory pathways compromised in ccRCC. In the present paper, we apply this method to identify dysregulated microRNA/mRNA mechanisms in ovarian cancer using data from The Cancer Genome Atlas (TCGA).
TCGA Microarray data was normalized and samples whose class labels (tumour or normal) were ambiguous with respect to consensus ensemble K-Means clustering were removed. Significantly anti-correlated and correlated genes/microRNA differentially expressed between tumour and normal samples were identified. TargetScan was used to identify gene targets of microRNA.
We identified novel microRNA/mRNA mechanisms in ovarian cancer. For example, the expression level of RAD51AP1 was found to be strongly anti-correlated with the expression of hsa-miR-140-3p, which was significantly down-regulated in the tumour samples. The anti-correlation signature was present separately in the tumour and normal samples, suggesting a direct causal dysregulation of RAD51AP1 by hsa-miR-140-3p in the ovary. Other pairs of potentially biological relevance include: hsa-miR-145/E2F3, hsa-miR-139-5p/TOP2A, and hsa-miR-133a/GCLC. We also identified sets of positively correlated microRNA/mRNA pairs that are most likely result from indirect regulatory mechanisms.
Our findings identify novel microRNA/mRNA relationships that can be verified experimentally. We identify both generic microRNA/mRNA regulation mechanisms in the ovary as well as specific microRNA/mRNA controls which are turned on or off in ovarian tumours. Our results suggest that the disease process uses specific mechanisms which may be significant for their utility as early detection biomarkers or in the development of microRNA therapies in treating ovarian cancers. The positively correlated microRNA/mRNA pairs suggest the existence of novel regulatory mechanisms that proceed via intermediate states (indirect regulation) in ovarian tumorigenesis.
PMCID: PMC3342161  PMID: 22452920
17.  Circulating microRNAs in esophageal squamous cell carcinoma: association with locoregional staging and survival 
Locoregional staging and prognostic information play a critical role in esophageal squamous cell carcinoma (ESCC) treatment strategies. Although microRNA (miRNA) is a promising marker for cancer detection, the relationship between circulating plasma miRNAs and ESCC remains unclear. Our study aims to investigate the association between circulating plasma miRNAs and tumor diagnosis or prognosis in ESCC patients. Plasma levels of miR-16, miR-21, miR-22, miR-126, miR-148b, miR-185, miR-221, miR-223, and miR-375 were evaluated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays from 38 ESCC patients prior to treatment and 19 healthy subjects. Differences in selected miRNAs and their diagnostic and prognostic value were examined. Levels of four of the selected miRNAs were found to be significantly higher in ESCC patients than in controls; namely, miR-16, miR-21, miR-185, and miR-375 (P < 0.050). In addition, the area under the receiver operating characteristic (ROC) curve (AUC) for miR-375 was 0.921 (95% confidence interval [CI] 0.817-0.976). Moreover, the expression levels of miR-16 were higher in patients with T3-4 tumors than in patients with T1-2 tumors (P = 0.020). Kaplan-Meier survival analysis showed that high expression levels of miR-16 and miR-21 in the plasma correlated significantly with shortened progression-free survival (PFS; P = 0.031 and P = 0.038, respectively) and overall survival (OS; P = 0.022 and P = 0.041, respectively) in ESCC patients. Four plasma miRNAs were identified that could potentially serve as novel diagnostic biomarkers for ESCC. Moreover, specific miRNAs, such as miR-16 and miR-21, can predict poor survival in ESCC.
PMCID: PMC4509208  PMID: 26221263
MicroRNA; esophageal squamous cell carcinoma; locoregional staging; prognosis
18.  Comprehensive microRNA Profiling of Prostate Cancer 
Journal of Cancer  2013;4(5):350-357.
MicroRNAs are small non-coding RNA molecules that have been shown to regulate the expression of genes linked to cancer. The relevance of microRNAs in the development, progression and prognosis of prostate cancer is not fully understood. It is also possible that these specific molecules may assist in the recognition of aggressive tumors and the development of new molecular targets. Our study investigated the importance of several microRNAs in cases of prostate cancer from 37 patients that were manually microdissected to obtain pure populations of tumor cells, normal epithelium and adjacent stroma. MicroRNA was extracted for PCR array profiling. Differentially expressed miRNAs for each case were used to compare tumor vs. normal epithelium and tumor-adjacent stroma samples.
Loss of 18 miRNAs (e.g.miR-34c, miR-29b, miR-212 and miR-10b) and upregulation of miR-143 and miR-146b were significantly found in all the tumors in comparison with normal epithelium and/or stroma (p≤ 0.001). A different signature was found in the high grade tumors (Gleason score ≥ 8) when compared with tumors Gleason score 6. Upregulation of miR-122, miR-335, miR-184, miR-193, miR-34, miR-138, miR-373, miR-9, miR-198, miR-144 and miR-215 and downregulation of miR-96, miR-222, miR-148, miR-92, miR-27, miR-125, miR-126, miR-27 were found in the high grade tumors.
MicroRNA profiling in prostate cancer appears to have unique expression patterns in comparison with normal tissue. These differential expressed miRNAs may provide novel diagnostic and prognostic tools that will assist in the recognition of prostate cancers with aggressive behavior.
PMCID: PMC3677622  PMID: 23781281
microRNA; Prostate Cancer; biomarkers.
19.  The expression profile of microRNAs in a model of 7,12-dimethyl-benz[a]anthrance-induced oral carcinogenesis in Syrian hamster 
Non-coding RNA molecules, such as microRNAs, may play an important role in carcinogenesis. Recent studies have indicated that microRNAs are involved in initiation and progression of various malignancies. However, little work has been done to compare the microRNA expression patterns in oral cancer. In this study, we constructed an animal model of oral squamous cell carcinoma to investigate expression profiles of microRNAs in oral carcinogenesis.
The animal model of oral squamous cell carcinoma was conducted by tri-weekly (Monday, Wednesday, and Friday) painting with 5% DMBA in acetone. Six Syrian hamsters, including three from the treated group and three from the control group, were used as a training group for microRNA microarray analysis. All microarray data were analyzed by Significance Analysis of Microarrays (SAM) and CLUSTER 3.0 software, and this result was further confirmed by qRT-PCR assay.
Seventeen microRNAs were differentially expressed in oral squamous cell carcinoma. Five microRNAs (hsa-miR-21, hsa-miR-200b, hsa-miR-221, hsa-miR-338, and mmu-miR-762) were significantly upregulated and twelve microRNAs (hsa-miR-16, hsa-miR-26a, hsa-miR-29a, hsa-miR-124a, hsa-miR-125b, mmu-miR-126-5p, hsa-miR-143, hsa-miR-145, hsa-miR-148b, hsa-miR-155, hsa-miR-199a, and hsa-miR-203) were down-regulated in cancer tissues. The expression levels of hsa-miR-21 and hsa-miR-16 seen with Stem-loop qRT-PCR were also seen in microarray analysis in all samples.
Our findings identified specific microRNA expression in oral squamous cell carcinoma and suggested that microRNAs have a role in oral carcinogenesis.
PMCID: PMC2687417  PMID: 19435529
20.  Downregulation of microRNA-382 is associated with poor outcome of esophageal squamous cell carcinoma 
AIM: To study the potential prognostic role of microRNA-382 (miR-382) in esophageal squamous cell carcinoma (ESCC).
METHODS: Forty six patients were divided into 2 groups according to postoperative survival time: the poor outcome group (28 patients), who showed early metastasis but no recurrence, and died within 1 year after surgery, 12 patients of the group received postoperative chemotherapy treatment that was given after early metastasis happening; the good outcome group (18 patients), who had no clinical metastasis and recurrence, and survived 5 years or more after surgery, all patients did not receive any postoperative treatment. Total RNA was extracted from the patients’ formalin-fixed and paraffin-embedded esophageal cancer tissues. miR-382 level was evaluated using high-throughput real-time quantitative polymerase chain reaction analysis. The correlation between miR-382 level and clinicopathologic features was analyzed through COX regression model, and Kaplan-Meier analysis was used to analyze the relationship between miR-382 level and patient survival time.
RESULTS: miR-382 was differentially expressed in the two groups. Overall the average miR-382 level in the ESCC patients with good outcome was 9.8 ± 3.8, while miR-382 level in the ESCC patients with poor outcome was 3.0 ± 0.8. The differences of miR-382 levels between two groups were significant (P < 0.05). Kaplan-Meier analysis results showed that miR-382 expression level generally had a significant reverse-correlation with ESCC patient survival time (P < 0.001), in which the patients with higher expressions of miR-382 had a longer survival time either among individuals with the same tumor stage or among the overall patients.
CONCLUSION: miR-382 levels are reverse-correlated with ESCC poor outcomes, suggesting that miR-382 could be a potential predictive biomarker for both prognosis and treatment of ESCC.
PMCID: PMC4462728  PMID: 26078564
Esophageal squamous cell carcinoma; miR-382; Metastasis; Outcome; Prognosis
21.  microRNA-195-Cdc42 axis acts as a prognostic factor of esophageal squamous cell carcinoma 
Background and purpose: Previous studies observed the downregulation of microRNA (miR)-195 in esophageal squamous cell carcinoma (ESCC) tissues, confirmed cell division cycle 42 (Cdc42) as one target gene of miR-195, and demonstrated that miR-195 may act as a tumor suppressor in ESCC by regulating Cdc42 expression. This study aimed to explore the association of miR-195 and Cdc42 combined expression with clinicopathologic factors and prognosis. Methods: Expression of miR-195 and Cdc42 mRNA in 98 pairs of ESCC and paracancerous tissues were detected using real-time quantitative RT-PCR. Results: miR-195 downregulation and Cdc42 upregulation were both prevalent in ESCC tissues, and negatively correlated with each other. In addition, miR-195 expression negatively correlated with TNM stage (P=0.008) and lymphatic metastasis (P=0.022), while Cdc42 expression positively correlated with TNM stage (P=0.011) and tumor differentiation (P=0.024). Moreover, combined expression of miR-195 and Cdc42 (miR-195/Cdc42) was found to be prognostic indicators for progression-free survival and overall survival of ESCC patients both in univariate and multivariate analyses. Conclusion: The main findings of this study indicate the involvement of miR-195-Cdc42 axis in the progression of ESCC and suggest that the combined aberrant expression of miR-195 and Cdc42 mRNA can serve as a promising unfavorable prognostic biomarker in ESCC.
PMCID: PMC4230103  PMID: 25400770
microRNA-195; cell division cycle 42; esophageal squamous cell carcinoma; real-time quantitative PCR; prognosis
22.  MicroRNA signatures in chemotherapy resistant esophageal cancer cell lines 
World Journal of Gastroenterology : WJG  2014;20(40):14904-14912.
AIM: To investigate expression of microRNA (miRNA) and potential targets in chemotherapy resistant esophageal cancer cell lines.
METHODS: An in-vitro model of acquired chemotherapy resistance in esophageal adeno- (EAC) and squamous cell carcinoma (ESCC) cells was used, and microRNA expression profiles for cisplatin or 5-fluorouracil (5-FU) resistant variants vs chemotherapy sensitive controls were compared using microarray and quantitative real-time polymerase chain reaction (PCR). The expression of chemotherapy-relevant genes potentially targeted by the dysregulated microRNAs in the chemotherapy resistant variants was also evaluated.
RESULTS: Chemotherapy resistant sublines were found to have specific miRNA signatures, and these miRNA signatures were different for the cisplatin vs 5-FU resistant cells from the same tumor cell line, and also for EAC vs ESCC cells with resistance to the same specific chemotherapy agent. Amongst others, miR-27b-3p, miR-193b-3p, miR-192-5p, miR-378 a-3p, miR-125a-5p and miR-18a-3p were dysregulated, consistent with negative posttranscriptional control of KRAS, TYMS, ABCC3, CBL-B and ERBB2 expression via these miRNAs.
CONCLUSION: The current study supports the hypothesis that microRNA expression has an impact on chemotherapy resistance in esophageal cancer.
PMCID: PMC4209553  PMID: 25356050
Esophageal cancer; MicroRNA; Chemotherapy; Resistance; Target
23.  MicroRNA-34b has an oncogenic role in esophageal squamous cell carcinoma 
Oncology Letters  2010;1(4):685-689.
Esophageal squamous cell carcinoma (ESCC) is a common malignancy and one of the more difficult diseases to diagnose in Japan due to its poor prognosis. MicroRNAs are small non-coding RNAs of 21–23 nucleotides that regulate gene expression. MicroRNA-34b (miR-34b) has been reported to be overexpressed in various types of cancer. However, its role in ESCC has yet to be extensively studied. The present study investigated the expression of miR-34b in 88 ESCC patients. The miR-34b expression in ESCC was significantly higher than that in the corresponding normal esophageal mucosa. It was more highly expressed in tumors with more advanced stages. However, its expression did not correlate with the p53 status. Transfection of anti-miR-34b to the ESCC cells suppressed cell growth in vitro. These results suggest an oncogenic role of miR in ESCC.
PMCID: PMC3436259  PMID: 22966364
microRNA-34b; esophageal squamous cell carcinoma; MTT assay
24.  MicroRNA Expression in Ileal Carcinoid Tumors: Down-regulation of MicroRNA-133a with Tumor Progression 
MicroRNAs are involved in cell proliferation, differentiation, and apoptosis and can function as tumor suppressor genes or oncogenes. The role of microRNAs in neuroendocrine tumors such as ileal carcinoids is largely unknown. We examined the differential expression of 95 microRNAs by RT-PCR using the QuantiMir System in eight matching primary and metastatic carcinoid tumors from the ileum. All microRNAs chosen for the QuantiMir System Array were based on their potential functions related to cancer biology, cell development and apoptosis. The expression of microRNAs for the samples was normalized to microRNA-197, and the matching primary and metastatic tumors were compared. There was down-regulation of microRNA-133a, 145, 146, 222 and 10b in all samples between the primary and matching metastatic tumors and up-regulation of microRNA-183, 488 and 19a + b in six of eight metastatic carcinoids compared to the primary tumors. MicroRNA-133a was further analyzed by TaqMan Real Time RT-PCR and Northern hybridization using six additional matching primary and metastatic samples which supported the PCR Array findings. There were significant differences in microRNA-133a expression with down-regulation in the metastasis compared to the primary in the eight original cases (p<0.009) and in the six additional cases used for validation (p<0.014). Laser capture microdissection and Real Time RT-PCR analysis using normal ileum found microRNA-133a expression in normal enterochromaffin cells. In situ hybridization in normal ileum showed that some of the mucosal endocrine cells expressed microRNA-133a. Both primary and metastatic ileal carcinoid tumors expressed microRNA-133a by in situ hybridization. These results provide information about novel marker microRNAs that may be used as biomarkers and/or therapeutic targets in intestinal carcinoid tumors.
PMCID: PMC2886953  PMID: 20037573
PCR array; carcinoids; enterochromaffin cells; RT-PCR; in situ hybridization
25.  microRNA-122 as a regulator of mitochondrial metabolic gene network in hepatocellular carcinoma 
A moderate loss of miR-122 function correlates with up-regulation of seed-matched genes and down-regulation of mitochondrially localized genes in both human hepatocellular carcinoma and in normal mice treated with anti-miR-122 antagomir.Putative direct targets up-regulated with loss of miR-122 and secondary targets down-regulated with loss of miR-122 are conserved between human beings and mice and are rapidly regulated in vitro in response to miR-122 over- and under-expression.Loss of miR-122 secondary target expression in either tumorous or adjacent non-tumorous tissue predicts poor survival of heptatocellular carcinoma patients.
Hepatocellular carcinoma (HCC) is one of the most aggressive human malignancies, common in Asia, Africa, and in areas with endemic infections of hepatitis-B or -C viruses (HBV or HCV) (But et al, 2008). Globally, the 5-year survival rate of HCC is <5% and about 600 000 HCC patients die each year. The high mortality associated with this disease is mainly attributed to the failure to diagnose HCC patients at an early stage and a lack of effective therapies for patients with advanced stage HCC. Understanding the relationships between phenotypic and molecular changes in HCC is, therefore, of paramount importance for the development of improved HCC diagnosis and treatment methods.
In this study, we examined mRNA and microRNA (miRNA)-expression profiles of tumor and adjacent non-tumor liver tissue from HCC patients. The patient population was selected from a region of endemic HBV infection, and HBV infection appears to contribute to the etiology of HCC in these patients. A total of 96 HCC patients were included in the study, of which about 88% tested positive for HBV antigen; patients testing positive for HCV antigen were excluded. Among the 220 miRNAs profiled, miR-122 was the most highly expressed miRNA in liver, and its expression was decreased almost two-fold in HCC tissue relative to adjacent non-tumor tissue, confirming earlier observations (Lagos-Quintana et al, 2002; Kutay et al, 2006; Budhu et al, 2008).
Over 1000 transcripts were correlated and over 1000 transcripts were anti-correlated with miR-122 expression. Consistent with the idea that transcripts anti-correlated with miR-122 are potential miR-122 targets, the most highly anti-correlated transcripts were highly enriched for the presence of the miR-122 central seed hexamer, CACTCC, in the 3′UTR. Although the complete set of negatively correlated genes was enriched for cell-cycle genes, the subset of seed-matched genes had no significant KEGG Pathway annotation, suggesting that miR-122 is unlikely to directly regulate the cell cycle in these patients. In contrast, transcripts positively correlated with miR-122 were not enriched for 3′UTR seed matches to miR-122. Interestingly, these 1042 transcripts were enriched for genes coding for mitochondrially localized proteins and for metabolic functions.
To analyze the impact of loss of miR-122 in vivo, silencing of miR-122 was performed by antisense inhibition (anti-miR-122) in wild-type mice (Figure 3). As with the genes negatively correlated with miR-122 in HCC patients, no significant biological annotation was associated with the seed-matched genes up-regulated by anti-miR-122 in mouse livers. The most significantly enriched biological annotation for anti-miR-122 down-regulated genes, as for positively correlated genes in HCC, was mitochondrial localization; the down-regulated mitochondrial genes were enriched for metabolic functions. Putative direct and downstream targets with orthologs on both the human and mouse microarrays showed significant overlap for regulations in the same direction. These overlaps defined sets of putative miR-122 primary and secondary targets. The results were further extended in the analysis of a separate dataset from 180 HCC, 40 cirrhotic, and 6 normal liver tissue samples (Figure 4), showing anti-correlation of proposed primary and secondary targets in non-healthy tissues.
To validate the direct correlation between miR-122 and some of the primary and secondary targets, we determined the expression of putative targets after transfection of miR-122 mimetic into PLC/PRF/5 HCC cells, including the putative direct targets SMARCD1 and MAP3K3 (MEKK3), a target described in the literature, CAT-1 (SLC7A1), and three putative secondary targets, PPARGC1A (PGC-1α) and succinate dehydrogenase subunits A and B. As expected, the putative direct targets showed reduced expression, whereas the putative secondary target genes showed increased expression in cells over-expressing miR-122 (Figure 4).
Functional classification of genes using the total ancestry method (Yu et al, 2007) identified PPARGC1A (PGC-1α) as the most connected secondary target. PPARGC1A has been proposed to function as a master regulator of mitochondrial biogenesis (Ventura-Clapier et al, 2008), suggesting that loss of PPARGC1A expression may contribute to the loss of mitochondrial gene expression correlated with loss of miR-122 expression. To further validate the link of miR-122 and PGC-1α protein, we transfected PLC/PRF/5 cells with miR-122-expression vector, and observed an increase in PGC-1α protein levels. Importantly, transfection of both miR-122 mimetic and miR-122-expression vector significantly reduced the lactate content of PLC/PRF/5 cells, whereas anti-miR-122 treatment increased lactate production. Together, the data support the function of miR-122 in mitochondrial metabolic functions.
Patient survival was not directly associated with miR-122-expression levels. However, miR-122 secondary targets were expressed at significantly higher levels in both tumor and adjacent non-tumor tissues among survivors as compared with deceased patients, providing supporting evidence for the potential relevance of loss of miR-122 function in HCC patient morbidity and mortality.
Overall, our findings reveal potentially new biological functions for miR-122 in liver physiology. We observed decreased expression of miR-122, a liver-specific miRNA, in HBV-associated HCC, and loss of miR-122 seemed to correlate with the decrease of mitochondrion-related metabolic pathway gene expression in HCC and in non-tumor liver tissues, a result that is consistent with the outcome of treatment of mice with anti-miR-122 and is of prognostic significance for HCC patients. Further investigation will be conducted to dissect the regulatory function of miR-122 on mitochondrial metabolism in HCC and to test whether increasing miR-122 expression can improve mitochondrial function in liver and perhaps in liver tumor tissues. Moreover, these results support the idea that primary targets of a given miRNA may be distributed over a variety of functional categories while resulting in a coordinated secondary response, potentially through synergistic action (Linsley et al, 2007).
Tumorigenesis involves multistep genetic alterations. To elucidate the microRNA (miRNA)–gene interaction network in carcinogenesis, we examined their genome-wide expression profiles in 96 pairs of tumor/non-tumor tissues from hepatocellular carcinoma (HCC). Comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR-122 is under-expressed in HCC and that increased expression of miR-122 seed-matched genes leads to a loss of mitochondrial metabolic function. Furthermore, the miR-122 secondary targets, which decrease in expression, are good prognostic markers for HCC. Transcriptome profiling data from additional 180 HCC and 40 liver cirrhotic patients in the same cohort were used to confirm the anti-correlation of miR-122 primary and secondary target gene sets. The HCC findings can be recapitulated in mouse liver by silencing miR-122 with antagomir treatment followed by gene-expression microarray analysis. In vitro miR-122 data further provided a direct link between induction of miR-122-controlled genes and impairment of mitochondrial metabolism. In conclusion, miR-122 regulates mitochondrial metabolism and its loss may be detrimental to sustaining critical liver function and contribute to morbidity and mortality of liver cancer patients.
PMCID: PMC2950084  PMID: 20739924
hepatocellular carcinoma; microarray; miR-122; mitochondrial; survival

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