LncRNA Erbb4-IR promotes esophageal squamous cell carcinoma (ESCC) by downregulating miR-145

Erbb4-IR is a recently identified lncRNA with pivotal functions in renal injury. The present study investigated the roles of Erbb4-IR in esophageal squamous cell carcinoma (ESCC). It was observed that Erbb4-IR was upregulated in tumor tissues of patients with ESCC. Plasma levels of Erbb4-IR in patients with ESCC were positively correlated with expression levels of Erbb4-IR in tumor tissues. MicroRNA-145 (miR-145) was downregulated in tumor tissues and inversely correlated with Erbb4-IR only in tumor tissues. Erbb4-IR overexpression led to downregulated miR-145, and increased rates of ECSS cell proliferation and decreased rates of ECSS cell apoptosis. Overexpression of miR-145 showed no significant effects on Erbb4-IR expression, but played an opposite role on cancer cell proliferation and apoptosis. In addition, miR-145 overexpression attenuated the effects of Erbb4-IR overexpression. Therefore, lncRNA Erbb4-IR may promote ESCC by downregulating miR-145.     

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.     

miR-760 exerts an antioncogenic effect in esophageal squamous cell carcinoma by negatively driving fat metabolism via targeting c-Myc

miR-760 is downregulated in various human tumors, and fat metabolism disorder correlates with tumor progression, especially anomalism of key fat metabolic enzymes that are positively modulated by c-Myc. The aim of our study is to elucidate the presumptive molecular mechanisms of miR-760-mediated esophageal squamous cell carcinoma (ESCC) cell function and to assess the therapeutic significance of miR-760 in ESCC patients. Quantitative real-time PCR (RT-qPCR) analysis indicated that miR-760 was significantly downexpressed in ESCC tissues and cell lines. Cell counting kit-8 (CCK-8) assay, colony formation assay, transwell assay, and flow cytometry denoted that induced ectopic overexpression of miR-760 dramatically inhibited ESCC cells proliferation, attenuated migration, and invasion facilitated apoptosis in vitro. Mechanistically, c-Myc predicted using bioinformatics was identified as a potential target gene of miR-760 by luciferase reporter assay. Furthermore, mRNA and protein expression levels of c-Myc and key fat metabolic enzymes were downregulated with miR-760 mimics. The above investigation results, responsible for the antineoplastic properties of miR-760 in ESCC, preliminarily highlighted that the hypothetical signal amongst miR-760, c-Myc, and key fat metabolic enzymes may develop a novel diagnostic marker, therapeutic target, and independent prognostic indicator.     

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.     

Plasma microRNA profiling highlights miR-1260b and miR-720 as novel diagnostic and prognostic biomarkers of esophageal squamous cell carcinoma

This paper was designed to explore the value of miRNAs as diagnostic biomarkers that may facilitate the early detection of esophageal squamous cell carcinoma (ESCC). Plasma miRNA profiles were defined via an array-based approach using samples from ESCC patients and healthy controls (n=5 each). Differentially expressed miRNAs in these samples were validated via qPCR in ESCC patients (n=96) and healthy controls (n=51), and the relationship between ESCC patient plasma miR-1260b and miR-720 levels and clinicopathological characteristics were additionally examined. In total, 12 plasma miRNAs that were differentially expressed between ESCC patients and healthy controls were identified via miRNA. Six of these miRNAs were subsequently validated, revealing that both miR-1260b and miR-720 were significantly differentially abundant in ESCC patients and controls, with miR-1260b being significantly upregulated in ESCC patients relative to controls (2.24, 1.41 respectively, P<0.001), while the opposite was observed with respect to miR-720 (0.66, 2.27 respectively, P=0.001). The use of both miR-720 and miR-1260b as a combined diagnostic tool was highly efficacious, yielding an AUC of 0.814, a sensitivity of 86.3%, and a specificity of 73.2% as a means of detecting ESCC patients. Elevated plasma miR-1260b level was also associated with a poorer patient prognosis when compared to patients with a low plasma miRNA level (P=0.021). This study has successfully developed a plasma miRNA biomarker signature of ESCC that may offer value as a diagnostic or prognostic tool when evaluating patients with ESCC.     

Eribulin upregulates miR-195 expression and downregulates Wnt3a expression in non-basal-like type of triple-negative breast cancer cell MDA-MB-231

Triple-negative breast cancer (TNBC), which does not show hormone sensitivity, is a poor prognosis disease without an established targeted treatment, so that establishing a therapeutic target for each subtype is desired. In addition, microRNA (miRNA), a non-cording RNA 19–25 nucleotide-longs in length, is known to be involved in regulating gene expression. We examined miRNA expression after exposure to eribulin, MDA-MB-231 cells, non-basal-like type of TNBC cell lines, and HCC1143 cells, basal-like type of TNBC cell lines. The activity of caspase-3 significantly increased compared to the control in MDA-MB-231, whereas no significant difference was observed in HCC1143. The expression level of 20-miRNAs significantly increased compared to the control in MDA-MB-231 after exposure to eribulin. The expression level of 6-miRNAs also significantly increased compared to the control in HCC1143. In these 2 cell types, miR-125b-1 and miR-195 were commonly expressed. While the expression level of miR-125b-1 decreased in both cells, the expression level of miR-195 increased in MDA-MB-231 and decreased in HCC1143. The expression level of miR-195 targeting Wnt3a significantly decreased compared to the control in MDA-MB-231, whereas it significantly increased in HCC1143. These results showed that exposure to eribulin highly increased the expression of miR-195 while it decreased the expression of Wnt3a in non-basal-like type of TNBC. Some miRNAs are known to regulate other signaling pathways involved in human pathogenesis by regulating the Wnt signaling pathway, and miRNA can act as a tumor-suppressing gene; therefore, miR-195 may serve as a therapeutic target in non-basal-like type of TNBC.     

miR-195 Inhibits EMT by Targeting FGF2 in Prostate Cancer Cells

Prostate cancer (PCa) is one of the leading causes of deaths in America. The major cause of mortality can be attributed to metastasis. Cancer metastasis involves sequential and interrelated events. miRNAs and epithelial-mesenchymal transition (EMT) are implicated in this process. miR-195 is downregulated in many human cancers. However, the roles of miR-195 in PCa metastasis and EMT remain unclear. In this study, data from Memorial Sloan Kettering Cancer Center (MSKCC) prostate cancer database were re-analysed to detect miR-195 expression and its roles in PCa. miR-195 was then overexpressed in castration-resistant PCa cell lines, DU-145 and PC-3. The role of miR-195 in migration and invasion in vitro was also investigated, and common markers in EMT were evaluated through Western blot analysis. A luciferase reporter assay was conducted to confirm the target gene of miR-195; were validated in PCa cells. In MSKCC data re-analyses, miR-195 was poorly expressed in metastatic PCa; miR-195 could be used to diagnose metastatic PCa by measuring the corresponding expression. Area under the receiver operating characteristic curve (AUC-ROC) was 0.705 (P = 0.017). Low miR-195 expression was characterised with a shorter relapse-free survival (RFS) time. miR-195 overexpression suppressed cell migration, invasion and EMT. Fibroblast growth factor 2 (FGF2) was confirmed as a direct target of miR-195. FGF2 knockdown also suppressed migration, invasion and EMT; by contrast, increased FGF2 partially reversed the suppressive effect of miR-195. And data from ONCOMINE prostate cancer database showed that PCa patients with high FGF2 expression showed shorter RFS time (P = 0.046). Overall, this study demonstrated that miR-195 suppressed PCa cell metastasis by downregulating FGF2. miR-195 restoration may be considered as a new therapeutic method to treat metastatic PCa.     

Cell-Specific Detection of miR-375 Downregulation for Predicting the Prognosis of Esophageal Squamous Cell Carcinoma by miRNA In Situ Hybridization

MicroRNAs (miRNAs) play important roles in the regulation of genes associated with cancer development and progression. By the more deeply characterization of miRNAs’ effect in cancer development, it requires a useful tool to investigate expression and distribution of a miRNA in cancer cells and tissues. To fulfill this application demand, we developed a miRNA in situ hybridization (MISH) approach using the 2′-Fluoro modified miRNA probe in combination with enzyme-labeled fluorescence (ELF) signal amplification approach. MISH was used to study expression of miR-375 in esophageal squamous cell carcinoma (ESCC) cell lines and tissues using a tissue microarray (TMA) containing 300 cases. The results showed that our MISH approach is a practical way to detect expression and distribution of a tested miRNA in both cultured cells and archive tissue sections. MISH results also showed that miR-375 was frequently downregulated in ESCCs, which was significantly associated with advanced clinical stage (p = 0.003) tumor metastasis (p = 0.04) and poor outcome (p = 0.04) of ESCC. Moreover, the accuracy of MISH results could be confirmed by QRT-PCR. Our results demonstrated that MISH is a useful and reliable tool to study miRNA expression in solid tumors. Downregulation of miR-375 can be used as a biomarker to predict the outcome of ESCC.     

Restoration of circPSMC3 sensitizes gefitinib-resistant esophageal squamous cell carcinoma cells to gefitinib by regulating miR-10a-5p/PTEN axis

Circular RNAs (circRNAs) has been shown to play an important role in the progression of various cancers. However, the function and underlying mechanisms of circRNAs affecting chemotherapy resistance in esophageal squamous cell carcinoma (ESCC) remain largely unknown. In this study, we used gefitinib-resistant (GR) ESCC cells to investigate the function of circPSMC3 and clarify the underlying mechanism in chemotherapy resistance in ESCC. The results suggested that circPSMC3 expression was downregulated, but miR-10a-5p was upregulated in ESCC tissues and cells, as well as in GR ESCC cells. CircPSMC3 overexpression increased the sensitivity of ESCC cells to gefitinib, as indicated by reduced half maximal inhibitory concentration value, increased apoptosis rate and cleaved caspase-3 protein expression. CircPSMC3 directly interacted with miR-10a-5p and inhibited the expression of miR-10a-5p. Phosphatase and tensin homolog (PTEN) was a direct target of miR-10a-5p and circPSMC3 promoted PTEN expression via decreasing miR-10a-5p level. Moreover, the effect of circPSMC3 on resistance of GR ESCC cells to gefitinib was remarkably reduced by restoration of miR-10a-5p and downregultion of PTEN. Taken together, these observations suggested that upregulation of circPSMC3 overcame resistance of GR ESCC cells to gefitinib by modulating the miR-10a-5p/PTEN axis, which provide a new therapeutic strategy for overcoming gefitinib resistance in ESCC.     

Epigenetic silencing of miR-203 in Kazakh patients with esophageal squamous cell carcinoma by MassARRAY spectrometry

Dysregulation of miR-203 by promoter methylation is associated with the development of various cancers. We aimed to explore the underlying link between promoter methylation and miR-203 expression in Kazakh esophageal squamous cell carcinoma (ESCC). MassARRAY® System spectrometry was used to quantitatively analyze the DNA methylation of 32 CpG sites within miR-203 in 99 Kazakh ESCC and 46 normal esophageal tissues (NETs) with similar population characteristics. We conducted real-time PCR to detect miR-203 expression levels and evaluated their association with methylation. Eleven CpG units within miR-203 promoter were frequently hypermethylated in ESCC compared with NETs (P < 0.05). The hypermethylation of several CpG units positively correlated with age, lower esophagus, constrictive type of ESCC, and moderately differentiated ESCC. Given the involvement of human papillomavirus (HPV) in etiology of ESCC was confirmed from our previous reports, herein we found that CpG units within miR-203 in HPV16-positive ESCC are more heavily methylated. Furthermore, miR-203 expression showed a nearly 4.5-fold decrease in ESCC than NETs (0.206 ± 0.336 vs. 0.908 ± 1.424, P < 0.001) and was significantly associated with lymph node metastasis (P = 0.012). The expression of miR-203 with 11 completely hypermethylated CpG units was approximately 6.5-fold lower than that with at least 1 unmethylated CpG unit (P < 0.001) and especially the CpG_15.16 and CpG_31.32 with higher methylation levels in ESCC tissues exhibited lower expression levels of miR-203, which indicated a reverse association between miR-203 methylation and expression. Hypermethylated miR-203 is a potential biomarker and targeted delivery of miR-203 could therefore serve as a preventive or therapeutic strategy for Kazakh ESCC.     

MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation

MicroRNAs (miRNAs) are small RNAs with diverse regulatory roles. The miR-124 miRNA is expressed in neurons in the developing and adult nervous system. Here we show that overexpression of miR-124 in differentiating mouse P19 cells promotes neurite outgrowth, while blocking miR-124 function delays neurite outgrowth and decreases acetylated α-tubulin. Altered neurite outgrowth also was observed in mouse primary cortical neurons when miR-124 expression was increased, or when miR-124 function was blocked. In uncommitted P19 cells, miR-124 expression led to disruption of actin filaments and stabilization of microtubules. Expression of miR-124 also decreased Cdc42 protein and affected the subcellular localization of Rac1, suggesting that miR-124 may act in part via alterations to members of the Rho GTPase family. Furthermore, constitutively active Cdc42 or Rac1 attenuated neurite outgrowth promoted by miR-124. To obtain a broader perspective, we identified mRNAs downregulated by miR-124 in P19 cells using microarrays. mRNAs for proteins involved in cytoskeletal regulation were enriched among mRNAs downregulated by miR-124. A miR-124 variant with an additional 5′ base failed to promote neurite outgrowth and downregulated substantially different mRNAs. These results indicate that miR-124 contributes to the control of neurite outgrowth during neuronal differentiation, possibly by regulation of the cytoskeleton.     

Altered miRNA Expression is Associated with Differentiation, Invasion, and Metastasis of Esophageal Squamous Cell Carcinoma (ESCC) in Patients from Huaian, China

Esophageal squamous cell carcinoma (ESCC) is the leading malignancy in Huaian, China. Recently, emerging studies have suggested that an aberrant microRNA (miRNA) expression signature exists in ESCC. However, there is discordant information available on specific miRNA expression in patients from different regions. In this study, we identified 12 miRNAs that are differentially expressed in patients with ESCC from Huaian, China. Among these miRNAs that displayed unique miRNA expression signatures, miR-1, miR-29c, miR-100, miR-133a, miR-133b, miR-143, miR-145, and miR-195 were downregulated, and miR-7, miR-21, miR-223, and miR-1246 were upregulated in cancerous tissue compared with the adjacent normal tissue. Bioinformatics analyses identified the major biological processes and signaling pathways that are targeted by these differentially expressed miRNAs. Accordingly, miR-29c, miR-100, miR-133a, and miR-133b were found to be involved in invasion and metastasis of ESCC, and miR-7 and miR-21 were found to be related to the differentiation of ESCC. Thus, our data present new evidence for the important roles of miRNAs in ESCC.     

microRNA-195 promotes apoptosis and suppresses tumorigenicity of human colorectal cancer cells

Deregulated microRNAs and their roles in cancer development have attracted much attention. In the present study, we analyzed the roles of miR-195 in colorectal cancer pathogenesis, as its participation in some other types of cancer has been suggested by previous reports. By comparing miR-195 expression in 81 human colorectal cancer tissues and matched non-neoplastic mucosa tissues, we found that miR-195 was downregulated in cancer tissues. And restoration of miR-195 in colorectal cancer cell lines HT29 and LoVo could reduce cell viability, promote cell apoptosis and suppress tumorigenicity. Moreover, important antiapoptotic Bcl-2 was identified to be directly targeted by miR-195, and miR-195 was further suggested to exert its proapoptotic function mainly through targeting Bcl-2 expression. Taken together, our study provides important roles of miR-195 in colorectal cancer pathogenesis and implicates its potential application in cancer therapy.     

Clinicopathological pattern and Annexin A2 and Cdc42 status in patients presenting with differentiation and lymphnode metastasis of esophageal squamous cell carcinomas

Annexin A2 and Cdc42 were identified by 2-dimensional electrophoresis (2-DE) and MALDI-TOF–MS between esophageal squamous cell carcinomas (ESCC) and corresponding normal esophagus mucosa in our previous study. To assess clinico-pathological pattern and Annexin A2 and Cdc42 status with respect to cell differentiation and lymphnode metastasis in patients with ESCC. The expression of Annexin A2 and Cdc42 in 22 pairs of fresh ESCC and matched tissues were detected by qRT-PCR and western blot, respectively. And it was further confirmed by immunohistochemistry with 175 pairs of formalin-fixed, paraffin-embedded ESCC. Results showed that Annexin A2 expression was significantly down-regulated, and Cdc42 was up-regulated in ESCC compared to matched control on both mRNA and protein level (P < 0.05), which was in accordance with our previous results on proteomics data. Additionally, Annexin A2 and Cdc42 expression was significantly correlated with lymphoid node metastasis (P < 0.05) and pathological differentiation (P < 0.05). Taken together, we proposed that the aberrant expression of Annexin A2 and Cdc42 played a role in carcinogenesis, differentiation and metastasis of ESCC, which implied its potential target for clinical biomarkers in differentiation and lymph node metastasis.