MiR-23a regulates TGF-β-induced epithelial-mesenchymal transition by targeting E-cadherin in lung cancer cells

Transforming growth factor-β (TGF-β)-induced epithelial-mesenchymal transition (EMT) has been shown to be related to the pathogenesis of various diseases including lung cancer. Recently, microRNAs (miRNA) have been recognized as a new class of genes involved in human tumorigenesis. MiR-23a/24/27a is a miRNA cluster located in chromosome 19p13.12, which can function as an oncogene in several human cancers. In this study, we analyzed miR-23a/24/27a expression in 10 non-small cell cancer (NSCLC) cell lines by real-time PCR analysis. Correlation between expression of these miRNAs and TGF-β/Smad signaling was evaluated. We found that miR-23a could be regulated by TGF-β1 in a Smad-dependent manner in A549 lung adenocarcinoma cells showing the EMT phenomenon. Knockdown of miR-23a partially restored E-cadherin expression under conditions of TGF-β1 stimulation. In contrast, overexpression of miR-23a could suppress E-cadherin expression and stimulate EMT. Furthermore, A549 cells with overexpressed miR-23a were more resistant to gefitinib compared to the parental cells. These findings suggest that miR-23a regulates TGF-β-induced EMT by targeting E-cadherin in lung cancer cells and may be useful as a new therapeutic target in NSCLC.     

MiR-101 functions as a tumor suppressor by directly targeting nemo-like kinase in liver cancer

Nemo-like kinase (NLK), an evolutionarily conserved MAP kinase-related kinase, has been reported to be involved in the development of hepatocellular carcinoma (HCC), but the underlying mechanisms leading to oncogenic NLK are poorly understood. A comprehensive microRNA (miRNA) profiling analysis on human HCC tissues identified four downregulated miRNAs that may target NLK. Ectopic expression of miRNA mimics suggested that miR-101 could suppress NLK in HCC cells. Notably, ectopic miR-101 expression repressed cancer cell growth and proliferation and imitated NLK knockdown effect on HCC cells. In conclusion, we suggest that miR-101 functions as a tumor suppressor by regulating abnormal NLK activity in liver.     

The microRNA-23b/27b/24-1 cluster is a disease progression marker and tumor suppressor in prostate cancer

Our recent study of microRNA (miRNA) expression signatures in prostate cancer (PCa) has revealed that all members of the miR-23b/27b/24-1 cluster are significantly downregulated in PCa tissues. The aim of this study was to investigate the effectiveness of these clustered miRNAs as a disease progression marker and to determine the functional significance of these clustered miRNAs in PCa. Expression of the miR-23b/27b/24-1 cluster was significantly reduced in PCa tissues. Kaplan-Meier survival curves showed that low expression of miR-27b predicted a short duration of progression to castration-resistant PCa. Gain-of-function studies using mature miR-23b, miR-27b, and miR-24-1 significantly inhibited cell proliferation, migration and invasion in PCa cells (PC3 and DU145). To identify the molecular targets of these miRNAs, we carried out gene expression and in silico database analyses. GOLM1 was directly regulated by miR-27b in PCa cells. Elucidation of the molecular targets and pathways regulated by the tumor-suppressive microRNAs should shed light on the oncogenic and metastatic processes in PCa.     

Methylation mediated silencing of MicroRNA-1 gene and its role in hepatocellular carcinogenesis

MicroRNAs (miR) are a class of small ( approximately 21 nucleotide) noncoding RNAs that, in general, negatively regulate gene expression. Some miRs harboring CGIs undergo methylation-mediated silencing, a characteristic of many tumor suppressor genes. To identify such miRs in liver cancer, the miRNA expression profile was analyzed in hepatocellular carcinoma (HCC) cell lines treated with 5-azacytidine (DNA hypomethylating agent) and/or trichostatin A (histone deacetylase inhibitor). The results showed that these epigenetic drugs differentially regulate expression of a few miRs, particularly miR-1-1, in HCC cells. The CGI spanning exon 1 and intron 1 of miR-1-1 was methylated in HCC cell lines and in primary human HCCs but not in matching liver tissues. The miR-1-1 gene was hypomethylated and activated in DNMT1-/- HCT 116 cells but not in DNMT3B null cells, indicating a key role for DNMT1 in its methylation. miR-1 expression was also markedly reduced in primary human hepatocellular carcinomas compared with matching normal liver tissues. Ectopic expression of miR-1 in HCC cells inhibited cell growth and reduced replication potential and clonogenic survival. The expression of FoxP1 and MET harboring three and two miR-1 cognate sites, respectively, in their respective 3'-untranslated regions, was markedly reduced by ectopic miR-1. Up-regulation of several miR-1 targets including FoxP1, MET, and HDAC4 in primary human HCCs and down-regulation of their expression in 5-AzaC-treated HCC cells suggest their role in hepatocarcinogenesis. The inhibition of cell cycle progression and induction of apoptosis after re-expression of miR-1 are some of the mechanisms by which DNA hypomethylating agents suppress hepatocarcinoma cell growth.     

Involvement of programmed cell death 4 in transforming growth factor-beta1-induced apoptosis in human hepatocellular carcinoma

The programmed cell death 4 (PDCD4) gene was originally identified as a tumor-related gene in humans and acts as a tumor-suppressor in mouse epidermal carcinoma cells. However, its function and regulatory mechanisms of expression in human cancer remain to be elucidated. We therefore investigated the expression of PDCD4 in human hepatocellular carcinoma (HCC) and the role of PDCD4 in human HCC cells. Downregulation of PDCD4 protein was observed in all HCC tissues tested compared with corresponding noncancerous liver, as revealed by Western blotting or immunohistochemical staining. Human HCC cell line, Huh7, transfected with PDCD4 cDNA showed nuclear fragmentation and DNA laddering characteristic of apoptotic cells associated with mitochondrial changes and caspase activation. Transforming growth factor-beta1 (TGF-beta1) treatment of Huh7 cells resulted in increased PDCD4 expression and occurrence of apoptosis, also concomitant with mitochondrial events and caspase activation. Transfection of Smad7, a known antagonist to TGF-beta1 signaling, protected cells from TGF-beta1-mediated apoptosis and suppressed TGF-beta1-induced PDCD4 expression. Moreover, antisense PDCD4 transfectants were resistant to apoptosis induced by TGF-beta1. In conclusion, these data suggest that PDCD4 is a proapoptotic molecule involved in TGF-beta1-induced apoptosis in human HCC cells, and a possible tumor suppressor in hepatocarcinogenesis.     

MicroRNA-25 functions as a potential tumor suppressor in colon cancer by targeting Smad7

Because it is a member of the miR-106b∼25 cluster, microRNA-25 (miR-25) is known to be dysregulated in human cancers. However, the expression and role of miR-25 in colon cancer remain unclear. In this study, miR-25 was found to be down-regulated in human colon cancer tissues when compared to those in matched, non-neoplastic mucosa tissues. Functional studies revealed that restoration of miR-25 expression inhibited cell proliferation and migration. In contrast, miR-25 inhibition could promote the proliferation and migratory ability of cells. Stable over-expression of miR-25 also suppressed the growth of colon cancer-cell xenografts in vivo. Furthermore, bioinformatic predictions and experimental validation were used to identify Smad7 as a direct target of miR-25. Functional reverse experiments indicated that the antitumor effects of miR-25 were probably mediated by its repression of Smad7. These results suggest that miR-25 may function as a tumor suppressor by targeting Smad7 in colon cancer. Thus, miR-25 may serve as a potential therapeutic agent or target for cancer therapy.     

Association between microRNA-527 and glypican-3 in hepatocellular carcinoma

The present study aimed to identify the specific microRNAs (miRNAs/miRs) and their corresponding target genes involved in hepatocellular carcinomas (HCCs). Microarray analysis was performed to examine the miRNA expression profiles of four paired HCC and corresponding non-cancerous (N) liver tissues using 985 miRNA probes. The Human miRNA Target database was used to identify the target genes of differentially expressed miRNAs between the HCC and N tissues. The protein expression levels of target genes in the HCC tissues and cell lines were evaluated using western blotting. miRNA-mediated suppression of target gene expression was evaluated by transiently transfecting the miRNA into the HCC cell lines. Of the 985 miRNAs evaluated, four miRNAs were differentially expressed (three upregulated and one downregulated miRNAs). Of these four miRNAs, miRNA-527 was highly downregulated in the HCC tissues. Glypican-3 (GPC-3) was predicted as a target gene of miRNA-527. Western blotting revealed that GPC-3 protein is highly expressed in the HCC tissues and HCC cell lines compared with N and normal cell lines. Transfection with miR-527 resulted in suppression of GPC-3 protein expression in the Cos7 cells. Furthermore, transfection with miR-527 also inhibited the intrinsic expression of GPC-3 in the Huh-7 cell line. This indicated that miR-527 in the HCC tissues may be an important novel miRNA that targets the GPC-3 gene expression. GPC-3, whose expression is regulated by miR-527, may be involved in the development and progression of HCC.     

Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma

We investigated the role of microRNAs (miRNAs) in the pathogenesis of human hepatocellular carcinoma (HCC). A genome-wide miRNA microarray was used to identify differentially expressed miRNAs in HCCs arisen on cirrhotic livers. Thirty-five miRNAs were identified. Several of these miRNAs were previously found deregulated in other human cancers, such as members of the let-7 family, mir-221, and mir-145. In addition, the hepato-specific miR-122a was found down-regulated in approximately 70% of HCCs and in all HCC-derived cell lines. Microarray data for let-7a, mir-221, and mir-122a were validated by Northern blot and real-time PCR analysis. Understanding the contribution of deregulated miRNAs to cancer requires the identification of gene targets. Here, we show that miR-122a can modulate cyclin G1 expression in HCC-derived cell lines and an inverse correlation between miR-122a and cyclin G1 expression exists in primary liver carcinomas. These results indicate that cyclin G1 is a target of miR-122a and expand our knowledge of the molecular alterations involved in HCC pathogenesis and of the role of miRNAs in human cancer.     

Loss of the Smad3 expression increases susceptibility to tumorigenicity in human gastric cancer

Loss of the tumor suppressive effect of transforming growth factor-beta (TGF-beta) has been commonly found at later stages in carcinogenic progression. Although the genes encoding TGF-beta receptors and Smads have been found genetically altered in certain human cancers, no mutation in Smad3 has been observed. Therefore, suppression of Smad3 expression may mediate key oncogenic properties of TGF-beta. First, we observed that 37.5% of human gastric cancer tissues showed low to undetectable levels of Smad3 and that in nine human gastric cancer cell lines examined, two showed deficient Smad3 expression. Introduction of Smad3 into human gastric cancer cells that did not express Smad3, restored TGF-beta responsiveness: induction of p21 and p15 gene expression, and growth inhibition in response to TGF-beta. Furthermore, these Smad3-expressing cells showed markedly decreased and delayed tumorigenicity in vivo. These findings suggest that Smad3 expression may have a critical role in tumor suppression in the early stages of gastric carcinogenesis.     

Lentiviral-mediated Smad4 RNAi induced anti-proliferation by p16 up-regulation and apoptosis by caspase 3 down-regulation in hepatoma SMMC-7721 cells

Transforming growth factor-beta (TGF-beta)-Smad signaling pathway participates in the regulation of a variety of cellular activities. Unlike the high incidences of Smad4 mutation or deletion in pancreatic cancer and gastrointestinal cancers, Smad4 gene is seldom mutated or deleted in hepatocellular carcinoma (HCC). The role of TGF-beta-Smad4 signaling pathway in leading to carcinogenesis of liver cells remains unknown. In this study, we succeeded in silencing Smad4 using lentiviral-mediated Smad4 RNA interference (RNAi). We investigated the role of Smad4 in TGF-beta1-induced cell proliferation and apoptosis of HCC cell line SMMC-7721. We determined cell proliferation, apoptosis, and expression of p21, p16, p53 and caspase 3. Results showed that TGF-beta1 not only had a significant anti-proliferation effect but also induced cellular apoptosis in SMMC-7721 cells. These effects induced by TGF-beta1 were almost completely blocked by the knockdown of Smad4. Western blot analysis revealed that p16 was up-regulated and caspase 3 was activated by silencing of Smad4, and the expression of p21 and wild-type p53 were not affected. These results suggest that TGF-beta1-induced cell growth inhibition by up-regulating p16 expression and cellular apoptosis by activating caspase 3 was Smad4-dependent. Additionally, the knock down of a specific gene using lentiviral-mediated RNAi appears to be a promising tool and strategy for analyzing endogenous gene function.     

MicorRNA 106b ∼ 25 cluster and gastric cancer

Conventional strategies for the early diagnosis and treatment of gastric cancer are not yet satisfactory, and it calls for better diagnosis and treatments based on a deeper understanding of the molecular mechanisms. It has been revealed that the number of verified human microRNA (miRNA) expression contribute to the initiation and progression of cancer. Among them, miR-106b ～ 25 cluster is of particular interest. The miRNA-106b ～ 25 cluster is composed of the highly conserved miRNA-106b, miRNA-93 and miRNA-25. The miRNA-106b ～ 25 polycistron exerted potential proliferative, anti-apoptotic and cell cycle-promoting effects on cancer cells. Over-expression of the miRNA-106b ～ 25 cluster is known to overcome TGF-beta mediated growth suppression via targeting p21 and Bim. This cluster can additionally target the inhibitory Smad7 protein and increase TGF-beta RI which is sufficient to induce epithelial-to-mesenchymal transition (EMT). MiRNA-93 can promote angiogenesis. The tumor suppressor genes RB and PTEN are the direct targets of miRNA-106b ～ 25. Especially, miRNA-106b ～ 25 clusters play an important role in oncogenesis of gastric cancer. Focus on the essential role in tumorgenisis and extremely low expression of miRNA-106b ～ 25 in normal tissues, it maybe an appropriate target of gastric cancer treatment and a novel biomarkers for detecting gastric cancer.     

Disruption of transforming growth factor-beta signaling through beta-spectrin ELF leads to hepatocellular cancer through cyclin D1 activation

Transforming growth factor-beta (TGF-beta) signaling members, TGF-beta receptor type II (TBRII), Smad2, Smad4 and Smad adaptor, embryonic liver fodrin (ELF), are prominent tumor suppressors in gastrointestinal cancers. Here, we show that 40% of elf(+/-) mice spontaneously develop hepatocellular cancer (HCC) with markedly increased cyclin D1, cyclin-dependent kinase 4 (Cdk4), c-Myc and MDM2 expression. Reduced ELF but not TBRII, or Smad4 was observed in 8 of 9 human HCCs (P<0.017). ELF and TBRII are also markedly decreased in human HCC cell lines SNU-398 and SNU-475. Restoration of ELF and TBRII in SNU-398 cells markedly decreases cyclin D1 as well as hyperphosphorylated-retinoblastoma (hyperphosphorylated-pRb). Thus, we show that TGF-beta signaling and Smad adaptor ELF suppress human hepatocarcinogenesis, potentially through cyclin D1 deregulation. Loss of ELF could serve as a primary event in progression toward a fully transformed phenotype and could hold promise for new therapeutic approaches in human HCCs.     

miR-30通过调节CD90表达抑制肝细胞肝癌

  目的  miRNA能通过转录后调节靶基因的表达量,并在致癌过程中发挥抑癌或促癌作用。研究发现miR-30家族在人类肿瘤中起着重要作用,并参与维持干细胞特性的上皮细胞间质化过程,本研究主要识别miR-30家族与肝细胞肝癌的关系。  方法  应用qRT-PCR实验方法检测93例肝细胞肝癌患者癌组织和癌旁正常组织中miR-30c,miR-30b和miR-30e表达水平,另对121例肝细胞肝癌患者癌组织进行miR-30家族的表达及CD90免疫组织化学表达检测,分析miR-30家族与CD90在肝细胞肝癌中的相关性。  结果  本研究发现miR-30c(P < 0.001)、miR-30b(P=0.004)和miR-30e(P < 0.001)与癌旁正常组织相比,癌组织中表达显著下调。CD90蛋白在癌组织中的表达水平显著高于癌旁正常组织(P=0.007)。MiR-30c(P=0.032)和miR-30e(P=0.015)在CD90阴性表达的患者中表达水平显著高于CD90阳性表达的患者。  结论  miR-30家族在肝细胞肝癌中可作为抑癌miRNA,并通过调节CD90蛋白来发挥这一作用。      

MicroRNA expression signature in gastric cancer

Objective: To identify the miRNA specific signature as novel diagnostic and prognostic tools for gastric cancer. Methods: miRNAs expression profiling of 3 normal gastric tissues, 24 malignant tissues, gastric cancer cell SGC7901 and normal gastric cell GES-1 were detected using microarray technology. The hierarchical clustering algorithm of the Cluster software was used to analyse the miRNAs expression of all samples. The expression levels of miR-433 and miR-9 which were significantly down-regulated in gastric cancer tissues and SGC7901 cells by microarray technology were validated by quantitative Real-time PCR (qRT-PCR).Results: Differential expressions of 26 individual miRNAs between normal samples (including 3 normal gastric tissues and GES-1 cells) and carcinomas (including 24 malignant tissues and SGC7901 cells) were discovered,19 of them showing down-regulation and 7 up-regulation in carcinoma samples. Hierarchical clustering of the cancer samples by their miRNA expression accurately separated the carcinomas from normal samples and further their histotypes of carcinomas. The expression levels of miR-433 and miR-9 were significantly down-regulated in gastric cancer tissues and SGC7901cells Conclusion: The differential expression of miR-433 and miR-9 may be used as a novel diagnostic tool for gastric cancer.     

人肝细胞肝癌和癌旁正常组织microRNA表达差异的分析

目的比较肝细胞肝癌和癌旁正常组织microRNA的表达差异,为研究肝癌的发病机理提供理论依据。方法用microRNA 微阵列芯片技术研究肝细胞肝癌细胞474种miRNAs表达谱,对比癌旁正常组织筛选差异表达miRNAs。结果获得213个差异表达（P<0.01）的miRNAs分子数据,与配对癌旁正常组织相比,其中上调表达的有116个,如miR-181,miR-21,let-7e等;下调表达的有97个,如miR-199,miR-451,miR-122等。结论miRNA可能成为肝癌的诊断工具,并对研究肝癌的致病机理提供新的理论依据。