Enhancer of zeste homolog 2 epigenetically silences multiple tumor suppressor microRNAs to promote liver cancer metastasis

Epigenetic alterations and microRNA (miRNA) deregulation are common in hepatocellular carcinoma (HCC). The histone H3 lysine 27 (H3K27) tri-methylating enzyme, enhancer of zeste homolog 2 (EZH2) mediates epigenetic silencing of gene expression and is frequently up-regulated in human cancers. In this study we aimed to delineate the implications of EZH2 up-regulation in miRNA deregulation and HCC metastasis. Expressions of a total of 90 epigenetic regulators were first determined in 38 pairs of primary HCCs and their corresponding nontumorous livers. We identified EZH2 and its associated polycomb repressive complex 2 (PRC2) as one of the most significantly deregulated epigenetic regulators in primary HCC samples. Up-regulation of EZH2 was next confirmed in 69.5% (41/59) of primary HCCs. Clinicopathologically, EZH2 up-regulation was associated with HCC progression and multiple HCC metastatic features, including venous invasion (P = 0.043), direct liver invasion (P = 0.014), and absence of tumor encapsulation (P = 0.043). We further demonstrated that knockdown of EZH2 in HCC cell lines reduced the global levels of tri-methylated H3K27, and suppressed HCC motility in vitro and pulmonary metastasis in a nude mouse model. By interrogating the miRNA expression profile in EZH2-knockdown cell lines and primary HCC samples, we identified a subset of miRNA that was epigenetically suppressed by EZH2 in human HCC. These included well-characterized tumor-suppressor miRNAs, such as miR-139-5p, miR-125b, miR-101, let-7c, and miR-200b. Pathway enrichment analysis revealed a common regulatory role of these EZH2-silenced miRNAs in modulating cell motility and metastasis-related pathways. Our findings suggest that EZH2 exerts its prometastatic function by way of epigenetic silencing of multiple tumor suppressor miRNAs. CONCLUSION: Our study demonstrated that EZH2 epigenetically silenced multiple miRNAs that negatively regulate HCC metastasis.     

Initial study of microRNA expression profiles of colonic cancer without lymph node metastasis

OBJECTIVE: To investigate the difference of microRNA expression profiles between colonic cancer without lymph node metastasis and the para-cancerous control, to identify the specific microRNA associated with the cancer and to predict the carcinogenetic mechanism of microRNA on the basis of these results. METHODS: The microRNA (miRNA) were extracted and isolated from six specimens, including colonic cancerous and para-cancerous ones, all of which were confirmed to be without lymph node metastasis. Agilent microRNA microarrays consisting of 723 probes were used for screening the expression differences of microRNA. Data were analyzed using feature extraction software. The expression level of differentially expressed microRNA using quantitative real-time polymerase chain reaction (RT-PCR) was validated. RESULTS: A total of 14 miRNAs were found to be associated with colonic cancer, in which the expression of miR-106b, miR-135b, miR-18a, miR-18b, miR-196b, miR-19a, miR-224, miR-335, miR-424, miR-20a*, miR-301b and miR-374a were up-regulated and the expression of miR-378 and miR-378* were downregulated in colonic cancer tissues, compared with the para-cancerous control. The expression level of miR-18a and miR-135b were validated in accordance with the results of RT-PCR. CONCLUSION: The miRNAs are differentially expressed between colonic tumor tissues and para-cancerous tissues. Many of these miRNAs are expected to participate in the process of multiple tumorigenesis. These miRNAs could play an important role in the carcinogenesis of colon. These results provide new insights in human colorectal cancer genesis.     

Alterations of epigenetics and microRNA in hepatocellular carcinoma

Studies have shown that alterations of epigenetics and microRNA (miRNA) play critical roles in the initiation and progression of hepatocellular carcinoma (HCC). Epigenetic silencing of tumor suppressor genes in HCC is generally mediated by DNA hypermethylation of CpG island promoters and histone modifications such as histone deacetylation, methylation of histone H3 lysine 9 (H3K9) and tri‐methylation of H3K27. Chromatin‐modifying drugs such as DNA methylation inhibitors and histone deacetylase inhibitors have shown clinical promise for cancer therapy. miRNA are small non‐coding RNA that regulate expression of various target genes. Specific miRNA are aberrantly expressed and play roles as tumor suppressors or oncogenes during hepatocarcinogenesis. We and other groups have demonstrated that important tumor suppressor miRNA are silenced by epigenetic alterations, resulting in activation of target oncogenes in human malignancies including HCC. Restoring the expression of tumor suppressor miRNA by inhibitors of DNA methylation and histone deacetylase may be a promising therapeutic strategy for HCC.     

MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3A and 3B

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate expression of many genes. Recent studies suggest roles of miRNAs in carcinogenesis. We and others have shown that expression profiles of miRNAs are different in lung cancer vs. normal lung, although the significance of this aberrant expression is poorly understood. Among the reported down-regulated miRNAs in lung cancer, the miRNA (miR)-29 family (29a, 29b, and 29c) has intriguing complementarities to the 3'-UTRs of DNA methyltransferase (DNMT)3A and -3B (de novo methyltransferases), two key enzymes involved in DNA methylation, that are frequently up-regulated in lung cancer and associated with poor prognosis. We investigated whether miR-29s could target DNMT3A and -B and whether restoration of miR-29s could normalize aberrant patterns of methylation in non-small-cell lung cancer. Here we show that expression of miR-29s is inversely correlated to DNMT3A and -3B in lung cancer tissues, and that miR-29s directly target both DNMT3A and -3B. The enforced expression of miR-29s in lung cancer cell lines restores normal patterns of DNA methylation, induces reexpression of methylation-silenced tumor suppressor genes, such as FHIT and WWOX, and inhibits tumorigenicity in vitro and in vivo. These findings support a role of miR-29s in epigenetic normalization of NSCLC, providing a rationale for the development of miRNA-based strategies for the treatment of lung cancer.     

肝癌转移相关的微小RNAs在不同转移潜能肝癌细胞系的定量研究

目的 研究与转移密切相关的微小RNAs(miRNAs)在不同转移潜能肝癌细胞系的表达水平,探讨其在肿瘤转移过程中的生物学功能.方法 提取细胞系MHCC97H、MHCC97L、HepG2、L02的总RNA,通过反转录获得特异miRNA(miR-122a、miR-124a、miR-148a、miR-148b、miR-15a、miR-219、miR-30c、miR-338、miR-34a、Let-7g、miR-9)的cDNA,并应用TaqMan MGB探针法对其进行定量检测.采用AB17500系统软件V1.3.1采集Ct值,并使用miRNA内参基因RNU6B校正,相对定量计算公式RQ=2-△Ct,A Ct=CtmiRNAs-CtRNu6B.数据均经SPSS13.0统计软件包处理,采用t检验或非参数检验. 结果 转移相关的miRNAs(miR-124a除外)在MHCC97H与MHCC97L中表达,差异均有统计学意义.HepG2中miR-30c、miR-338、miR-34a和Let-g的表达水平明显高于L02,分别为miR-30c(8.41±0.40比6.82±0.29),miR-338(3.14±0.29比-2.36±0.32),miR-34a(0.71±0.40比-2.95±0.26),Let-7g(-4.07±0.55比-6.98±0.56),t值依次为2.948,12.656,7.484,3.684,P值均＜0.05,差异有统计学意义.而miR-148b,miR-9的表达则显著低于正常肝细胞,分别为miR-148b(1.96±0.51比3.76±0.28),miR-9(-4.38±0.86比-1.10±0.53),t值依次为-3.073,-3.324,P值均＜0.05,差异有统计学意义.miR-148家族中miR-48b在所测细胞系中的表达(5.46±1.21)均显著强于miR-148a的表达(1.29±0.35),Z=-5.097,P=3×10-7,差异有统计学意义.结论 可以利用肝癌细胞系列细胞平台进一步研究肝癌转移相关miRNAs在肿瘤转移过程中的生物学功能.     

MicroRNA silencing of tumor suppressor DLC-1 promotes efficient hepatitis C virus replication in primary human hepatocytes

MicroRNAs (miRNAs) are approximately 22-nucleotide noncoding RNAs that constitute silencers of target gene expression. Aberrant expression of miRNA has been linked to a variety of cancers, including hepatocellular carcinoma (HCC). Hepatitis C virus (HCV) infection is considered a major cause of chronic liver disease and HCC, although the mechanism of virus infection-associated hepatocarcinogenesis remains unclear. We report a direct role of miRNAs induced in HCV-infected primary human hepatocytes that target the tumor suppressor gene DLC-1 (a Rho GTPase-activating protein), which is frequently deleted in HCC, and other solid human tumors. MicroRNA miR-141 that targets DLC-1 was accentuated in cells infected with HCV genotypes 1a, 1b, and 2a. We present several lines of evidence that efficient HCV replication requires miR-141-mediated suppression of DLC-1. An increase in miR-141 correlated with the inhibition of DLC-1 protein in HCV-infected cells. Depletion of miR-141 with oligonucleotides complementary to the miRNAs inhibited virus replication, whereas artificially increased levels of intracellular miR-141 enhanced HCV replication. HCV-infected hepatocytes showed enhanced cell proliferation that can be countered by overexpression of DLC-1. CONCLUSION: The collective results of this study suggest a novel mechanism of HCV infection-associated miRNA-mediated regulation of a tumor suppressor protein that has the ability to influence cell proliferation and HCV infection-mediated liver cancer.     

MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer

BACKGROUND AND AIMS: microRNAs (miRNAs) are short noncoding RNAs that regulate gene expression negatively. Although a role for aberrant miRNA expression in cancer has been postulated, the pathophysiologic role and relevance of aberrantly expressed miRNA to tumor biology has not been established. METHODS: We evaluated the expression of miRNA in human hepatocellular cancer (HCC) by expression profiling, and defined a target gene and biologically functional effect of an up-regulated miRNA. RESULTS: miR-21 was noted to be highly overexpressed in HCC tumors and cell lines in expression profiling studies using a miRNA microarray. Inhibition of miR-21 in cultured HCC cells increased expression of the phosphatase and tensin homolog (PTEN) tumor suppressor, and decreased tumor cell proliferation, migration, and invasion. In contrast-enhanced miR-21 expression by transfection with precursor miR-21 increased tumor cell proliferation, migration, and invasion. Moreover, an increase in cell migration was observed in normal human hepatocytes transfected with precursor miR-21. PTEN was shown to be a direct target of miR-21, and to contribute to miR-21 effects on cell invasion. Modulation of miR-21 altered focal adhesion kinase phosphorylation and expression of matrix metalloproteases 2 and 9, both downstream mediators of PTEN involved in cell migration and invasion. CONCLUSIONS: Aberrant expression of miR-21 can contribute to HCC growth and spread by modulating PTEN expression and PTEN-dependent pathways involved in mediating phenotypic characteristics of cancer cells such as cell growth, migration, and invasion.     

微RNA-29家族降解PTEN mRNA促进非小细胞肺癌细胞存活与淋巴结侵袭

目的:探索微RNA(microRNA,miRNA/miR)-29家族对淋巴结侵袭性非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞增殖、侵袭的影响及潜在的分子机制.方法:TCGA数据库分析磷酸酶和张力蛋白同源基因(phosphatase and tension homology de...     

The role of miR-148a in gastric cancer

Purpose

Gastric cancer is one of the most common malignant diseases worldwide, although much progress has been achieved in recent years, the early diagnosis and treatment for gastric cancer are not yet satisfactory and, thus the prognosis is still poor. MicroRNAs (miRNAs) can regulate a variety of physiological and developmental processes, it has been revealed that many miRNAs contribute the initiation and progression of various cancers. MiR-148a is one of the most important miRNAs in gastric cancer, and the aim of this paper is to provide an overview of various roles of miR-148a in gastric cancer.

Methods and results

We searched studies in electronic databases. MiR-148a was down-regulated in gastric cancer tissues and cell lines, which was resulted from the hypermethylation in its promoter region. Furthermore, miR-148a could regulate several different target genes and pathways involving tumor proliferation, invasion and metastasis.

Conclusion

MiR-148a may serve as a novel biomarker for the diagnosis and as a new therapeutic target in gastric cancer.     

miR-29对胃腺癌细胞侵袭力的影响

目的探讨miR-29对胃腺癌细胞侵袭力的影响,以期为胃癌的转移、侵袭机制提供线索。方法采用实时荧光定量PCR技术检测46例患者胃腺癌标本及癌旁组织中miR-29表达,分析与患者临床病理特征的关系。结果 PCR反应检测结果显示miR-29a、miR-29b、miR-29c在胃癌组织中相对含量分别为14.13±5.78、9.81±1.95、16.77±2.04,在癌旁组织中分别为31.64±10.26、26.88±5.03、52.57±8.02,胃癌组织中的miR-29相对含量明显低于癌旁组织(P0.05);将胃癌组织标本与癌旁组织标本中的miR-29相对含量计算比值(C/P),患者不同性别、年龄、TNM分期、浸润深度、分化程度时C/P值相比,差异无统计学意义(P0.05);存在淋巴结转移时miR-29a C/P值明显低于无淋巴结转移时(P0.05),存在远处转移时miR-29b、miR-29c C/P值明显低于无远处转移时(P0.05)。结论胃腺癌组织中miR-29表达明显低于癌旁组织,miR-29a在淋巴结转移及miR-29b、miR-29c在远处转移患者胃腺癌组织中表达降低,提示其可能与胃癌的侵袭及转移有关。     

MYC stimulates EZH2 expression by repression of its negative regulator miR-26a

The MYC oncogene, which is commonly mutated/amplified in tumors, represents an important regulator of cell growth because of its ability to induce both proliferation and apoptosis. Recent evidence links MYC to altered miRNA expression, thereby suggesting that MYC-regulated miRNAs might contribute to tumorigenesis. To further analyze the impact of MYC-regulated miRNAs, we investigated a murine lymphoma model harboring the MYC transgene in a Tet-off system to control its expression. Microarray-based miRNA expression profiling revealed both known and novel MYC targets. Among the miRNAs repressed by MYC, we identified the potential tumor suppressor miR-26a, which possessed the ability to attenuate proliferation in MYC-dependent cells. Interestingly, miR-26a was also found to be deregulated in primary human Burkitt lymphoma samples, thereby probably being of clinical relevance. Although today only few miRNA targets have been identified in human disease, we could show that ectopic expression of miR-26a influenced cell cycle progression by targeting the bona fide oncogene EZH2, a Polycomb protein and global regulator of gene expression yet unknown to be regulated by miRNAs. Thus, in addition to directly targeting protein-coding genes, MYC modulates genes important to oncogenesis via deregulation of miRNAs, thereby vitally contributing to MYC-induced lymphomagenesis.     

DNA methylation disturbances as novel therapeutic target in lung cancer: preclinical and clinical results

The prognosis of lung cancer is very much limited by the difficulties of diagnosing early stage disease amenable to surgery. Thus, novel diagnostic and therapeutic approaches are urgently needed for this common type of cancer. Recently, epigenetic alterations of tumor cells have been defined for a multitude of tissues and genes. Thus, promoter hypermethylation of tumor suppressor genes, and other targets of neoplasia-associated methylation disturbances, have become the most frequent recurrent alteration in solid tumors and hematologic neoplasia. In lung cancer, several sets of genes including the tumor suppressor gene p16, the DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT), E-cadherin and retinoic acid receptor beta have been shown to be frequently methylated and inactivated. Distinct methylation patterns can provide molecular distinctions between different histologic subtypes of lung cancer. Gene hypermethylation in lung cancer is an early event associated with exposure to tobacco-specific carcinogens. Highly sensitive detection of hypermethylated DNA in sputum and peripheral blood offers a powerful tool for detecting lung cancer at an early stage. Epigenetic alterations in cancer, as opposed to genetic lesions, are potentially reversible. Thus, hypermethylation has been studied as a therapeutic target for agents which revert this epigenotype. The most advanced drugs to inhibit methylation are two azanucleosides, decitabine and its ribonucleoside analogue 5-azacytidine. In vitro, demethylating agents given at low doses reactivate tumor suppressor genes, and in mouse models, the development of lung cancer can be retarded. This effect is more powerful when histone acetylation, as a second epigenetic silencing mechanism, is also inhibited pharmacologically (HDAC inhibitors). Clinical trials of both groups of agents have been performed, and novel demethylating agents which are not incorporated into DNA offer further perspectives for epigenetic therapy of lung cancer and other malignancies.     

胰腺癌组织c-MET表达与循环miR-34a、miR-449水平的相关性

目的 探讨胰腺癌组织织间质表皮转化因子(c-MET)的表达与循环miR-34a、miR-449水平的相关性及其临床意义。方法 收集2015年3月至2017年3月间嘉兴医学院附属第二医院行手术治疗并经病理证实的41例胰腺癌患者的临床资料。通过免疫组织化学染色检测胰腺癌组织及其匹配的癌旁正常胰腺组c-MET表达,根据测定结果将患者分为c-MET阳性组与c-MET阴性组。采集患者手术前和手术后3个月外周血,应用荧光定量PCR法检测循环miR-34a和miR-449水平。分析癌组织c-MET表达与患者临床病理参数、预后及循环miR-34a、miR-449水平的关系。分析循环miR-34a和miR-449水平对胰腺癌TNM分期、淋巴结转移和预后的评估价值。结果 胰腺癌组织c-MET阳性率明显高于癌旁正常组织(63.4%比24.4%),差异有统计学意义(P<0.05)。与c-MET阴性患者比较,c-MET阳性患者TNMⅢ/Ⅳ期者居多(73.1%比33.4%),淋巴结转移率高(76.9%比46.7%),生存时间短(29.5个月比35.0个月),生存率低(38.5%比53.3%),差异均有统计学意义(P值均<0.05)。术前c-MET阳性患者循环miR-34a、miR-449水平明显低于c-MET阴性患者(0.228±0.068比0.524±0.106、0.252±0.063比0.432±0.094,P<0.05),术后c-MET阳性患者循环miR-449水平仍明显低于c-MET阴性患者(0.414±0.088比0.512±0.114,P<0.05),但两组间miR-34a水平的差异无统计学意义。术前循环miR-34a、miR-449水平对胰腺癌TNM分期、淋巴结转移及预后有预测价值(P<0.05)。结论 miR-34a、miR-449靶向结合胰腺癌组织c-MET,有可能成为潜在的胰腺癌标志物。