循环miRNAs在肺癌诊断和治疗中的研究进展

肺癌是全世界男性和女性癌症死亡的第一原因。尽管近些年对癌症信号通路的了解和在新的治疗方法开发上有了新进展,但是肺癌的不良预后和高复发率依然存在。循环miRNAs在许多实体瘤（如肺癌）中,可作为稳定表达的非侵入性诊断标志物。一些miRNAs异常可能与肿瘤细胞常规化疗的耐药性有关,影响着肿瘤细胞的药物敏感性。这些miRNAs的调控,通过使用miRNAs类似物,可以调节关键基因网络和信号通路,通过抑制肿瘤细胞增殖来提高抗癌治疗的疗效,增加药物敏感性。因此,miRNAs疗法提供了新的抗肿瘤方法:更有效的个性化治疗、提高药物疗效、预测不同抗癌药物的反应。本综述的目的是为肺癌循环miRNA提供一个作为潜在诊断生物标志物、大规模临床筛选诊断的可行性评估以及miRNAS抗癌耐药机制对肺癌的治疗的概述。最后,miRNA分析的局限性和潜在性需要今后进一步探索。     

肺癌A549细胞上皮-间质转化及其对微小RNA表达的影响

目的 探讨肺癌A549细胞上皮-间质转化(EMT)对微小RNA(miRNA)表达的影响.方法 采用不同浓度的转化生长因子β1(TGF-β1)诱导肺癌A549细胞发生EMT,应用相差显微镜观察A549细胞形态学变化,采用Western blot法检测EMT相关标记蛋白表达的变化,应用miRNA芯片检测EMT前后miRNA表达的变化,应用荧光定量逆转录聚合酶链反应(RT-PCT)验证芯片结果的可靠性.结果 肺癌A549细胞发生EMT后,细胞形态拉长,细胞间连接变得疏松.肺癌A549细胞发生EMT后,上皮标记蛋白E-钙黏附素(E-cadherin)表达降低,而间质标记波形蛋白(Vimentin)和纤维连接蛋白(Fibronectin)表达上调.miRNA芯片获得51个miRNA在诱导前后有统计学意义(P＜0.05),且表达差异在2倍以上.18个表达上调,33个表达下调,其中mir-33a和mir-193a-3p的表达经诱导后分别下调了92.8%和86.5%;荧光定量RT-PCR检测mir-33a和mir-193a-3p的表达经诱导后分别下调了73.1%和56.6%.结论 EMT可影响肺癌A549细胞中miRNA的表达变化,miRNA可能通过EMT调节肺癌侵袭转移.

Abstract：

Objective To investigate the effect of epithelial-mesenchymal transition (EMT) on the expression of microRNAs (miRNAs) in lung cancer A549 cells. Methods Transforming growth factor beta1 (TGF-β1) in different concentrations was used to induce EMT in lung cancer A549 cells. The morphological changes were observed under phase-contrnst microscope. The changes of EMT-related proteins were analyzed by Western blot. The changes of miRNAs expression after EMT were detected by microRNA (miRNA) array. Real time quantitative RT-PCR was applied to verify the reliability of miRNA array results.Results The lung cancer A549 cells became elongated and the cell-cell junction became loose after EMT.The epithelial protein marker E-cadherin was down-regulated and the mesenchymal protein markers vimentin and fibronectin up-regulated. There were 51 miRNAs showing statistically significunt changes of expression more than double (P ＜ 0. 05 ) after EMT. Among them 18 were up-regulated and 33 down-regulated. Of them, mir-33a was down-regulated by 92.8% and mir-193a-3p by 86.5%. Real time quantitative RT-PCR showed that mir-33a was down-regulated by 73.1% and mir-193a-3p by 56.6%. Conclusion Epithelial-mesenchymal trnsition has effects on the expression of miRNAs, and miRNAs may regulate the invasion and metastasis of lung cancer cells via EMT.     

MicroRNAs and metastasis-related gene expression in Egyptian breast cancer patients

Aim and background: MicroRNAs (miRNAs) are a class of naturally occurring small noncoding RNAs that regulate gene expression, cell growth, differentiation and apoptosis by targeting mRNAs for translational repression or cleavage. The present study was conducted to study miRNAs in Egyptian breast cancer (BC) and their relation to metastasis, tumor invasion and apoptosis in addition to their association with the ER and PR statuses. Methods: Real Time RT-PCR was performed to identify the miRNA expression level of eight miRNAs and eight metastatic-related genes in 40 breast cancer samples and their adjacent non-neoplastic tissues. The expression levels of each miRNA relative to U6 RNA were determined using the 2–ΔCT method. Also, miRNA expression profiles of the BC and their corresponding ANT were evaluated. Results: The BC patients showed an up-regulation in miRNAs (mir-155, mir-10, mir-21 and mir-373) with an upregulation in MMP2, MMp9 and VEGF genes. We found down regulation in mir-17p, mir-126, mir-335, mir-30b and also TIMP3, TMP1 and PDCD4 genes in the cancer tissue compared to the adjacent non-neoplastic tissues. Mir -10b, mir -21, mir-155 and mir373 and the metastatic genes MMP2, MMP9 and VEGF were significantly associated with an increase in tumor size (P < 0.05). No significant difference was observed between any of the studied miRNAs regarding lymph node metastasis. Mir-21 was significantly over-expressed in ER-/PR- cases. Conclusion: Specific miRNAs (mir-10, mir-21, mir-155, mir-373, mir-30b, mir-126, mir-17p, mir-335) are associated with tumor metastasis and other clinical characteristics for BC, facilitating identification of individuals who are at risk.     

循环血液外泌体miRNA在卵巢癌发生发展中作用及其诊断价值的研究进展

卵巢癌（ovarian cancer,OC）是女性恶性肿瘤死亡的主要原因。由于卵巢癌无症状发展,缺乏早期诊断标志物,大多数患者在晚期才被诊断出来。早期检测卵巢癌可显著提高总生存率,在过去的几十年里,微小RNA（miRNA）在癌症的发展中起着重要的作用,因此引起了极大的关注。miRNA可以在循环血液中稳定存在(如包裹在外泌体中),并可通过外泌体的分泌和转移在肿瘤细胞之间和肿瘤细胞微环境的沟通中发挥重要的作用。此外,外泌体miRNA在卵巢癌中的表达是失调的,可能反映肿瘤的恶性特征。因此评估外泌体来源的循环miRNA可能会为卵巢癌提供一类新的非侵袭性生物标志物。本综述概述了有关外泌体miRNA在卵巢癌发生发展过程中的作用以及循环血液外泌体miRNA作为卵巢癌早期诊断潜在生物标志物的现状。     

MicroRNAs,TGF-β signaling,and the inflammatory microenvironment in cancer

Inflammatory cells and mediators form a major part of the tumor microenvironment and play important roles in the regulation of cancer initiation, tumor cell proliferation, and metastasis. MicroRNAs (miRNAs) play important roles in several physiological and pathological processes, including the regulation of the inflammatory microenvironment in cancer. Transforming growth factor-β (TGF-β) is an inflammation-related cytokine that functions in both tumor suppression and promotion; however, its underlying molecular mechanisms remain unclear. Recent evidence indicates an association between miRNAs and TGF-β signaling, providing new insight into the nature of the inflammatory microenvironment in cancer. The present review is an overview of the interaction between miRNAs and inflammatory cytokines, with emphasis on the cross talk between TGF-β signaling and miRNAs and their influence on cancer cell behavior. The emerging roles of miRNAs in cancer-related inflammation and the potential to target miRNA signaling pathways for cancer therapy are also discussed.     

Detection of circulating microRNAs with Ago2 complexes to monitor the tumor dynamics of colorectal cancer patients during chemotherapy

Because of the different forms of circulating miRNAs in plasma, Argonaute2 (Ago2)-miRNAs and extracellular vesicles (EV-miRNAs), we examined the two forms of extracellular miRNAs in vitro and developed a unique methodology to detect circulating Ago2-miRNAs in small volumes of plasma. We demonstrated that Ago2-miR-21 could be released into the extracellular fluid by active export from viable cancer cells and cytolysis in vitro. As miR-21 and miR-200c were abundantly expressed in both metastatic liver sites and primary lesions, we evaluated Ago2-miR-21 as a candidate biomarker of both active export and cytolysis while Ago2-miR-200c as a biomarker of cytolysis in plasma obtained from colorectal cancer (CRC) patients before treatment and in a series of plasma obtained from CRC patients with liver metastasis who received systemic chemotherapy. The measurement of Ago2-miR-21 allowed us to distinguish CRC patients from subjects without CRC. The trend in ΔCt values for Ago2-miR-21 and -200c during chemotherapy could predict tumor response to ongoing treatment. Thus, capturing circulating Ago2-miRNAs from active export can screen patients with tumor burdens, while capturing them from passive release by cytolysis can monitor tumor dynamics during chemotherapy treatment.     

Splenectomy reduces lung metastases and tumoral and metastatic niche inflammation

The immune microenvironment plays a crucial role in supporting tumor growth and metastasis. Tumor-associated macrophages (TAMs) and neutrophils (TANs) are essential components of this microenvironment and affect tumor growth and progression in almost all solid neoplasms. Furthermore, TAMs, TANs and tumor-infiltrating dendritic cells (TIDCs) are found to infiltrate specific distant organs to prepare them as a site for metastatic cell seeding, forming the pre-metastatic niche. The spleen was identified as a major reservoir and source of circulating and tumor infiltrating immune cells. However, discrepancies about its role in supporting tumor growth exist. Thus, here we investigated the role of splenectomy in primary tumor and metastatic growth, and in the formation of an inflammatory niche. In a murine 4T1 and E0771 breast and Panc02 pancreatic cancer model, our results show that while splenectomy reduces the number of infiltrating TAMs, TANs and TIDCs within primary tumors, it does not affect its growth. In line, fewer TAMs, TANs and TIDCs accumulate in the metastatic microenvironment after splenectomy. Interestingly though, this affected metastatic growth depending on the metastatic route/site. The number of hematogenous breast cancer lung metastases was reduced after splenectomy but no effect was observed in breast or pancreatic lymph node metastases. Moreover, we observed that the immune composition of the pre-metastatic niche in lungs of breast cancer bearing mice was altered, and that this could cause the reduction of metastases. Altogether, our results highlight that splenectomy affects the immune microenvironment not only of primary tumors but also of pre-metastatic and metastatic sites.     

Clinical significance of circulating miRNA detection in lung cancer

Lung cancer is the most common cancer in the world and the leading cause of tumor death among males. MicroRNAs (miRNAs) are single-stranded RNAs of approximately 22 nucleotides and constituted a new class of gene regulators in humans. As a novel class of emerging biomarkers, the aberrant expression of miRNA has been detected in various tumors. miRNAs are secreted into circulation by microvesicles from the broken tumor cells and act as either oncogenes or tumor suppressors in tumor tissues. In this review, we summarized different circulating miRNAs and their expression level as well as predictable values in lung cancer patients which were investigated in recent 5 years. Circulating miRNAs are found to be dysregulated and have association with clinicopathological parameters and overall survival in lung cancer patients. In conclusion, circulating miRNAs have the potential for distinguishing lung cancer patients from healthy individuals, with the advantages of stabilities, noninvasiveness and cost-effectiveness.     

MicroRNA 345, a methylation-sensitive microRNA is involved in cell proliferation and invasion in human colorectal cancer

Aberrant methylation has been shown to trigger the inactivation of tumor suppressor genes during tumorigenesis. MicroRNAs (miRNAs) have been found deregulated in human colorectal cancer (CRC), and some of them may function as tumor suppressor genes. Here, we investigated CpG island promoter hypermethylation as a potential mechanism underlying miRNA disruption and identifed methylation-sensitive miRNAs that might repress CRC development. We compared differential expression of miRNAs after 5-aza-2'-deoxycitidine (5-aza-dC) treatment using microarrays. DNA methylation status of the candidate miRNA was analyzed. The candidate miRNA was transfected into CRC cells and growth-suppressive mechanisms were explored. Luciferase reporter assay and western blot were used to identify the target genes of the candidate miRNA. The expression of mir-345 was significantly increased after 5-aza-dC treatment. DNA methylation analyses of mir-345 showed high methylation levels in tumor versus normal tissues. Expression of mir-345 was significantly down-regulated in 51.6% of CRC tissues compared with corresponding non-cancerous tissues. Low expression of mir-345 was associated with lymph node metastasis and worse histological type. Increased mir-345 function was sufficient to suppress colon cancer cell proliferation and invasiveness in vitro. Furthermore, we identified BCL2-associated athanogene 3 (BAG3), an anti-apoptosis protein, to be a target of mir-345. These results suggested as a methylation-sensitive miRNA in CRC, mir-345 may play an important role of antineoplastic as a growth inhibitor in the development of CRC.     

Reverse of non‐small cell lung cancer drug resistance induced by cancer‐associated fibroblasts via a paracrine pathway

The tumor microenvironment orchestrates the sustained growth, metastasis and recurrence of cancer. As an indispensable component of the tumor microenvironment, cancer‐associated fibroblasts (CAF) are considered as an essential synthetic machine producing various tumor components, leading to cancer sustained stemness, drug resistance and tumor recurrence. Here, we developed a sustainable primary culture of lung cancer cells fed with lung cancer‐associated fibroblasts, resulting in enrichment and acquisition of drug resistance in cancer cells. Moreover, IGF2/AKT/Sox2/ABCB1 signaling activation in cancer cells was observed in the presence of CAF, which induces upregulation of P‐glycoprotein expression and the drug resistance of non‐small cell lung cancer cells. Our results demonstrated that CAF cells constitute a mechanism for cancer drug resistance. Thus, traditional chemotherapy combined with insulin‐like growth factor 2 (IGF2) signaling inhibitor may present an innovative therapeutic strategy for non‐small cell lung cancer therapy.     

Genome structure-based screening identified epigenetically silenced microRNA associated with invasiveness in non-small-cell lung cancer

MicroRNA (miRNA) expression is frequently altered in human cancers. To search for epigenetically silenced miRNAs in non-small-cell lung cancer (NSCLC), we mapped human miRNAs on autosomal chromosomes and selected 55 miRNAs in silico. We treated six NSCLC cell lines with the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) and determined the expressions of the 55 miRNAs. Fourteen miRNAs were decreased in the cancer cell lines and were induced after 5-aza-CdR treatment. After a detailed DNA methylation analysis, we found that mir-34b and mir-126 were silenced by DNA methylation. Mir-34b was silenced by the DNA methylation of its own promoter, whereas mir-126 was silenced by the DNA methylation of its host gene, EGFL7. A chromatin immunoprecipitation assay revealed H3K9me2 and H3K9me3 in mir-34b and EGFL7, and H3K27me3 in EGFL7. The overexpression of mir-34b and mir-126 decreased the expression of c-Met and Crk, respectively. The 5-aza-CdR treatment of lung cancer cell line resulted in increased mir-34b expression and decreased c-Met protein. We next analyzed the DNA methylation status of these miRNAs using 99 primary NSCLCs. Mir-34b and mir-126 were methylated in 41 and 7% of all the cases, respectively. The DNA methylation of mir-34b was not associated with c-Met expression determined by immunohistochemistry, but both mir-34b methylation (p = 0.007) and c-Met expression (p = 0.005) were significantly associated with lymphatic invasion in a multivariate analysis. The DNA methylation of mir-34b can be used as a biomarker for an invasive phenotype of lung cancer.     

Systemic inflammation increases cancer cell adhesion to hepatic sinusoids by neutrophil mediated mechanisms

Interactions between endothelial selectins and selectin ligands expressed on tumor cells have been implicated in the binding of circulating metastatic cancer cells to the vascular endothelium during extravasation. Moreover, there is mounting evidence that inflammatory environments can accelerate the progression of metastasis by neutrophil mediated mechanisms. In this study, a physiologically relevant in vivo model of early metastasis coupled with intravital microscopy was used to visualize the trafficking of tumor cells within the liver vasculature in real time. Using GFP‐labeled Lewis lung carcinoma subline H‐59 cells, we show here that disrupting the interactions between endothelial selectins and tumor cell selectin ligands diminished tumor cell recruitment to the liver. Furthermore, systemic inflammation induced by intravenous injection of lipopolysaccharide significantly enhanced the metastatic potential of these lung carcinoma cells by increasing their propensity to adhere to the liver sinusoidal endothelium. Confocal microscopy revealed frequent colocalization of cancer cells with neutrophils and neutrophil depletion in vivo significantly attenuated the lipopolysaccharide‐induced increase in H‐59 cell adhesion. Although direct selectin–selectin ligand interactions contributed significantly to tumor cell adhesion to sinusoidal endothelial cells, we show here that in addition, interactions between adherent neutrophils within the inflamed sinusoids and circulating tumor cells may further increase tumor cell arrest in the liver. © 2009 UICC     

Blood cell origin of circulating microRNAs: a cautionary note for cancer biomarker studies

Circulating, cell-free microRNAs (miRNAs) hold great promise as a new class of cancer biomarkers due to their surprisingly high stability in plasma, association with disease states, and ease of sensitive measurement. Yet little is known about the origin of circulating miRNAs in either healthy or sick people or what factors influence levels of circulating miRNA biomarkers. Of 79 solid tumor circulating miRNA biomarkers reported in the literature, we found that 58% (46 of 79) are highly expressed in one or more blood cell type. Plasma levels of miRNA biomarkers expressed by myeloid (e.g., miR-223, miR-197, miR-574-3p, and let-7a) and lymphoid (e.g., miR-150) blood cells tightly correlated with corresponding white blood cell counts. Plasma miRNA biomarkers expressed by red blood cells (e.g., miR-486-5p, miR-451, miR-92a, and miR-16) could not be correlated to red cell counts due to limited variation in hematocrit in the cohort studied but were significantly increased in hemolyzed specimens (20- to 30-fold plasma increase; P < 0.0000001). Finally, in a patient undergoing autologous hematopoietic cell transplantation, plasma levels of myeloid- and lymphoid-expressed miRNAs (miR-223 and miR-150, respectively) tracked closely with changes in corresponding blood counts. We present evidence that blood cells are a major contributor to circulating miRNA and that perturbations in blood cell counts and hemolysis can alter plasma miRNA biomarker levels by up to 50-fold. Given that a majority of reported circulating miRNA cancer biomarkers are highly expressed in blood cells, we suggest caution in interpretation of such results as they may reflect a blood cell-based phenomenon rather than a cancer-specific origin.     

Modulation of neutrophil granulocytes in the tumor microenvironment: Mechanisms and consequences for tumor progression

Accumulating evidence indicates a critical role of myeloid cells in the pathophysiology of human cancers. In contrast to the well-characterized tumor-associated macrophages (TAMs), the significance of granulocytes in cancer has only recently begun to emerge. Increased numbers of neutrophil granulocytes have been observed both in the peripheral blood and in the tumor tissues of patients with different types of cancer. Importantly, these studies linked neutrophils to poor clinical outcome in cancer patients which suggests that these cells might have important tumor-promoting activities. Indeed, a number of functional in vitro and in vivo studies demonstrated that tumors stimulated neutrophils to promote angiogenesis and immunosuppression, as well as migration, invasion and metastasis of the tumor cells. Therefore, it became necessary to understand the mechanisms modulating the changes in the biology and functions of neutrophils in the context of the tumor microenvironment.In this review we will discuss several functions of neutrophils that might contribute to tumor progression. Furthermore, we will address in detail the cellular and molecular mechanisms that control modulation of neutrophils in the tumor microenvironment, such as recruitment to the tumor site (chemotaxis), prolonged survival and enhanced release of protumoral mediators.     

Plasma microRNA signature is associated with risk stratification in prostate cancer patients

The aim of this study was to establish a unique expression profile of circulating cell‐free microRNAs (miRNAs) capable of differentiating between prostate cancer (PCa) patients with high‐risk and intermediate‐risk Gleason scores. MiRNA expression profiles were determined in plasma samples from 79 treatment‐naïve PCa patients, 1–2 follow‐up samples after radical prostatectomy (RP) from 51 out of the 79 PCa patients, and 33 healthy men, using a quantitative real‐time PCR‐based array containing 48 selected miRNAs. We identified 27 up‐ and 2 downregulated plasma miRNAs in PCa patients compared with healthy men. Most of the upregulated miRNA levels were also associated with increasing PSA levels and Gleason scores. Particularly, the levels of miR‐16 (p = 0.002), miR‐148a (p = 0.006) and miR‐195 (p = 0.006) significantly correlated with high‐risk Gleason scores, whereby miR‐148a (p = 0.003) was also significantly associated with increasing PSA values. The high miRNA levels before RP remained increased in the postsurgical plasma samples. Our findings show a network of deregulated plasma miRNAs. In particular, miR‐16, miR‐148a and miR‐195 are involved in the regulation of the PI3K/Akt signaling pathway. These miRNAs may be promising therapeutic targets for high‐risk PCa stratification.     

Extracellular Vesicles As miRNA Nano-Shuttles: Dual Role in Tumor Progression

Tumor-derived extracellular vesicles (EVs) have a pleiotropic role in cancer, interacting with target cells of the tumor microenvironment, such as fibroblasts, immune and endothelial cells. EVs can modulate tumor progression, angiogenic switch, metastasis, and immune escape. These vesicles are nano-shuttles containing a wide spectrum of miRNAs that contribute to tumor progression. MiRNAs contained in extracellular vesicles (EV-miRNAs) are disseminated in the extracellular space and are able to influence the expression of target genes with either tumor suppressor or oncogenic functions, depending on both parental and target cells. Metastatic cancer cells can balance their oncogenic potential by expressing miRNAs with oncogenic function, whilst exporting miRNAs with tumor suppressor roles out of the cells. Importantly, treatment of cancer cells with specific natural and chemical compounds could induce the elimination of miRNAs with oncogenic function, thereby reducing their aggressiveness. In this review, we discuss the mechanisms by which EV-miRNAs, acting as miRNAs with oncogenic or tumor suppressor functions, could contribute to cancer progression.     

外泌体miRNAs在乳腺癌肿瘤微环境中的研究进展

乳腺癌是全球女性最常发生的恶性肿瘤,患者死亡的主要原因是复发、转移和耐药性的出现。研究已经证明,外泌体介导癌细胞与肿瘤微环境之间的信息交流,外泌体携带的miRNAs通过差异表达于乳腺癌细胞,在微环境中影响癌基因表达的调控,介导乳腺癌细胞的信号通路,调节癌细胞周期进程以及重塑肿瘤相关成纤维细胞等,从而促进乳腺癌的发生、发展和转移;另外外泌体介导中和、药物外排和免疫系统抑制三种主要机制导致耐药性。未来,各种类型乳腺癌中差异表达的miRNAs有望成为临床诊断和预后的相关生物标志物,及抗肿瘤治疗的新靶点。     

miRNA调节免疫检查点在肿瘤治疗中的研究进展

肿瘤发生和发展的主要机制之一是免疫反应对癌症特异性抗原的沉默,癌症免疫监视的缺陷可能发生在肿瘤进展的任何阶段。肿瘤微环境中,对T淋巴细胞有活化或抑制作用的免疫检查点分子的异常表达可引起肿瘤细胞的免疫耐受或逃逸。靶向免疫检查点分子(如PD-1及其配体PD-L1等)已被证实是治疗多种类型癌症的新方向。微小RNA(miRNAs)在肿瘤微环境中有重要作用,研究表明一些肿瘤中miRNA的表达特异性高,其对免疫应答特别是早期调节有非常重要的作用。因此,miRNA可能成为癌症治疗中调节免疫检查点的理想分子。多种miRNA在癌细胞中异常表达,为癌症治疗提供了新的机遇,但这些miRNA的确切功能及其与免疫检查点分子间的相互作用还在探索阶段。本综述总结了近来关于miRNA作为免疫检查点分子调节剂的研究结果及其在临床实践中治疗癌症的潜在应用。