Genome-wide microRNA expression analysis of clear cell renal cell carcinoma by next generation deep sequencing

MicroRNAs (miRNAs), non-coding RNAs regulating gene expression, are frequently aberrantly expressed in human cancers. Next-generation deep sequencing technology enables genome-wide expression profiling of known miRNAs and discovery of novel miRNAs at unprecedented quantitative and qualitative accuracy. Deep sequencing was performed on 11 fresh frozen clear cell renal cell carcinoma (ccRCC) and adjacent non-tumoral renal cortex (NRC) pairs, 11 additional frozen ccRCC tissues, and 2 ccRCC cell lines (n?=?35). The 22 ccRCCs patients belonged to 3 prognostic sub-groups, i.e. those without disease recurrence, with recurrence and with metastatic disease at diagnosis. Thirty-two consecutive samples (16 ccRCC/NRC pairs) were used for stem-loop PCR validation. Novel miRNAs were predicted using 2 distinct bioinformatic pipelines. In total, 463 known miRNAs (expression frequency 1-150,000/million) were identified. We found that 100 miRNA were significantly differentially expressed between ccRCC and NRC. Differential expression of 5 miRNAs was confirmed by stem-loop PCR in the 32 ccRCC/NRC samples. With respect to RCC subgroups, 5 miRNAs discriminated between non-recurrent versus recurrent and metastatic disease, whereas 12 uniquely distinguished non-recurrent versus metastatic disease. Blocking overexpressed miR-210 or miR-27a in cell line SKCR-7 by transfecting specific antagomirs did not result in significant changes in proliferation or apoptosis. Twenty-three previously unknown miRNAs were predicted in silico. Quantitative genome-wide miRNA profiling accurately separated ccRCC from (benign) NRC. Individual differentially expressed miRNAs may potentially serve as diagnostic or prognostic markers or future therapeutic targets in ccRCC. The biological relevance of candidate novel miRNAs is unknown at present.     

肾透明细胞癌预后相关miRNAs的生物信息学分析

目的寻找可作为肾透明细胞癌（ccRCC）生物标志物的miRNA,以及ccRCC与正常组织间miRNA差异表达情况。 方法利用TCGA数据库下载ccRCC中miRNA表达数据,分析肿瘤与正常组织间差异表达miRNA。使用Kaplan-Meier曲线对患者进行生存分析,筛选出表达情况与临床预后相关的miRNA。通过生物信息学对miRNA的靶基因进行预测,然后运用FunRich软件和ClueGO对靶基因进行GO和KEGG富集分析。 结果通过TCGA数据库分析发现,ccRCC较正常组织差异表达miRNA共54个,其中上调33个,下调21个。通过生存分析发现hsa-miR-21和hsa-miR-155与患者预后相关,P≤0.05。进一步通过Perl软件在Targetscan、miRDB、miRTarBase、miRPath这四个数据库中预测miRNA靶基因并将结果取交集,共发现129个靶基因。GO和KEGG分析结果表明,这些靶基因主要与转录因子活性、信号转导以及FoxO、TNF等信号通路密切相关。 结论通过生物信息学分析发现了ccRCC与正常组织的差异表达miRNA;其中hsa-miR-21和hsa-miR-155与患者总体生存率相关,并通过调控靶基因参与相关的信号通路进而影响ccRCC的发生发展进程,提示hsa-miR-21和hsa-miR-155可能是ccRCC潜在的生物标志物。     

Deep Sequencing of RNA from Three Different Extracellular Vesicle (EV) Subtypes Released from the Human LIM1863 Colon Cancer Cell Line Uncovers Distinct Mirna-Enrichment Signatures

Secreted microRNAs (miRNAs) enclosed within extracellular vesicles (EVs) play a pivotal role in intercellular communication by regulating recipient cell gene expression and affecting target cell function. Here, we report the isolation of three distinct EV subtypes from the human colon carcinoma cell line LIM1863 – shed microvesicles (sMVs) and two exosome populations (immunoaffinity isolated A33-exosomes and EpCAM-exosomes). Deep sequencing of miRNA libraries prepared from parental LIM1863 cells/derived EV subtype RNA yielded 254 miRNA identifications, of which 63 are selectively enriched in the EVs - miR-19a/b-3p, miR-378a/c/d, and miR-577 and members of the let-7 and miR-8 families being the most prominent. Let-7a-3p*, let-7f-1-3p*, miR-451a, miR-574-5p*, miR-4454 and miR-7641 are common to all EV subtypes, and 6 miRNAs (miR-320a/b/c/d, miR-221-3p, and miR-200c-3p) discern LIM1863 exosomes from sMVs; miR-98-5p was selectively represented only in sMVs. Notably, A33-Exos contained the largest number (32) of exclusively-enriched miRNAs; 14 of these miRNAs have not been reported in the context of CRC tissue/biofluid analyses and warrant further examination as potential diagnostic markers of CRC. Surprisingly, miRNA passenger strands (star miRNAs) for miR-3613-3p*, -362-3p*, -625-3p*, -6842-3p* were the dominant strand in A33-Exos, the converse to that observed in parental cells. This finding suggests miRNA biogenesis may be interlinked with endosomal/exosomal processing.     

MicroRNA-153-5p promotes the proliferation and metastasis of renal cell carcinoma via direct targeting of AGO1

MicroRNAs (miRNAs) have been demonstrated to affect the biological processes of cancers and showed great potential for prognostic biomarkers. In this study, we screened differentially expressed miRNAs in ccRCC based on three dimensions of metastasis, prognosis, and differential expression compared to normal tissue using bioinformatics algorithms. MiR-153-5p was identified as a candidate miRNA to promote ccRCC occurrence and progression. Clinically, we found that miR-153-5p was significantly upregulated and related to unfavorable clinical features in ccRCC. Besides, miR-153-5p served as an independent prognostic biomarker. Functionally, miR-153-5p depletion remarkably inhibited the proliferation and metastasis of ccRCC via the phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Furthermore, AGO1 was proved to be a direct target of miR-153-5p. AGO1 is associated with favorable clinical features and exhibited independent prognostic value in ccRCC. Besides, we observed that AGO1 knockdown significantly promoted tumor proliferation and metastasis. Downregulation of AGO1 partly abolished the oncogenic effects of miR-153-5p knockdown. Furthermore, miR-153-5p combined with AGO1 showed more robust prognostic significance in ccRCC. In conclusion, we found that the newly identified miR-153-5p/AGO1 axis was responsible for tumor occurrence and progression via PI3K/Akt signaling, which may therefore provide promising therapeutic targets and prognostic biomarkers for patients with ccRCC.Subject terms: Cancer screening, miRNAs     

miR-29a/b Enhances Cell Migration and Invasion in Nasopharyngeal Carcinoma Progression by Regulating SPARC and COL3A1 Gene Expression

Nasopharyngeal carcinoma (NPC) is a malignant tumor associated with a genetic predisposition, Epstein-Barr virus infection and chromosomal abnormalities. Recently, several miRNAs have been shown to target specific mRNAs to regulate NPC development and progression. However, the involvement of miRNAs in processes leading to NPC migration and invasion remains to be elucidated. We predicted that miR-29a/b are associated with dysregulated genes controlling NPC through an integrated interaction network of miRNAs and genes. miR-29a/b over-expression in NPC cell lines had no significant effect on proliferation, whereas miR-29b mildly increased the percentage of cells in the G1 phase with a concomitant decrease in the percentage of cells in S phase. Furthermore, we demonstrated that miR-29a/b might be responsible for increasing S18 cell migration and invasion, and only COL3A1 was identified as a direct target of miR-29b despite the fact that both SPARC and COL3A1 were inhibited by miR-29a/b over-expression. Meanwhile, SPARC proteins were increased in metastatic NPC tissue and are involved in NPC progression. Unexpectedly, we identified that miRNA-29b expression was elevated in the serum of NPC patients with a high risk of metastasis. The 5-year actuarial overall survival rates in NPC patients with high serum miR-29b expression was significantly shorter than those with low serum miR-29b expression; therefore, serum miR-29b expression could be a promising prognostic marker.     

Serum exosomal miR-210 as a potential biomarker for clear cell renal cell carcinoma

Exosomal microRNAs (miRNAs) are suggested to reflect molecular changes occurring in their cells of origin and are potential indicators in the early detection of cancers. This study aimed to determine whether certain exosomal miRNAs from tumor tissue can be used as noninvasive biomarkers for clear cell renal cell carcinoma (ccRCC). Based on ccRCC miRNA expression profiles and the literature, we selected six miRNAs (miR-210, miR-224, miR-452, miR-155, miR-21, and miR-34a) and analyzed their expression in tissues, sera, and serum exosomes through quantitative real-time polymerase chain reaction in hypoxia-induced (with CoCl₂) renal cell lines. miR-210, miR-224, miR-452, miR-155, and miR-21 were upregulated in tumor tissues compared with normal tissues. Serum miR-210 and miR-155 levels were higher in patients with ccRCC than in healthy controls (HCs). Furthermore, only exosomal miR-210 was significantly upregulated in patients with ccRCC than in HCs. Moreover, receiver operating characteristic (ROC) curve analysis revealed an area under the ROC curve of 0.8779 (95% confidence interval, 0.7987-0.9571) and a sensitivity and specificity of 82.5% and 80.0%, respectively. Moreover, exosomal miR-210 was upregulated at an advanced stage, and Fuhrman grade and metastasis decreased significantly one month after surgery. Acute hypoxia exposure activates miR-210 and release of exosomes with upregulated miR-210 in both normal and tumor RCC cell lines and interferes with vacuole membrane protein 1 mRNA expression, especially in the metastatic ccRCC cell line. In conclusion, Serum exosomal miR-210 originating from tumor tissue has potential as a novel noninvasive biomarker for the detection and prognosis of ccRCC.     

Genome-wide screen for aberrantly expressed miRNAs reveals miRNA profile signature in breast cancer

Dysregulation in the expression of miRNAs contributes to the occurrence and development of many human cancers. We herein attempted to obtain the potential association between miRNA expression profile and breast cancer by applying high-throughput sequencing technology. Small RNAs from seven paired tumor and adjacent normal tissue samples were sequenced. To determine the miRNA expression profiles in tissues and sera, another five equally pooled serum samples from 20 patients and 30 normal women were sequenced. Despite a similar number in abundantly expressed miRNAs across samples, we detected varying miRNA expression profiles. Some miRNAs showed inconsistent or opposite dysregulation trends across different tumor tissues, including some abundantly expressed miRNA gene clusters and gene families. Wilcoxon sign-rank test for paired samples analysis revealed that abnormal miRNAs showed a higher level of variation across the seven tumor samples. We also completely surveyed abnormal miRNAs expressed in tumor and serum tissues in the mixed datasets based on the relative expression levels. Most of these miRNAs were significantly down-regulated in tumor samples, but nine abnormal miRNAs (miR-18a, 19a, 20a, 30a, 103b, 126, 126*, 192, 1287) were consistently expressed in tumor tissues and serum samples. Based on experimentally validated target mRNAs, functional enrichment analysis indicated that these abnormal miRNAs and miRNA groups (miRNA gene clusters and gene families) have important roles in multiple biological processes. Dynamic miRNA expression profiles, various abnormal miRNA profiles and complexity of the miRNA regulatory network reveal that the miRNA expression profile is a potential biomarker for classifying or detecting human disease.     

MicroRNA Expression Profiling in Clear Cell Renal Cell Carcinoma: Identification and Functional Validation of Key miRNAs

ObjectiveThis study aims to profile dysregulated microRNA (miRNA) expression in clear cell renal cell carcinoma (ccRCC) and to identify key regulatory miRNAs in ccRCC.ConclusionsThis study identified 11 commonly dysregulated miRNAs in ccRCC, three of which (miR-199a-5p, miR-22 and miR-429) may represent key miRNAs involved in the pathogenesis of ccRCC. Further studies suggested that miR-199a-5p plays an important role in inhibition of cell invasion of ccRCC cells by suppressing expression of TGFBR1 and JunB.     

Identification of a 4-microRNA signature for clear cell renal cell carcinoma metastasis and prognosis

Renal cell carcinoma (RCC) metastasis portends a poor prognosis and cannot be reliably predicted. Early determination of the metastatic potential of RCC may help guide proper treatment. We analyzed microRNA (miRNA) expression in clear cell RCC (ccRCC) for the purpose of developing a miRNA expression signature to determine the risk of metastasis and prognosis. We used the microarray technology to profile miRNA expression of 78 benign kidney and ccRCC samples. Using 28 localized and metastatic ccRCC specimens as the training cohort and the univariate logistic regression and risk score methods, we developed a miRNA signature model in which the expression levels of miR-10b, miR-139-5p, miR-130b and miR-199b-5p were used to determine the status of ccRCC metastasis. We validated the signature in an independent 40-sample testing cohort of different stages of primary ccRCCs using the microarray data. Within the testing cohort patients who had at least 5 years follow-up if no metastasis developed, the signature showed a high sensitivity and specificity. The risk status was proven to be associated with the cancer-specific survival. Using the most stably expressed miRNA among benign and tumorous kidney tissue as the internal reference for normalization, we successfully converted his signature to be a quantitative PCR (qPCR)-based assay, which showed the same high sensitivity and specificity. The 4-miRNA is associated with ccRCC metastasis and prognosis. The signature is ready for and will benefit from further large clinical cohort validation and has the potential for clinical application.     

Altered miRNA expression in canine retinas during normal development and in models of retinal degeneration

Background

Although more than 246 loci/genes are associated with inherited retinal diseases, the mechanistic events that link genetic mutations to photoreceptor cell death are poorly understood. miRNAs play a relevant role during retinal development and disease. Thus, as a first step in characterizing miRNA involvement during disease expression and progression, we examined miRNAs expression changes in normal retinal development and in four canine models of retinal degenerative disease.

Results

The initial microarray analysis showed that 50 miRNAs were differentially expressed (DE) early (3 vs. 7 wks) in normal retina development, while only 2 were DE between 7 and 16 wks, when the dog retina is fully mature. miRNA expression profiles were similar between dogs affected with xlpra2, an early-onset retinal disease caused by a microdeletion in RPGRORF15, and normal dogs early in development (3 wks) and at the peak of photoreceptor death (7 wks), when only 2 miRNAs were DE. However, the expression varied much more markedly during the chronic cell death stage at 16 wks (118 up-/55 down-regulated miRNAs). Functional analyses indicated that these DE miRNAs are associated with an increased inflammatory response, as well as cell death/survival. qRT-PCR of selected apoptosis-related miRNAs (“apoptomirs”) confirmed the microarray results in xlpra2, and extended the analysis to the early-onset retinal diseases rcd1 (PDE6B-mutation) and erd (STK38L-mutation), as well as the slowly progressing prcd (PRCD-mutation). The results showed up-regulation of anti-apoptotic (miR-9, -19a, -20, -21, -29b, -146a, -155, -221) and down-regulation of pro-apoptotic (miR-122, -129) apoptomirs in the early-onset diseases and, with few exceptions, also in the prcd-mutants.

Conclusions

Our results suggest that apoptomirs might be expressed by diseased retinas in an attempt to counteract the degenerative process. The pattern of expression in diseased retinas mirrored the morphology and cell death kinetics previously described for these diseases. This study suggests that common miRNA regulatory mechanisms may be involved in retinal degeneration processes and provides attractive opportunities for the development of novel miRNA-based therapies to delay the progression of the degenerative process.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-172) contains supplementary material, which is available to authorized users.     

Identification of Novel AR-Targeted MicroRNAs Mediating Androgen Signalling through Critical Pathways to Regulate Cell Viability in Prostate Cancer

MicroRNAs (miRNAs) have been recognized as significantly involved in prostate cancer (PCa). Since androgen receptor (AR) plays a central role in PCa carcinogenesis and progression, it is imperative to systematically elucidate the causal association between AR and miRNAs, focusing on the molecular mechanisms by which miRNAs mediate AR signalling. In this study, we performed a series of time-course microarrays to observe the dynamic genome-wide expressions of mRNAs and miRNAs in parallel in hormone-sensitive prostate cancer LNCaP cells stimulated by androgen. Accordingly, we introduced Response Score to identify AR target miRNAs, as well as Modulation Score to identify miRNA target mRNAs. Based on theoretical identification and experimental validation, novel mechanisms addressing cell viability in PCa were unravelled for 3 miRNAs newly recognized as AR targets. (1) miR-19a is directly up-regulated by AR, and represses SUZ12, RAB13, SC4MOL, PSAP and ABCA1, respectively. (2) miR-27a is directly up-regulated by AR, and represses ABCA1 and PDS5B. (3) miR-133b is directly up-regulated by AR, and represses CDC2L5, PTPRK, RB1CC1, and CPNE3, respectively. Moreover, we found miR-133b is essential to PCa cell survival. Our study gives certain clues on miRNAs mediated AR signalling to cell viability by influencing critical pathways, especially by breaking through androgen’s growth restriction effect on normal prostate tissue.     

Role of Deregulated microRNAs in Breast Cancer Progression Using FFPE Tissue

MicroRNAs (miRNAs) contribute to cancer initiation and progression by silencing the expression of their target genes, causing either mRNA molecule degradation or translational inhibition. Intraductal epithelial proliferations of the breast are histologically and clinically classified into normal, atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). To better understand the progression of ductal breast cancer development, we attempt to identify deregulated miRNAs in this process using Formalin-Fixed, Paraffin-Embedded (FFPE) tissues from breast cancer patients. Following tissue microdissection, we obtained 8 normal, 4 ADH, 6 DCIS and 7 IDC samples, which were subject to RNA isolation and miRNA expression profiling analysis. We found that miR-21, miR-200b/c, miR-141, and miR-183 were consistently up-regulated in ADH, DCIS and IDC compared to normal, while miR-557 was uniquely down-regulated in DCIS. Interestingly, the most significant miRNA deregulations occurred during the transition from normal to ADH. However, the data did not reveal a step-wise miRNA alteration among discrete steps along tumor progression, which is in accordance with previous reports of mRNA profiling of different stages of breast cancer. Furthermore, the expression of MSH2 and SMAD7, two important molecules involving TGF-β pathway, was restored following miR-21 knockdown in both MCF-7 and Hs578T breast cancer cells. In this study, we have not only identified a number of potential candidate miRNAs for breast cancer, but also found that deregulation of miRNA expression during breast tumorigenesis might be an early event since it occurred significantly during normal to ADH transition. Consequently, we have demonstrated the feasibility of miRNA expression profiling analysis using archived FFPE tissues, typically with rich clinical information, as a means of miRNA biomarker discovery.     

Ultra-deep sequencing reveals the microRNA expression pattern of the human stomach

Background

While microRNAs (miRNAs) play important roles in tissue differentiation and in maintaining basal physiology, little is known about the miRNA expression levels in stomach tissue. Alterations in the miRNA profile can lead to cell deregulation, which can induce neoplasia.

Methodology/Principal Findings

A small RNA library of stomach tissue was sequenced using high-throughput SOLiD sequencing technology. We obtained 261,274 quality reads with perfect matches to the human miRnome, and 42% of known miRNAs were identified. Digital Gene Expression profiling (DGE) was performed based on read abundance and showed that fifteen miRNAs were highly expressed in gastric tissue. Subsequently, the expression of these miRNAs was validated in 10 healthy individuals by RT-PCR showed a significant correlation of 83.97% (P<0.05). Six miRNAs showed a low variable pattern of expression (miR-29b, miR-29c, miR-19b, miR-31, miR-148a, miR-451) and could be considered part of the expression pattern of the healthy gastric tissue.

Conclusions/Significance

This study aimed to validate normal miRNA profiles of human gastric tissue to establish a reference profile for healthy individuals. Determining the regulatory processes acting in the stomach will be important in the fight against gastric cancer, which is the second-leading cause of cancer mortality worldwide.     

胃癌中显著下调的miR-433的作用靶点研究

目的为了筛选胃癌中miRNAs的表达标记,验证胃癌相关miRNAs的作用靶点,建立一种新的诊断和治疗胃癌的方法。方法运用基因芯片技术检测3个正常胃组织标本,24个胃癌组织标本,胃癌细胞SGC7901和正常胃黏膜细胞GES-1中328个miRNAs的表达情况。用以上方法检测出在胃癌组织和SGC7901中,miR-433的表达水平显著下调。为了确保结果的准确性,采用实时荧光定量PCR对其进行验证。并用基因克隆和Western印迹方法分析miR-433的作用靶点。结果共有26个miRNAs在胃癌标本（包括24个胃癌组织和SGC7901）中异常表达。其中19个miRNAs下调,7个miRNAs上调。实时荧光定量PCR检测出miR-433在胃癌标本中的表达水平显著下调,该结果和基因芯片检测结果一致。另外,在本实验中发现miR-433与Grb2（growth factor receptor—bound protein 2）的表达呈负相关。结论胃癌相关miRNAs已进行了初步筛选。其中,miR-433可能是胃癌中的标记性miRNAs之一,Grb2是其作用靶点。这为建立新的以miRNAs为基础的诊断和治疗胃癌的方法提供了相关信息。     

Hippo Signaling Mediates Proliferation,Invasiveness, and Metastatic Potential of Clear Cell Renal Cell Carcinoma

Recent work has identified dysfunctional Hippo signaling to be involved in maintenance and progression of various human cancers, although data on clear cell renal cell carcinoma (ccRCC) have been limited. Here, we provide evidence implicating aberrant Hippo signaling in ccRCC proliferation, invasiveness, and metastatic potential. Nuclear overexpression of the Hippo target Yes-associated protein (YAP) was found in a subset of patients with ccRCC. Immunostaining was particularly prominent at the tumor margins and highlighted neoplastic cells invading the tumor-adjacent stroma. Short hairpin RNA-mediated knockdown of YAP significantly inhibited proliferation, migration, and anchorage-independent growth of ccRCC cells in soft agar and led to significantly reduced murine xenograft growth. Microarray analysis of YAP knockdown versus mock-transduced ccRCC cells revealed down-regulation of endothelin 1, endothelin 2, cysteine-rich, angiogenic inducer, 61 (CYR61), and c-Myc in ccRCC cells as well as up-regulation of the cell adhesion molecule cadherin 6. Signaling pathway impact analysis revealed activation of the p53 signaling and cell cycle pathways as well as inhibition of mitogen-activated protein kinase signaling on YAP down-regulation. Our data suggest CYR61 and c-Myc as well as signaling through the endothelin axis as bona fide downstream effectors of YAP and establish aberrant Hippo signaling as a potential therapeutic target in ccRCC.