Characterization Of microRNA Expression Research Articles

Characterization of microRNA expression in B cells derived from Japanese black cattle naturally infected with bovine leukemia virus by deep sequencing.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL), a malignant B cell lymphoma. However, the mechanisms of BLV-associated lymphomagenesis remain poorly understood. Here, after deep sequencing, we performed comparative analyses of B cell microRNAs (miRNAs) in cattle infected with BLV and those without BLV. In BLV-infected cattle, BLV-derived miRNAs (blv-miRNAs) accounted for 38% of all miRNAs in B cells. Four of these blv-miRNAs (blv-miR-B1-5p, blv-miR-B2-5p, blv-miR-B4-3p, and blv-miR-B5-5p) had highly significant positive correlations with BLV proviral load (PVL). The read counts of 90 host-derived miRNAs (bta-miRNAs) were significantly down-regulated in BLV-infected cattle compared to those in uninfected cattle. Only bta-miR-375 had a positive correlation with PVL in BLV-infected cattle and was highly expressed in the B cell lymphoma tissue of EBL cattle. There were a few bta-miRNAs that correlated with BLV tax/rex gene expression; however, BLV AS1 expression had a significant negative correlation with many of the down-regulated bta-miRNAs that are important for tumor development and/or tumor suppression. These results suggest that BLV promotes lymphomagenesis via AS1 and blv-miRNAs, rather than tax/rex, by down-regulating the expression of bta-miRNAs that have a tumor-suppressing function, and this downregulation is linked to increased PVL.

Combined MicroRNA In Situ Hybridization and Immunohistochemical Detection of Protein Markers.

MicroRNAs are short (18-23 nucleotides) noncoding RNAs involved in posttranscriptional regulation of gene expression through their specific binding to the 3'UTR of mRNAs. MicroRNAs can be detected in tissues using specific locked nucleic acid (LNA)-enhanced probes. The characterization of microRNA expression in tissues by in situ detection is often crucial following a microRNA biomarker discovery phase in order to validate the candidate microRNA biomarker and allow better interpretation of its molecular functions and derived cellular interactions. The in situ hybridization data provides information about contextual distribution and cellular origin of the microRNA. By combining microRNA in situ hybridization with immunohistochemical staining of protein markers, it is possible to precisely characterize the microRNA-expressing cells and to identify the potential microRNA targets. This combined technology can also help to monitor changes in the level of potential microRNA targets in a therapeutic setting. In this chapter, we present a fluorescence-based detection method that allows the combination of microRNA in situ hybridization with immunohistochemical staining of one and, in this updated version of the paper, two protein markers detected with primary antibodies raised in the same host species.

Characterization of microRNA expression in primary human colon adenocarcinoma cells (SW480) and their lymph node metastatic derivatives (SW620).

Background and objectiveMetastasis is the major cause of cancer-related deaths in patients with colon cancer, however, the exact molecular mechanism is unclear. MicroRNAs (miRNAs) play an important role in the pathogenesis and progression of cancer. Therefore, in this study, we aimed to identify differentially expressed miRNAs in two colon carcinoma cell lines: SW480, derived from primary colon carcinoma and SW620, derived from lymph node metastasis, which were obtained from the same patient.Materials and methodsThree independent samples of cancer cells were collected from SW480 and SW620 cells, respectively. An miRNA microarray platform, miRCURY LNA™ microRNA array with 1,223 probes containing 3,000 capture probes, was used to determine the miRNA expression profiles of these two cell lines. Differentially expressed miRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR).ResultsThe raw data were submitted to the Gene Expression Omnibus database (GSE72412). Thirteen miRNAs were differentially expressed between SW480 and SW620 cells, of which, seven miRNAs (hsa-miR-920, hsa-miR-636, hsa-miR-766-3p, hsa-miR-545-5p, hsa-miR-195-3p, hsa-miR-125a-3p, and hsa-miR-196b-3p) were found to be upregulated and six miRNAs (hsa-miR-3613-3p, hsa-miR-29b-3p, hsa-miR-1297, hsa-miR-141-5p, hsa-miR-200c-3p, and hsa-miR-141-3p) were found to be downregulated. Target analysis of the predicted miRNAs showed that these genes were primarily involved in protein binding, cell adhesion, and cancer metastasis. Furthermore, qRT-PCR validated the results of miRNA microarray.ConclusionThis is the first systematic analysis of the differences of miRNAs between SW480 and SW620 cells. The results provide useful information to explore potential biomarkers of miRNAs for predicting colon cancer metastasis.

Abstract 1961: Characterization of MicroRNA Expression during the Development of Resistance to a mTOR-Inhibitor in renal cell carcinoma

Abstract Introduction: Second line therapy of metastatic renal cell carcinoma (RCC) using the mTOR inhibitor RAD001 (everolimus) is hampered by the appearance of drug resistance. The cellular mechanisms leading to drug resistance are not yet clarified. Although the influence of miRNAs is proven for other systemic treatments, their impact on mTOR inhibitors is not investigated. Therefore, we analyzed miRNA alterations in correlation to development of resistance to RAD001 in RCC cell culture models. Material and Methods: The RCC cell line 786-O was treated with 1μM RAD001 for up to 20 passages after developing sunitinib resistance. TotalRNA was isolated at defined intervals. Resistance-development as well as drug efficacy during the treatment was monitored by proliferation assays. MiRNA Microarrays (Agilent Technologies) were performed using RAD001 sensitive and RAD001 resistant cells to identify differently expressed miRNAs. Results were validated by qRT PCR. Functional analyses are performed after transfection of RAD001 sensitive cells with miRNA candidates. Results: The Microarray analyses revealed a miRNA signature including 9 differently expressed miRNAs in RAD001 resistant cells compared to sensitive cells. QRT-PCR results showed that especially miR-302e was strongly up-regulated by the factor of four. At present, functional analyses of miR-302e are ongoing. Conclusion: To our knowledge this is the first study analyzing miRNA alterations during the development of resistance to mTOR inhibitors. We have shown that miR-302e expression is associated with RAD001 resistance in RCC cells. Since miR-302e is a known tumor suppressor modulating radio sensitivity and drug sensitivity in several tumors we are proving now if miR-302e can regulate the mTOR inhibitor resistance of RCC. Citation Format: Joana Heinzelmann, Franziska Bauer, Gerhard Unteregger, Darja Schendel, Anne Weiland, Michael Stöckle, Kerstin Junker. Characterization of MicroRNA Expression during the Development of Resistance to a mTOR-Inhibitor in renal cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1961.

Identification and characterization of microRNA expression in Ginkgo biloba L. leaves

Ginkgo biloba L., an extant primitive gymnosperm species, occupies an important position in the evolution of the plant kingdom. Furthermore, its leaves contain a large number of active, medicinally valuable compounds; therefore, it is also an important tree economically. MicroRNAs (miRNAs) are important regulators of gene expression implicated in controlling plant development and environmental interaction. Extensive studies of miRNAs have been performed in angiosperms, but no reports have focused on small RNAs in the basal gymnosperm G. biloba. Here, we used Illumina next-generation sequencing HiSeq™ 2000 platform to identify and quantitatively profile small RNAs in G. biloba leaves. A total of 13,414,548 and 12,293,511 reads, containing 202 and 201 known miRNAs, belonging to 82 and 78 families, were sequenced from two small RNA libraries constructed from female and male leaves, respectively. We also identified 174/174 novel miRNAs in the female/male leaves of G. biloba. Relative expression levels of the miRNAs and the precursor sequences of several of the known and novel miRNAs were further validated by subcloning and quantitative real-time PCR (qRT-PCR). Target predictions of the 380 and 340 known miRNAs, from female and male leaves, resulted in 2116 and 2002 target genes, while those of the 155 and 158 novel miRNAs resulted in 596 and 793 target genes, respectively. KEGG annotation analysis revealed that high-ranked miRNA-target genes are implicated in plant-pathogen interactions, plant hormone signal transduction, and flavonoid biosynthesis. Taken together, we identified a large number of miRNAs in mature female and male G. biloba leaves for the first time. We ascertained that the regulatory networks of the miRNAs are involved in many different primary biological processes based on potential target designations.

Characterization of microRNA expression in serous ovarian carcinoma.

Serous ovarian cancer is a major gynecologic malignancy with a poor 5‑year survival rate. However, little is known regarding the behavior and genetics of ovarian tumorigenesis. MicroRNAs (miRNAs) have been shown to be dysregulated in ovarian carcinomas. To assess the miRNA expression profiles in serous ovarian cancer, we defined the patterns of miRNA expression in 100 formalin‑fixed, paraffin‑embedded ovarian cancer tissues blocks as well as 50 corresponding normal oviduct tissues using miRNA microarray. MiRNA expression profiling showed that 63 miRNAs were downregulated and 43 miRNAs were upregulated in serous ovarian cancer tissues compared with control tissues. The expression of five dysregulated miRNAs was validated using quantitative polymerase chain reaction (RT‑qPCR). GO term and pathway analysis revealed that the biological process of the cell cycle was significantly enriched and the MAPK signaling pathway was highly involved in the progression of ovarian cancer. The results suggested that the aberrant expression of miRNAs is involved in ovarian carcinogenesis and thus these miRNAs may function as diagnostic and prognostic biomarkers.

Characterization of microRNA expression in bone cells

Characterization of microRNA expression in bone cells

Characterization of microRNA expression in bovine adipose tissues: a potential regulatory mechanism of subcutaneous adipose tissue development

BackgroundMicroRNAs (miRNAs), a family of small non-coding RNA molecules, appear to regulate animal lipid metabolism and preadipocyte conversion to form lipid-assimilating adipocytes (i.e. adipogenesis). However, no miRNA to date has been reported to modulate adipogenesis and lipid deposition in beef cattle.ResultsThe expression patterns of 89 miRNAs including four bovine specific miRNAs in subcutaneous adipose tissues from three groups of crossbred steers differing in backfat thickness were compared using qRT-PCR analysis. Eighty-six miRNAs were detectable in all samples, with 42 miRNAs differing among crossbreds (P < 0.05) and 15 miRNAs differentially expressed between tissues with high and low backfat thickness (P < 0.05). The expression levels of 18 miRNAs were correlated with backfat thickness (P < 0.05). The miRNA most differentially expressed and the most strongly associated with backfat thickness was miR-378, with a 1.99-fold increase in high backfat thickness tissues (r = 0.72).ConclusionsMiRNA expression patterns differed significantly in response to host genetic components. Approximately 20% of the miRNAs in this study were identified as being correlated with backfat thickness. This result suggests that miRNAs may play a regulatory role in white adipose tissue development in beef animals.

Abstract LB-356: Characterization of microRNA expression of clear cell renal cell carcinoma by whole genome small RNA deep sequencing using human tissue cohort

Abstract Introduction: MicroRNA (miRNA) is a group of small non-coding regulating RNA and has shown altered expression in cancer. With great advantages in test specificity and capability to detect novel (unknown) miRNA and other small RNA targets, the next-generation deep sequencing technology is replacing microarray platforms, at least in the discovery phase. Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney malignancies and one of the leading causes of death. Characterizing miRNA expression of ccRCC will enhance the understanding of its tumorigenesis and progression and will, therefore, lead to the development of cancer-specific molecular therapy. In this study, we analyzed miRNA expression using a human ccRCC cohort by whole genome small RNA deep sequencing. We characterized the miRNA expression in association with CCRCC and further validated the altered miRNA expression in a larger clinical cohort by RT-PCR. A group of tumor associated novel miRNAs have been discovered. Design: (1) Deep sequencing of whole genome small RNA (17-52 nucleotides in size) in a 6-sample frozen CCRCC cohort (3 benign and 3 ccRCC samples) using Illumina system (Solexa) was performed. (2) The miRNA expression of benign kidney and ccRCC samples was quantitatively compared. (3) 8 miRNAs, which were randomly selected from the list of top 20 aberrantly expressed miRNAs detected by deep sequencing, were tested using ABI RT-PCR technology in a 38-sample ccRCC cohort (9 benign kidney and 29 different stage ccRCC samples) for validation. (4) Analysis of unknown small RNAs was performed to find tumor associated novel miRNAs. Results: (1) Using a deep sequencing technology, we detected miRNAs and other small RNAs in frozen human samples of ccRCC cohort and successfully profiled miRNA expression in association with tumor. (2) We validated the altered expression of 8 selected miRNA targets in the 38-sample clinical ccRCC cohort using an RT-PCR method. (3) We discovered a group of novel miRNAs associated with ccRCC. Conclusions: We performed whole genome small RNA deep sequencing of human ccRCC tissue with further RT-PCR validation in a clinical sample cohort. We have characterized miRNA expression and discovered a group of novel miRNAs in association with ccRCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-356.