miR-215 Overexpression Research Articles

Role of acidosis-sensitive microRNAs in gene expression and functional parameters of tumors in vitro and in vivo.

Background: The acidic extracellular environment of tumors has been shown to affect the malignant progression of tumor cells by modulating proliferation, cell death or metastatic potential. The aim of the study was to analyze whether acidosis-dependent miRNAs play a role in the signaling cascade from low pH through changes in gene expression to functional properties of tumors in vitro and in vivo.Methods: In two experimental tumor lines the expression of 13 genes was tested under acidic conditions in combination with overexpression or downregulation of 4 pH-sensitive miRNAs (miR-7, 183, 203, 215). Additionally, the impact on proliferation, cell cycle distribution, apoptosis, necrosis, migration and cell adhesion were measured.Results: Most of the genes showed a pH-dependent expression, but only a few of them were additionally regulated by miRNAs in vitro (Brip1, Clspn, Rif1) or in vivo (Fstl, Tlr5, Txnip). Especially miR-215 overexpression was able to counteract the acidosis effect in some genes. The impact on proliferation was cell line-dependent and most pronounced with overexpression of miR-183 and miR-203, whereas apoptosis and necrosis were pH-dependent but not influenced by miRNAs. The tumor growth was markedly regulated by miR-183 and miR-7. In addition, acidosis had a strong effect on cell adhesion, which could be modulated by miR-7, miR-203 and miR-215.Conclusions: The results indicate that the acidosis effect on gene expression and functional properties of tumor cells could be mediated by pH-dependent miRNAs. Many effects were cell line dependent and therefore do not reflect universal intracellular signaling cascades. However, the role of miRNAs in the adaptation to an acidic environment may open new therapeutic strategies.

MiR-215-5p Reduces Liver Metastasis in an Experimental Model of Colorectal Cancer through Regulation of ECM-Receptor Interactions and Focal Adhesion.

Background: Growing evidence suggests that miR-215-5p is a tumor suppressor in colorectal cancer (CRC); however, its role in metastasis remains unclear. This study evaluates the effects of miR-215 overexpression on the metastatic potential of CRC. Methods: CRC cell lines were stably transfected with miR-215-5p and used for in vitro and in vivo functional analyses. Next-generation sequencing and RT-qPCR were performed to study changes on the mRNA level. Results: Overexpression of miR-215-5p significantly reduced the clonogenic potential, migration, and invasiveness of CRC cells in vitro and tumor weight and volume, and liver metastasis in vivo. Transcriptome analysis revealed mRNAs regulated by miR-215-5p and RT-qPCR confirmed results for seven selected genes. Significantly elevated levels of CTNNBIP1 were also observed in patients' primary tumors and liver metastases compared to adjacent tissues, indicating its direct regulation by miR-215-5p. Gene Ontology and KEGG pathway analysis identified cellular processes and pathways associated with miR-215-5p deregulation. Conclusions: MiR-215-5p suppresses the metastatic potential of CRC cells through the regulation of divergent molecular pathways, including extracellular-matrix-receptor interaction and focal adhesion. Although the specific targets of miR-215-5p contributing to the formation of distant metastases must be further elucidated, this miRNA could serve as a promising target for CRC patients' future therapeutic strategies.

CircHIPK3 regulates melanoma cell behaviors by binding with miR-215–5p to upregulate YY1

ObjectTo investigate the role of circHIPK3 in melanoma. MethodsBioinformatics analysis and experiments including RT-qPCR, Pearson's correlation analysis, luciferase reporter, Western blot, and RIP assays were applied to explore the function and mechanism of circHIPK3 in melanoma. ResultsCircHIPK3 expression was strikingly upregulated while miR-215–5p was downregulated in melanoma tissues and cell lines. Pearson's correlation analysis unveiled circHIPK3 expression was positively correlated with Ki-67 (a marker of proliferation), which implied that circHIPK3 may play a vital role in the progression of melanoma. In mechanism, luciferase reporter and RIP assays validated that circHIPK3 was able to bind with miR-215–5p. Moreover, we confirmed that overexpression of circHIPK3 could facilitate cell proliferation and depress cell apoptosis in melanoma while overexpression of miR-215–5p exerted opposite effects. Besides, our findings indicated that miR-215–5p overexpression significantly reversed the circHIPK3 overexpressing-mediated promotive effect on cell proliferation and inhibitory effect on cell apoptosis. Furthermore, we found that miR-215–5p could directly target YY1. Upregulation of YY1 could notably offset the inhibitory effect of circHIPK3 downregulation on cell proliferation and the promotive effect on cell apoptosis. ConclusionOur study corroborated that circHIPK3 regulated melanoma cell behaviors via the miR-215–5p/YY1 axis, which might provide a novel insight for the treatment of melanoma patients.

MiR-215 Inhibits Colorectal Cancer Cell Migration and Invasion via Targeting Stearoyl-CoA Desaturase.

Background This study was aimed at exploring the effects of miR-215 and its target gene stearoyl-CoA desaturase (SCD) on colorectal cancer (CRC) cell migration and invasion. Methods Here, we analyzed the relationship between miR-215 and SCD, as well as the regulation of miR-215 on CRC cells. We constructed wild-type and mutant plasmids of SCD to identify whether SCD was a target gene of miR-215 by using a luciferase reporter assay. The expression of miR-215 and SCD was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. MTT, wound healing, and Transwell assays were applied to determine the effect of miR-215 on CRC cell proliferation, migration, and invasion. Results It was found that miR-215 expression was significantly decreased in CRC tissue while SCD was highly expressed compared with those in adjacent normal tissue. The luciferase reporter assay indicated that SCD was a direct target gene of miR-215. Functional analysis revealed that miR-215 overexpression significantly inhibited CRC cell proliferation, migration, and invasion in vitro. In addition, the result of rescue experiments showed that overexpression of SCD could promote the proliferation, migration, and invasion of CRC cells, and the carcinogenic effect of SCD could be inhibited by miR-215. Conclusions Taken together, our findings suggested that miR-215 could inhibit CRC cell migration and invasion via targeting SCD. The result could eventually contribute to the treatment for CRC.

Hepatic miR‑215 target Rictor and modulation of hepatic insulin signalling in rats.

Increasing evidence has suggested that hepatic lipid accumulation is associated with hepatic insulin resistance; however, the underlying mechanism is yet to be determined. It was demonstrated that the levels of microRNA-215 (miR-215) expression in the liver of rats fed a high-fat diet were significantly increased compared with rats on a control diet. Additionally, it was revealed via luciferase assays and western blotting that miR-215 targets rapamycin-insensitive companion of mammalian target of rapamycin (Rictor), an important protein in the hepatic insulin signalling pathway. Following overexpression of miR-215 in the H4IIE rat hepatocarcinoma cell line, it was reported that the intracellular insulin signalling pathway was inhibited; conversely, inhibition of miR-215 expression induced this pathway. Furthermore, it was demonstrated via reverse transcription-quantitative polymerase chain reaction analysis that free fatty acids promoted the expression of miR-215. The present study provided a novel mechanistic insight into the association between nonalcoholic fatty liver and hepatic insulin resistance.

Tumor suppressor miR-215 counteracts hypoxia-induced colon cancer stem cell activity

Cancer stem cells, also known as tumor-initiating cells (TICs), are a population of aggressive and self-renewing cells that are responsible for the initiation and progression of many cancers, including colorectal carcinoma. Intratumoral hypoxia, i.e. reduced oxygen supply following uncontrolled proliferation of cancer cells, is thought to support TIC activity by inducing specific hypoxia-responsive mechanisms that are not yet entirely understood. Using previously established and fully characterized patient-derived TIC cultures, we could observe increased sphere and colony formation under hypoxic conditions. Mechanistically, microRNA (miRNA)-profiling experiments allowed us to identify miR-215 as one of the main hypoxia-induced miRNAs in primary colon TICs. Through stable overexpression of miR-215, followed by a set of functional in vitro and in vivo investigations, miR-215 was pinpointed as a negative feedback regulator, working against the TIC-promoting effects of hypoxia. Furthermore, we could single out LGR5, a bona fide marker of non-neoplastic intestinal stem cells, as a downstream target of hypoxia/miR-215 signaling. The strong tumor- and TIC-suppressor potential of miR-215 and the regulatory role of the hypoxia/miR-215/LGR5 axis may thus represent interesting points of attack for the development of innovative anti-CSC therapy approaches.

Retracted Article: Knockdown of long non-coding RNA OIP5-AS1 suppresses cell proliferation and migration in ox-LDL-induced human vascular smooth muscle cells (hVMSCs) through targeting miR-152-3p/PAPPA axis.

Emerging evidence has demonstrated that long non-coding RNA Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) is associated with cellular behaviors among malignant tumors. However, the role of OIP5-AS1 in atherosclerosis remains largely undefined. The aim of this study was to explore the expression and role of OIP5-AS1 in a cell model of atherosclerosis, as well as the underlying mechanism. We found that expression of OIP5-AS1 was upregulated in human vascular smooth muscle cells (hVMSCs) under oxidized low density lipoprotein (ox-LDL) administration, and knockdown of OIP5-AS1 suppressed cell viability (CCK-8) and proliferating cell nuclear antigen (PCNA) protein level in ox-LDL-treated hVMSCs, as well as inhibited cell migration rate (wound healing assay) and protein expression of matrix metalloproteinase (MMP)-2 and MMP-9. Mechanically, OIP5-AS1 functioned as competing endogenous RNA (ceRNA) to positively regulate PAPPA expression through sponging miRNA-152-3p (miR-152), and pregnancy-associated plasma protein A (PAPPA) was identified as a downstream target gene for miR-152. Moreover, expression of miR-152 was downregulated and PAPPA was upregulated in ox-LDL-treated hVMSCs. Similarly to OIP5-AS1 knockdown, miR-215 overexpression could inhibit cell proliferation and migration of hVMSCs administrated by ox-LDL, which was abated by PAPPA upregulation. Moreover, miR-215 downregulation partially reversed the suppressive role of OIP5-AS1 knockdown as well. In conclusion, knockdown of OIP5-AS1 suppressed ox-LDL-treated hVMSC proliferation and migration presumably through targeting miR-152/PAPPA axis, suggesting a novel OIP5-AS1/miR-152/PAPPA pathway in atherogenesis.

MiR‑215 promotes epithelial to mesenchymal transition and proliferation by regulating LEFTY2 in endometrial cancer.

Endometrial cancer (EC) is the most common gynecological tumor in developed countries with an increasing incidence. Left-right determination factor 2 (LEFTY2), a suppressor of cell proliferation and tumor growth, is a negative regulator of EC progression. The roles of LEFTY2 are emerging; however, the regulatory mechanisms of its expression have not been well understood. MicroRNA (miR)-215 as an oncogene serves an important role in tumorigenesis by regulating target genes. In the present study, it was demonstrated that overexpression of miR-215 promoted epithelial to mesenchymal transition (EMT), colony formation and DNA synthesis in EC HEC-1A cells and its expression was upregulated in EC tissues. Using online miR target prediction software, it was revealed that LEFTY2 is predicted as a target of miR-215. Using western blot analysis and immunofluorescence assays, it was demonstrated that overexpression of miR-215 markedly downregulated LEFTY2 protein expression levels in HEC-1A cells and LEFTY2 protein expression was downregulated in EC tissues, which was inversely correlated with miR-215 expression. Furthermore, the present study indicated that overexpression of LEFTY2 protein promoted mesenchymal to epithelial transition and sensitized HEC-1A cells to cisplatin treatment. In addition, it was revealed that the overexpression of LEFTY2 inhibited colony formation and DNA synthesis in HEC-1A cells. Thus, miR-215 may promote EMT and proliferation by regulating LEFTY2 in EC.

MicroRNA-215 suppresses the proliferation, migration and invasion of non-small cell lung carcinoma cells through the downregulation of matrix metalloproteinase-16 expression.

The present study investigated the expression of microRNA (miR)-215 in non-small cell lung carcinoma (NSCLC) at tissue and cellular levels, as well as its biological functions and mechanism of action. A total of 56 patients with NSCLC were included in the present study. NSCLC tissues and tumor-adjacent normal tissues were resected and collected. Reverse transcription-quantitative polymerase chain reaction was used to measure the expression of miR-215. Following transfection with miR-215 mimics, A549 cell proliferation, migration and invasion were determined using a Cell Counting Kit-8 and Transwell assay. Western blotting was employed to measure the expression of matrix metalloproteinase (MMP)-16 protein. A dual-luciferase reporter assay was conducted to determine the existence of a direct interaction between miR-215 and the MMP-16 gene. Reduced expression of miR-215 in NSCLC was closely associated with lymphatic metastasis and TNM staging. Overexpression of miR-215 inhibited the proliferation of A549 cells in vitro. Upregulated expression of miR-215 inhibited the migration and invasion of A549 cells in vitro. miR-215 exerted its biological functions possibly by regulating the expression of MMP-16. Elevated expression of MMP-16 promoted the proliferation, migration and invasion of A549 cells. miR-215 regulated the proliferation, migration and invasion of A549 cells by binding with the seed 3′-untranslated region of MMP-16 mRNA. The present study demonstrates that reduced expression of miR-215 in NSCLC is negatively associated with lymphatic metastasis and TNM staging. In addition, miR-215 acts as a tumor suppressor gene by inhibiting the proliferation, migration and invasion of NSCLC cells via the downregulation of MMP-16 expression.

Upregulation of miR-215 exerts neuroprotection effects against ischemic injury via negative regulation of Act1/IL-17RA signaling.

This study investigated the role of miR-215 and nuclear factor-κB activator (Act)1 and their mechanisms of action in ischemic stroke. Cell viability was examined with the 3-(4,5-dimethythiazol-. 2-yl)-2,5-diphenyl tetrazolium bromide assay; cell apoptosis was detected by flow cytometry; and mRNA and protein expression was assessed by quantitative real-time PCR and western blotting, respectively. A mouse model of middle cerebral artery occlusion (MCAO) was treated with or without miR-215 mimic to verify the in vitro results. The relationship between miR-215 and interleukin (IL)-17 was evaluated in human peripheral blood from 29 patients. Act1 was upregulated whereas miR-215 was downregulated in ischemic stroke. Overexpression of miR-215 by transfection of a mimic repressed Act1 protein levels in vitro and in vivo, although the luciferase assay showed that miR-215 did not directly bind to the 3' untranslated region of Act1. MiR-215 overexpression inhibited cell apoptosis and autophagy. Increasing miR-215 levels reduced ischemic infarction and improved neurological deficit, while loss of miR-215 phenocopied the effects of IL-17. Upregulation of miR-215 exerts neuroprotection against ischemic injury by negatively regulating Act1/IL-17 receptor A signaling. These findings provide potential therapeutic targets for the treatment of ischemic stroke.

MiR-215 functions as an oncogene in high-grade glioma by regulating retinoblastoma 1.

To investigate the roles of miR-215 in high-grade glioma and to clarify the regulation of retinoblastoma 1 (RB1) by miR-215. miR-215 is frequently up-regulated in high-grade glioma tissues. Increased miR-215 expression is significantly associated with World Health Organization grade (P<0.01) tumor size (P<0.05) and poor prognosis (P<0.01). Over-expression of miR-215 promoted cell proliferation and knockdown of miR-215 inhibited cell proliferation in vitro. RB1 was identified as a direct and functional target of miR-215. RB1 is generally down-regulated in glioma tissues and its expression inversely correlated with miR-215, which is up-regulated in high-grade glioma tissues, and its expression was negatively correlated with miR-215. The new miR-215/RB1 axis provides new insights into the molecular mechanism and treatment for glioma.

MicroRNA-215 acts as a tumor suppressor in breast cancer by targeting AKT serine/threonine kinase 1.

There are accumulating reports that microRNAs are dysregulated in a number of human cancer types, and that they may function as tumor suppressors or oncogenes in tumorigenesis and tumor development. microRNA-215 (miR-215) has been identified as a tumor suppressor in epithelial ovarian, pancreatic, non-small cell lung and colon cancer, whereas it may act as an oncogene in gastric and cervical cancer. The role of miR-215 in breast cancer carcinogenesis and progression has yet to be elucidated. In the present study, the expression level of miR-215 was determined in breast cancer tissues and cell lines using the reverse transcription-quantitative polymerase chain reaction. The effects of miR-215 overexpression on proliferation and the invasive capacity of breast cancer cells were assessed using MTT and cell invasion assays. The results revealed that miR-215 was significantly downregulated in breast cancer tissues and cell lines. Restoration of miR-215 expression inhibited the proliferation and invasion of breast cancer cells. The underlying molecular mechanism for the suppression of proliferation and invasion by miR-215 was investigated. AKT serine/threonine kinase 1 (AKT1) was validated as a novel direct target of miR-215, and the effect of AKT1 small interfering RNA mimicked the effect of miR-215 overexpression in breast cancer cells. These results indicated that miR-215 acted as a tumor suppressor, and that its downregulation in tumor tissues may contribute to the carcinogenesis and progression of breast cancer, indicating that miR-215 may be a novel therapeutic target for the treatment of breast cancer.

MiR-215 suppresses proliferation and migration of non-small cell lung cancer cells

The expression of microRNA-215 (miR-215) in non-small cell lung cancer (NSCLC) tissues and the effects of miR-215 on the proliferation and migration of NSCLC cells were investigated. qRT-PCR was used to detect the expression of miR-215 in NSCLC tissues and paired normal tumor-adjacent lung tissues; MTT assay, transwell assay and soft-agar assay were used in vitro to evaluate the role of miR-215 on proliferation, migration and cell clonality on NSCLC cells, after transfecting miR-215 mimics to NSCLC cell line A549 or miR-215 to H1299. miR-215 was significantly decreased in NSCLC tissues compared to the paired normal tissues; Overexpression of miR-215 in A549 cells resulted in reduction of the cell proliferation, migration and cell clonality, while downregulation of miR-215 in H1299 cells could promote cell proliferation, migration and clonality. In conclusion, miR-215 was downregulated in NSCLC tissues and may play a key role in the development of NSCLC.

MicroRNA-215 suppresses cell proliferation, migration and invasion of colon cancer by repressing Yin-Yang 1

Colorectal cancer is one of the most common malignant tumors worldwide with rising incidence. MicroRNAs are small non-coding RNAs that implicate in multiple physiological or pathological processes. The aberrant expression of miRNA-215 (miR-215) has been illustrated in various types of cancers. However, the expression of miR-215 in human colon cancer and the biological roles of it remain largely unknown. We conducted this study to explore the expression and the function of miR-215 in human colon cancer. The results showed that miR-215 was remarkably downregulated in colon cancer tissues and cell lines. Overexpression of miR-215 by miR-215 mimic significantly inhibited colon cancer cell proliferation, migration and invasion while knockdown of miR-215 by miR-215 inhibitor exerted reverse effects. Furthermore, we newly identified Yin-Yang 1(YY1) as a direct target of miR-215 which could rescue the effects of miR-215 on colon cancer cells. In summary, our investigation revealed that miR-215 was downregulated in colon cancer and it suppressed colon cancer cell proliferation, migration and invasion by directly targeting YY1.

MiR-215 functions as a tumor suppressor and directly targets ZEB2 in human non-small cell lung cancer.

MicroRNA-215 (miR-215) has previously been demonstrated to be dysregulated in a number of human malignancies and to be correlated with tumor progression. However, the expression and function of miR-215 in non-small cell lung cancer (NSCLC) has remained to be elucidated. Therefore, the present study aimed to investigate the effects of miR-215 in NSCLC tumorigenesis and development. Reverse transcription-quantitative polymerase chain reaction was used to evaluate miR-215 expression in NSCLC cell lines and primary tumor tissues. The association between miR-215 expression and certain clinicopathological factors was also determined, and the effects of miR-215 on the biological behavior of NSCLC cells were investigated. In addition, the potential regulatory function of miR-215 on zinc finger E-box-binding homeobox 2 (ZEB2) expression was examined. miR-215 expression was significantly downregulated in NSCLC cell lines and clinical specimens. Reduced miR-215 expression was significantly associated with lymph node metastasis and advanced TNM stage. Overexpression of miR-215 inhibited NSCLC cell proliferation, invasion and migration, and promoted cell apoptosis in vitro, and suppressed tumorigenicity in vivo. Furthermore, luciferase reporter assay analysis identified ZEB2 as a direct target of miR-215. These findings indicated that miR-215 may act as a tumor suppressor in NSCLC and may serve as a novel therapeutic agent for miR-based therapy.

MiR-215 overexpression distinguishes ampullary carcinomas from pancreatic carcinomas

AbstractDistinguishing ampullary carcinoma from pancreatic carcinoma is important because of their different prognoses. microRNAs are differentially expressed according to the tissue of origin. However, there is rare research on the differential diagnosis between the two types of cancers by microRNA in periampullary cancers. The present study was undertaken to compare microRNA profiles between ampullary and pancreatic carcinomas using microarrays. miR-215 was most significantly overexpressed in ampullary carcinomas; whereas the expressions of miR-134 and miR-214 were significantly lower in ampullary carcinomas than in pancreatic carcinomas. When these discriminatory microRNAs were applied to liver metastases, they were correctly predicted for the tissue of origin. Although this study is limited by small sample size, striking difference in microRNA expression and concordant expression of discriminating microRNAs in primary tumors and metastases suggest that these novel discriminatory microRNAs warrant future validation.

The CDX1–microRNA-215 axis regulates colorectal cancer stem cell differentiation

The transcription factor caudal-type homeobox 1 (CDX1) is a key regulator of differentiation in the normal colon and in colorectal cancer (CRC). CDX1 activates the expression of enterocyte genes, but it is not clear how the concomitant silencing of stem cell genes is achieved. MicroRNAs (miRNAs) are important mediators of gene repression and have been implicated in tumor suppression and carcinogenesis, but the roles of miRNAs in differentiation, particularly in CRC, remain poorly understood. Here, we identified microRNA-215 (miR-215) as a direct transcriptional target of CDX1 by using high-throughput small RNA sequencing to profile miRNA expression in two pairs of CRC cell lines: CDX1-low HCT116 and HCT116 with stable CDX1 overexpression, and CDX1-high LS174T and LS174T with stable CDX1 knockdown. Validation of candidate miRNAs identified by RNA-seq in a larger cell-line panel revealed miR-215 to be most significantly correlated with CDX1 expression. Quantitative ChIP-PCR and promoter luciferase assays confirmed that CDX1 directly activates miR-215 transcription. miR-215 expression is depleted in FACS-enriched cancer stem cells compared with unsorted samples. Overexpression of miR-215 in poorly differentiated cell lines causes a decrease in clonogenicity, whereas miR-215 knockdown increases clonogenicity and impairs differentiation in CDX1-high cell lines. We identified the genome-wide targets of miR-215 and found that miR-215 mediates the repression of cell cycle and stemness genes downstream of CDX1. In particular, the miR-215 target gene BMI1 has been shown to promote stemness and self-renewal and to vary inversely with CDX1. Our work situates miR-215 as a link between CDX1 expression and BMI1 repression that governs differentiation in CRC.

MicroRNA-215 functions as a tumor suppressor and directly targets ZEB2 in human pancreatic cancer.

It has been shown that microRNA-215 (miR-215) is dysregulated in several human malignancies, and this correlates with tumor progression. However, its expression and function in pancreatic cancer is still unclear. The aim of this study was to explore the effects of miR-215 on pancreatic cancer formation and progression. Using quantitative RT-PCR, we detected miR-215 expression in pancreatic cancer cell lines and primary tumor tissues. The association of miR-215 expression with clinicopathological factors and prognosis was also analyzed. We then observed the effects of miR-215 on the biological behavior of pancreatic cancer cells. Lastly, the potential regulatory function of miR-215 on ZEB2 expression was investigated. miR-215 expression levels were significantly downregulated in pancreatic cancer samples and cell lines. Decreased miR-215 expression was significantly associated with large tumor size, advanced TNM stage, lymph node metastasis, vessel invasion, and lower overall survival. Multivariate regression analysis corroborated that downregulation of miR-215 was an independent unfavorable prognostic factor. Overexpression of miR-215 inhibited pancreatic cancer cell proliferation, invasion, and migration; promoted cell apoptosis in vitro; and suppressed tumorigenicity in vivo. Further, ZEB2 was confirmed as a direct target of miR-215 by using a luciferase reporter assay. These findings indicate that miR-215 may act as a tumor suppressor in pancreatic cancer cells, and could serve as a novel therapeutic target for miR-based therapy.

Abstract 5247: Comprehensive and systematic identification of miR-215 targets in colon cancer via a novel translation capture immunoprecipitation (TrIP) approach

Abstract The important roles that non-coding miRNAs play in cancer development, makes the investigation of the various genes they target particularly crucial. In addition to targeting mRNA transcripts for degradation, miRNA can also alter gene expression at the translational level to achieve rapid regulation. It has been a technical challenge to study translational control using a small number of cells. We have overcome this hurdle by developing a translational capture immunoprecipitation approach (TrIP) to investigate RNA binding proteins or non-coding RNA regulation at the translational level from a small numbers of cells. The new approach is based on the fact that nascent translated polypeptides are associated with hsp70 chaperones along with actively translating mRNA transcript complexes, called polyribosomes. We are able to isolate actively translating mRNA from free cytoplasmic mRNA by immunoprecipitation of hsp70 associated polyribosome complexes. In this study, we utilize this novel approach to identify both degraded and non-degraded mRNA transcripts targeted by miR-215 in colon cancer. Following miR-215 overexpression in colon cancer cells, actively translating mRNA transcripts were isolated using the TrIP technique and purified following standard RNA isolation techniques. We were able to identify additional non-degraded mRNA targets regulated by miR-215 using a TrIP with transcriptome-wide microarray based approach, when compared to a generic approach based on steady state total RNA that can only identify degraded targets. Specific targets were independently validated using qRT-PCR analysis. We further validated miR-215 targets at the protein level with a comprehensive and high-throughput quantitative SILAC proteomics analysis. Protein targets were further validated using Western blotting with select positive control targets including TYMS, DTL and DHFR. This approach offers the unique opportunity to understand not only the mRNA targets a miRNA may target for degradation, but also those it regulates at the translational level, providing unique insight into the actions of miRNAs. Interest in this type of thorough analysis of different miRNAs or RNA binding proteins will likely continue to grow as the important roles miRNA play in cancer are further investigated. Citation Format: Andrew Fesler, Xiao Xu, Emily Chen, Jingfang Ju. Comprehensive and systematic identification of miR-215 targets in colon cancer via a novel translation capture immunoprecipitation (TrIP) approach. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5247. doi:10.1158/1538-7445.AM2014-5247

Use of miRNA profiling in metastatic renal cell carcinoma to reveal a tumor suppressor effect for mir-215.

4633 Background: Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney. Metastatic RCC is difficult to treat. The five-year survival rate for metastatic RCC is &lt;10%. Recently, microRNAs (miRNAs) have been shown to have a role in cancer metastasis and potential as prognostic biomarkers in cancer. Methods: We performed a miRNA microarray to identify a miRNA signature characteristic of metastatic compared to primary RCC. Results were validated by quantitative real time PCR. Target prediction analysis and gene expression profiling identified many of the dysregulated miRNAs could target genes involved in tumor metastasis. The effect of miR-215 on cellular migration and invasion was shown in a RCC cell line model. Results: We identified 65 miRNAs that were significantly altered in metastatic when compared to primary RCC. Nine (14%) miRNAs had increased expression while 56 (86%) miRNAs showed decreased expression. miR-10b, miR-196a, and miR-27b were the most downregulated while miR-638, miR-1915, and miR-149* were the most upregulated. A non-supervised 2D-cluster analysis showed that a sub-group of the primary tumors clustered under the metastatic arm with a group of miRNAs that follow the same pattern of expression suggesting they have an inherited aggressive signature. We validated our results by examining the expressions of miR-10b, miR-126, miR-196a, miR-204, and miR-215, in two independent cohorts of patients. We also showed that overexpression of miR-215 decreased cellular migration and invasion in a RCC cell line model. In addition, through gene expression profiling, we identified direct and indirect targets of miR-215 that can contribute to tumor metastasis. Conclusions: Our analysis showed that miRNAs are altered in metastatic RCC and can contribute to kidney cancer metastasis through different biological processes. Dysregulated miRNAs represent potential prognostic biomarkers and may have therapeutic applications in kidney cancer.