|Description||EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit|
|Overview||RNA-binding protein immunoprecipitation (RIP) is the RNA analog of the more well-known ChIP application (chromatin immunoprecipitation), which identifies DNA targets of DNA-binding proteins in an in-vivo cellular context. RIP can be used to identify specific RNA molecules (of many types) associated with specific nuclear or cytoplasmic binding proteins. These experiments involve immunoprecipitation of endogenously formed complexes of RNA-binding proteins and co-isolation of any RNA species associated with that RNA-binding protein. Purification of these RNA species allows interrogation and identification of mRNAs (and potentially non-coding RNAs associated with them) and can be directly measured using down stream applications including quantitative reverse transcription polymerase chain reaction (RT-PCR), microarray analysis (RIP-chip) and “deep-sequencing” or 2nd-generation sequencing based platforms (RIP-Seq).
Features & Benefits:
-Protein A/G magnetic beads, optimized to bind nucleic acid-protein immune complexes
-RNAse inhibitors and RNAse-free reagents
-Positive and negative controls
|Background Information||Gene regulation plays a critical role in complex cellular processes such as development, differentiation, and cellular response to environmental changes. In addition to transcriptional regulation of gene expression by transcription factors, cells utilize post-transcriptional regulatory mechanisms. One such mechanism involves use of certain RNA-binding proteins (RBPs) to temporally and coordinately regulate the rate of mRNA translation of functionally related gene
products. While the regulation of gene expression by transcription factors has been well studied over time, the post-transcriptional regulation of mRNAs by RBPs and the role of non-coding RNAs in this process is a relatively nascent field that remains to be thoroughly explored.
|Materials Required but Not Delivered||Magna Grip™ Rack 8 well ( 20-400) (Now Available!) or similar magnetic rack.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Upon receipt, store components at the temperatures indicated on the labels. Kit components are stable for 6 months from date of shipment when stored as directed.|
|Material Size||12 assays|
|Material Package||RIP Kit capacity: 12 RNA-binding protein immunoprecipitation assays|
EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit MSDS
|Reference overview||Application||Pub Med ID|
|MiR-155-5p controls colon cancer cell migration via post-transcriptional regulation of Human Antigen R (HuR).|
Al-Haidari, A; Algaber, A; Madhi, R; Syk, I; Thorlacius, H
Cancer Lett 421 145-151 2018
Colorectal cancer (CRC) is the third most common cancer and a significant cause of cancer-related deaths worldwide. Metastasis is the worst prognostic factor for patients with CRC. HuR (ELAVL1) is overexpressed in CRC and has been reported to promote colon cancer growth by targeting RNA in the cell cytoplasm. Herein, the role of miR-155-5p in regulating HuR expression and cell migration was examined in colon cancer cells. MiR-155-5p knockdown in serum-starved colon cancer cells decreased both colon cancer cell chemotaxis and cytoplasmic expression of HuR. Bioinformatics analysis predicted two putative binding sites in the AU-rich elements (AREs) at the 3'-UTR of HuR mRNA. MiR-155-5p binding to HuR was verified using specific target site blockers and functionally validated by use of RNA immunoprecipitation assays, showing that miR-155-5p-dependent regulation of HuR expression is mediated by AREs. Targeting AREs with a specific blocker inhibited colon cancer cell migration. Taken together, these novel findings demonstrate that AREs mediate miR-155-5p positive regulation of HuR mRNA levels and translation as well as migration in colon cancer cells, suggesting that targeting miR-155-5p and/or Hur might be useful therapeutic strategies against colon cancer metastasis.
|MALAT1/miR-101-3p/MCL1 axis mediates cisplatin resistance in lung cancer.|
Wang, H; Wang, L; Zhang, G; Lu, C; Chu, H; Yang, R; Zhao, G
Oncotarget 9 7501-7512 2018
In this study, we investigated the mechanism by which lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) mediates cisplatin resistance in lung cancer. Lung cancer patients with high MALAT1 levels were associated with cisplatin resistance and low overall survival. Moreover, cisplatin-resistant A549/DDP cells showed higher MALAT1 expression than cisplatin-sensitive lung cancer cells (A549, H460, H1299 and SPC-A1). Dual luciferase reporter and RNA immunoprecipitation assays showed direct binding of miR-101-3p to MALAT1. MALAT1 knockdown in lung cancer cells resulted in miR-101-3p upregulation and increased cisplatin sensitivity. In addition, miR-101-3p decreased myeloid cell leukemia 1 (MCL1) expression by binding to the 3'-untranslated region (3'-UTR) of its mRNA. These results demonstrate that MALAT1/miR-101-3p/MCL1 signaling underlies cisplatin resistance in lung cancer.
|Long Non-coding RNA LINC00339 Stimulates Glioma Vasculogenic Mimicry Formation by Regulating the miR-539-5p/TWIST1/MMPs Axis.|
Guo, J; Cai, H; Liu, X; Zheng, J; Liu, Y; Gong, W; Chen, J; Xi, Z; Xue, Y
Mol Ther Nucleic Acids 10 170-186 2018
Glioma is recognized as a highly angiogenic malignant brain tumor. Vasculogenic mimicry (VM) greatly restricts the therapeutic effect of anti-angiogenic tumor therapy for glioma patients. However, the molecular mechanisms of VM formation in glioma remain unclear. Here, we demonstrated that LINC00339 was upregulated in glioma tissue as well as in glioma cell lines. The expression of LINC00339 in glioma tissues was positively correlated with glioma VM formation. Knockdown of LINC00339 inhibited glioma cell proliferation, migration, invasion, and tube formation, meanwhile downregulating the expression of VM-related molecular MMP-2 and MMP-14. Furthermore, knockdown of LINC00339 significantly increased the expression of miR-539-5p. Both bioinformatics and luciferase reporter assay revealed that LINC00339 regulated the above effects via binding to miR-539-5p. Besides, overexpression of miR-539-5p resulted in decreased expression of TWIST1, a transcription factor known to play an oncogenic role in glioma and identified as a direct target of miR-539-5p. TWIST1 upregulated the promoter activities of MMP-2 and MMP-14. The in vivo study showed that nude mice carrying tumors with knockdown of LINC00339 and overexpression of miR-539-5p exhibited the smallest tumor volume through inhibiting VM formation. In conclusion, LINC00339 may be used as a novel therapeutic target for VM formation in glioma.
|LncRNA CASC9 promotes esophageal squamous cell carcinoma metastasis through upregulating LAMC2 expression by interacting with the CREB-binding protein.|
Liang, Y; Chen, X; Wu, Y; Li, J; Zhang, S; Wang, K; Guan, X; Yang, K; Bai, Y
Cell Death Differ 0 2018
Esophageal squamous cell carcinoma (ESCC) is the main subtype of esophageal cancer. Long noncoding RNAs (lncRNAs) are thought to play a critical role in cancer development. Recently, lncRNA CASC9 was shown to be dysregulated in many cancer types, but the mechanisms whereby this occurs remain largely unknown. In this study, we found that CASC9 was significantly upregulated in ESCC tissues, with further analysis revealing that elevated CASC9 expression was associated with ESCC prognosis and metastasis. Furthermore, we found that CASC9 knockdown significantly repressed ESCC migration and invasion in vitro and metastasis in nude mice in vivo. A microarray analysis and mechanical experiments indicated that CASC9 preferentially affected gene expression linked to ECM-integrin interactions, including LAMC2, an upstream inducer of the integrin pathway. We demonstrated that LAMC2 was consistently upregulated in ESCC and promoted ESCC metastasis. LAMC2 overexpression partially compromised the decrease of cell migration and invasion capacity in CASC9 knockdowns. In addition, we found that both CASC9 and LAMC2 depletion reduced the phosphorylation of FAK, PI3K, and Akt, which are downstream effectors of the integrin pathway. Moreover, the reduction in phosphorylation caused by CASC9 depletion was rescued by LAMC2 overexpression, further confirming that CASC9 exerts a pro-metastatic role through LAMC2. Mechanistically, RNA pull-down and RNA-binding protein immunoprecipitation (RIP) assay indicated that CASC9 could bind with the transcriptional coactivator CREB-binding protein (CBP) in the nucleus. Chromatin immunoprecipitation (ChIP) assay additionally illustrated that CASC9 increased the enrichment of CBP and H3K27 acetylation in the LAMC2 promoter, thereby upregulating LAMC2 expression. In conclusion, we demonstrate that CASC9 upregulates LAMC2 expression by binding with CBP and modifying histone acetylation. Our research reveals the prognostic and pro-metastatic roles for CASC9 in ESCC, suggesting that CASC9 could serve as a biomarker for prognosis and a target for metastasis treatment.
|Studying the mechanism of PLAGL2 overexpression and its carcinogenic characteristics based on 3'-untranslated region in colorectal cancer.|
Su, C; Li, D; Li, N; Du, Y; Yang, C; Bai, Y; Lin, C; Li, X; Zhang, Y
Int J Oncol 0 2018
Pleomorphic adenoma gene like-2 (PLAGL2) is a zinc finger protein transcription factor, which is upregulated and serves an oncogenic function in multiple human malignancies, including colorectal cancer (CRC). First, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression levels of PLAGL2 in CRC tissues and normal tissues. Then, bioinformatics analysis, RT-qPCR, western blotting, luciferase reporter assays and RNA-binding protein immunoprecipitation assays were performed to explore whether the underlying mechanisms, including copy number variation (CNV), microRNAs (miRNAs/miRs) and RNA-binding proteins (RBPs) led to the abnormal expression of PLAGL2. Finally, cell counting kit-8 assays, Transwell assays and xenograft models were used to detect carcinogenesis-associated characteristics based on the 3'-untranslated region (3'-UTR) of PLAGL2. In the present study, PLAGL2 was revealed to be upregulated in CRC tissues compared with normal CRC tissues. CNV was one of the causes leading to the upregulation of PLAGL2. miRNA, including downregulated miR-486-5p, and RBPs, including upregulated human antigen R (HuR), were other key underlying causes. In addition, PLAGL2 3'-UTR was revealed to promote the progression of CRC in vitro and in vivo, and to regulate the expression of C-MYC and CD44. To conclude, these results suggested that high expression of PLAGL2 in CRC was associated with CNV, miR-486-5p and HuR expression, whose 3'-UTR may promote colon carcinogenesis and serve as a novel potential biomarker for CRC therapies.
|miR-296-3p Negatively Regulated by Nicotine Stimulates Cytoplasmic Translocation of c-Myc via MK2 to Suppress Chemotherapy Resistance.|
Deng, X; Liu, Z; Liu, X; Fu, Q; Deng, T; Lu, J; Liu, Y; Liang, Z; Jiang, Q; Cheng, C; Fang, W
Mol Ther 26 1066-1081 2018
This study aimed to identify mechanisms by which microRNA 296-3p (miR-296-3p) functions as a tumor suppressor to restrain nasopharyngeal carcinoma (NPC) cell growth, metastasis, and chemoresistance. Mechanistic studies revealed that miR-296-3p negatively regulated by nicotine directly targets the oncogenic protein mitogen-activated protein kinase-activated protein kinase-2 (Mapkapk2) (MK2). Suppression of MK2 downregulated Ras/Braf/Erk/Mek/c-Myc and phosphoinositide-3-kinase (PI3K)/Akt/c-Myc signaling and promoted cytoplasmic translocation of c-Myc, which activated miR-296-3p expression by a feedback loop. This ultimately inhibited cell cycle progression, epithelial-to-mesenchymal transition (EMT), and chemoresistance of NPC. In addition, nicotine as a key component of tobacco was observed to suppress miR-296-3p and thus elevate MK2 expression by inducing PI3K/Akt/c-Myc signaling. In clinical samples, reduced miR-296-3p as an unfavorable factor was inversely correlated with MK2 and c-Myc expression. These results reveal a novel mechanism by which miR-296-3p negatively regulated by nicotine directly targets MK2-induced Ras/Braf/Erk/Mek/c-Myc or PI3K/AKT/c-Myc signaling to stimulate its own expression and suppress NPC cell proliferation and metastasis. miR-296-3p may thus serve as a therapeutic target to reverse chemotherapy resistance of NPC.
|LncRNA PVT1 regulates triple-negative breast cancer through KLF5/beta-catenin signaling.|
Tang, J; Li, Y; Sang, Y; Yu, B; Lv, D; Zhang, W; Feng, H
Oncogene 0 2018
Recent molecularly targeted approach gains advance in breast cancer treatment. However, the estimated 5-year survival rate has not met the desired expectation for improvement, especially for patients with triple-negative breast cancer (TNBC). Here we report that the lncRNA PVT1 promotes KLF5/beta-catenin signaling to drive TNBC tumorigenesis. PVT1 is upregulated in clinical TNBC tumors. Using genetic approaches targeting PVT1 in TNBC cells, we found that PVT1 depletion inhibited cell proliferation, colony formation, and orthotopic xenograft tumor growth. Mechanistically, PVT1 binds with KLF5 and increases its stability via BAP1, which upregulates beta-catenin signaling, resulting in enhanced TNBC tumorigenesis. PVT1, KLF5, and beta-catenin were also revealed to be co-expressed in clinical TNBC samples. Our findings uncover a new singaling pathway to mediate TNBC, and provide PVT1 as a new target for improving treatment of TNBC.
|Imsnc761 and DDX6 synergistically suppress cell proliferation and promote apoptosis via p53 in testicular embryonal carcinoma cells.|
Duan, Z; Ping, P; Wang, G; Zhang, X; Sun, F
Biosci Rep 0 2018
Intermediate-size noncoding RNAs (imsncRNAs) have been shown to play important regulatory roles in the development of several eukaryotic organisms. In this research, we selected imsnc761 as a research target. Expression analyses in a previous study showed that imsnc761 was downregulated in maturation-arrested testis tissue compared with the level in normal controls. In this study, we found that imsnc761 could interact with DEAD-box helicase 6 (DDX6) to induce NT2 cell apoptosis and proliferation inhibition via the p53 pathway. This interaction between imsnc761 and DDX6 also inhibited mitochondrial function and specific gene transcription and translation. To facilitate further research, we used label-free quantification method to analyse the associated differences in KEGG pathways and biological processes. This confirmed the changes of several specific pathways, which matched our molecular experimental results.
|LncRNA DICER1-AS1 promotes the proliferation, invasion and autophagy of osteosarcoma cells via miR-30b/ATG5.|
Gu, Z; Hou, Z; Zheng, L; Wang, X; Wu, L; Zhang, C
Biomed Pharmacother 104 110-118 2018
Osteosarcoma is a prevalent primary malignant tumor and long non-coding RNAs (lncRNAs) have been validated to modulate the osteosarcoma tumorigenesis. In present study, our research team investigates the role of a novel identified lncRNA DICER1-AS1 on the tumor progression and autophagy. Results showed that lncRNA DICER1-AS1 was up-regulated in osteosarcoma cells using microarray analysis and RT-PCR. Cellular functional experiments revealed that DICER1-AS1 knockdown suppressed the proliferation, migration, invasion and autophagy of osteosarcoma cells in vitro. Besides, DICER1-AS1 knockdown inhibited the protein expression levels of ATG5, LC3-II and Beclin 1, suggesting the inhibition on the autophagy of osteosarcoma cells. Moreover, miR-30b was verified to target 3'-UTR of DICER1-AS1 and ATG5 using bioinformatics tools and luciferase reporter assay or RNA-immunoprecipitation (RIP). Western blot showed that ATG5 protein expression was decreased in DICER1-AS1 knockdown and miR-30b mimics transfected cells, while increased in miR-30b inhibitor transfected cells, presenting a negative correlation with miR-30b and a positive correlation with DICER1-AS1. Finally, xenograft assay in vivo indicated that DICER1-AS1 knockdown inhibited the osteosarcoma tumor growth and protein expression level of ATG5. In summary, all the results conclude that DICER1-AS1 regulates the proliferation, invasion and autophagy of osteosarcoma via miR-30b/ATG5 axis, providing a novel insight for osteosarcoma tumorigenesis.
|LncRNA TUG1 sponges miR-204-5p to promote osteoblast differentiation through upregulating Runx2 in aortic valve calcification.|
Yu, C; Li, L; Xie, F; Guo, S; Liu, F; Dong, N; Wang, Y
Cardiovasc Res 114 168-179 2018
Emerging evidence indicates that long non-coding RNAs (lncRNAs) play a vital role in cardiovascular physiology and pathology. Although the lncRNA TUG1 is implicated in atherosclerosis, its function in calcific aortic valve disease (CAVD) remains unknown.In this study, we found that TUG1 was highly expressed in human aortic valves and primary valve interstitial cells (VICs). Moreover, TUG1 knockdown induced inhibition of osteoblast differentiation in CAVD both in vitro and in vivo. Mechanistically, silencing of TUG1 increased the expression of miR-204-5p and subsequently inhibited Runx2 expression at the post-transcriptional level. Importantly, TUG1 directly interacted with miR-204-5p and downregulation of miR-204-5p efficiently reversed the suppression of Runx2 induced by TUG1 short hairpin RNA (shRNA). Thus, TUG1 positively regulated the expression of Runx2, through sponging miR-204-5p, and promoted osteogenic differentiation in CAVD.All together, the evidence generated by our study elucidates the role of lncRNA TUG1 as a miRNA sponge in CAVD, and sheds new light on lncRNA-directed diagnostics and therapeutics in CAVD.
|RNA-Binding Protein Immunoprecipitation|
|White Paper - The Message in the Marks: Deciphering Cancer Epigenetics|
|Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit|