17-700 Sigma-AldrichMagna RIP™ RNA-Binding Protein Immunoprecipitation Kit

The identification and characterization of long non-coding RNAs (lncRNAs) in diverse biological process has currently developed rapidly. LncRNA-PVT1, located adjacent to the MYC locus on chromosomal region 8q24, has been reported to be associated with many biological processes. However, the function and mechanism of PVT1 in pancreatic carcinoma (PC) is poorly understood. In this present study, we first measured the level of PVT1 in the PC cell lines and tissues by quantitative real-time PCR (qRT-PCR), and then employed loss-of-function and gain-of-function approaches to explore the association between PVT1 expression levels and PC cell proliferation/migration ability. Furthermore, bioinformatics analysis was utilized to show that PVT1 contains binding site for miR-448 and an inverse correlation between PVT1 and miR-448 was obtained in PC specimens. Additionally, dual luciferase reporter assay, RNA-binding protein immunoprecipitation (RIP) and applied biotin-avidin pulldown system were applied to further confirm that PVT1 directly bind with microRNA binding site harboring in the PVT1 sequence. Then, SERBP1 was identified as a target of miR-448 according to the gene expression array analysis of PC clinical samples. Together, we revealed that PVT1 functions as an endogenous "sponge" by competing for miR-448 binding to regulate the miRNA target SERBP1 and, therefore, promotes the proliferation and migration of PC cells.

Rheumatoid arthritis (RA) is a systemic and chronic inflammatory disease. Synoviocyte migration and invasion were found to be essential to the pathology of RA. Upregulation of long noncoding RNA ZFAS1 has been observed in cancers and promotes cell migration and invasion. To date, the functions and mechanisms of ZFAS1 in RA have not been revealed. In this study, we analyzed expression pattern of ZFAS1 in RA patients and found that ZFAS1 expression was increased in synovial tissue and fibroblast-like synoviocytes (FLS) from RA patients (RA-FLS) compared with that in healthy donors. Functional assays showed that silence of ZFAS1 suppressed RA-FLS migration and invasion, while overexpression of ZFAS1 showed the opposite effect. Further investigation demonstrated that ZFAS1 directly interacted with miR-27a and decreased miR-27a expression. ZFAS1 promotes RA-FLS migration and invasion in an miR-27a-dependent manner. Taken together, the present study provides the first evidence that ZFAS1 promotes cell migration and invasion through miR-27a in RA-FLS, suggesting that ZFAS1 may be an effective therapeutic target for RA patients.

We aim to investigate the interaction between the EZH2 and the long noncoding RNA (lncRNA) SPRY4-IT1. We also explore their respective effects on human lung adenocarcinoma (LA) cell invasion and migration. Both LA and adjacent normal tissues were obtained from 256 LA patients. SPTY4-IT expression and EZH2 mRNA expressions in tissues and cells were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The siRNAs against SPRY4-IT1 and EZH2 were co-transfected into A549 and H1975 cells. The interaction between SPRY4-IT1 and EZH2 was determined using a RNA pull-down assay and a RNA immunoprecipitation (RIP) assay. A Transwell assay and scratch assay were used to evaluate the cell migration and invasion abilities. The expressions of E-cadherin and Vimentin in the epithelial-mesenchymal transition (EMT) and EZH2 protein expression were detected through western blotting. SPRY4-IT1 expression was observed to be significantly lower, while the expression of EZH2 was higher in the LA tissues than in the adjacent normal tissues. Compared with the HBE cell line, expressions of SPRY4-IT1 in each human LA cell line had decreased, with the lowest observed reduction in the A549 cell line, while EZH2 mRNA and protein expression increased in each human LA cell lines. After SPRY4-IT1-siRNA was transfected into A549 and H1975 cells, invasion and migration abilities were enhanced, in addition to a reduction in the expression of E-cadherin, while expressions of Vimentin exhibited an increased rate. Consequently, we find that EZH2 promotes LA cell invasion and metastasis by inhibiting SPRY4-IT1 expression.

MRPL33 gene encodes a large mitoribosomal subunit protein, which may be involved in mitochondrial translation. Although two splice variants of MRPL33 have been described, its splicing regulation remains elusive. Here we observed that inclusion of alternative exon 3 was greatly promoted in a panel of human cancer cells. Depletion of the exon 3-containing long isoform of MRPL33 (MRPL33-L) led to impaired proliferation and increased apoptosis in cancer cell lines and in a xenograft model. MRPL33-L knockdown could also induce mitochondrial dysfunction including increased accumulation of reactive oxygen species, decreased ATP production and 16 S rRNA levels. We further showed that alternative splicing of MRPL33-L pre-mRNA is regulated by hnRNPK and that knocking down hnRNPK could phenocopy MRPL33-L depletion. More importantly, overexpression of MRPL33-L could increase tumorigenic potential of hnRNPK-depleted cancer cells, likely indicating that hnRNPK mediates tumorigenesis through splicing regulation of MRPL33 pre-mRNA. Finally, we found that inclusion of MRPL33 exon 3 was promoted in human colorectal cancer tissues and this was correlated with hnRNPK levels. In summary, our findings underscore the biological significance of MRPL33-L and hnRNPK in the tumor formation and identifies hnRNPK as a critical splicing regulator of MRPL33 pre-mRNA in cancer cells.

RNA-binding proteins (RBP) are important facilitators of post-transcriptional gene regulation. We have previously established that nuclear overexpression of the RBP Musashi-2 (MSI2) during male germ cell maturation is detrimental to sperm cell development and fertility. Herein we determine the genes and pathways impacted by the upregulation of Msi2. Microarray analysis and qPCR confirmed differential gene expression in factors fundamental to the cell cycle, cellular proliferation, and cell death. Similarly, comparative protein expression analysis via iTRAQ, immunoblot, and immunolocalization, identified differential expression and localization of important regulators of transcription, translation, RNA processing, and spermatogenesis. Specifically, the testis-expressed transcription factor, Tbx1, and the piRNA regulator of gamete development, Piwil1, were both found to be targeted for translational repression by MSI2. This study provides key evidence to support a fundamental role for MSI2 in post-transcriptional regulation during male gamete development.

Oral squamous cell carcinoma (OSCC), as the most common type of oral cancer, is responsible for almost 3% of all malignant tumors worldwide. Non-coding RNAs such as lncRNAs and microRNAs have been involved in many cancers including OSCC. Recently, lncRNA metastasis-associated lung adenocarcinoma transcript-1 (MALAT1) has been reported to play an oncogenic role in OSCC metastasis. However, the underlying mechanism of MALAT1 in regulating OSCC progression remains unclear. The aim of this study was to investigate the specific role of MALAT1 in OSCC development. It was observed that MALAT1 was upregulated in OSCC cell lines. Inhibition of MALAT1 can prevent OSCC proliferation while overexpressing MALAT1 promoted OSCC progression. In addition, bioinformatics search was used to identify that miR-125b was a direct target of MALAT1, which indicated a negative correlation between MALAT1 and miR-125b. Besides these, STAT3 was predicted as a binding target of miR-125b in OSCC. Overexpression of MALAT1 was able to suppress the tumor inhibitory effect of miR-125b mimics via upregulating STAT3. Moreover, the function of MALAT1 in OSCC development was further investigated by using in vivo assays. The established nude mice models revealed that downregulated MALAT1 greatly inhibited OSCC tumor growth and reversely upregualated MALAT1 promoted OSCC development via miR-125b/STAT3 axis, respectively. In conclusion, MALAT1 can function as a competing endogenous RNA (ceRNA) to modulate STAT3 expression by absorbing miR-125b in OSCC and could be used as a novel therapeutic target in OSCC diagnosis and treatment.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy. Long non-coding RNAs (lncRNAs) are important regulators in pathological processes, yet their potential roles in PDAC are poorly understood. Here, we identify a fundamental role for a novel lincRNA, linc00511, in the progression of PDAC. Linc00511 levels in PDAC tissue specimens and cell lines were examined by quantitative real-time PCR. Corresponding adjacent non-neoplastic tissues were used as controls. The function of linc00511 in PDAC cell lines was determined by RNA interference approach in vitro and in vivo. Fluorescence in situ hybridization (FISH) was used to characterize linc00511 expression in PDAC cells. Insights of the mechanism of competitive endogenous RNAs (ceRNAs) were obtained from bioinformatic analysis, luciferase assays and RIP assays. The association between the linc00511/hsa-miR29b-3p axis and VEGFA was verified by Western blotting assay. Immunohistochemistry was performed to evaluate the expression of VEGFA in PDAC samples. The aberrant up-regulation of linc00511 was detected in PDAC cell lines and patient specimens compared with controls. An increase in linc00511 expression indicates the adverse clinical pathological characteristics and poor prognosis. Functionally, linc00511 depletion in PDAC cells decreased proliferation, migration, invasion and endothelial tube formation. Mechanistically, linc00511 could up-regulate VEGFA via its competing endogenous RNA (ceRNA) activity on hsa-miR-29b-3p. In summary, our results define an important axis controlling proliferation, invasion and tumour angiogenesis in PDAC. Linc00511 is a novel lncRNA that plays a significant regulatory role in the pathogenesis and progression of PDAC. Thus, Linc00511 represents a new prognostic biomarker to predict clinical outcome of PDAC patients after surgery and may serve as a potential therapeutic target for PDAC treatment.

Purpose: Emerging studies demonstrate that long noncoding RNAs (lncRNA) participate in the regulation of various cancers. In the current study, a novel lncRNA-TTN-AS1 has been identified and explored in esophageal squamous cell carcinoma (ESCC).Experimental Design: To discover a new regulatory circuitry in which RNAs crosstalk with each other, the transcriptome of lncRNA-miRNA-mRNA from ESCC and adjacent nonmalignant specimens were analyzed using multiple microarrays and diverse bioinformatics platforms. The functional role and mechanism of a novel lncRNA-TTN-AS1 were further investigated by gain-of-function and loss-of-function assays in vivo and in vitro An ESCC biomarker panel, consisting of lncRNA-TTN-AS1, miR-133b, and FSCN1, was validated by qRT-PCR and in situ hybridization using samples from 148 patients.Results:lncRNA-TTN-AS1 as an oncogene is highly expressed in ESCC tissues and cell lines, and promotes ESCC cell proliferation and metastasis. Mechanistically, lncRNA-TTN-AS1 promotes expression of transcription factor Snail1 by competitively binding miR-133b, resulting in the epithelial-mesenchymal transition (EMT) cascade. Moreover, lncRNA-TTN-AS1 also induces FSCN1 expression by sponging miR-133b and upregulation of mRNA-stabilizing protein HuR, which further promotes ESCC invasion cascades. We also discovered and validated a clinically applicable ESCC biomarker panel, consisting of lncRNA-TTN-AS1, miR-133b, and FSCN1, that is significantly associated with overall survival and provides additional prognostic evidence for ESCC patients.Conclusions: As a novel regulator, lncRNA-TTN-AS1 plays an important role in ESCC cell proliferation and metastasis. The lncRNA-TTN-AS1/miR133b/FSCN1 regulatory axis provides bona fide targets for anti-ESCC therapies. Clin Cancer Res; 24(2); 486-98. ©2017 AACR.

The function and the mechanism of long non-coding RNAs (lncRNAs) on the osteogenic differentiation of maxillary sinus membrane stem cells (MSMSCs) remain largely unknown.The expression of lnc-NTF3-5 and Runt-related transcription factor 2 (RUNX2), Osterix (OSX), and Alkaline Phosphatase (ALP) was examined by quantitative real-time PCR (qRT-PCR) in MSMSCs during the process osteogenic differentiation. Then the function of lnc-NTF3-5 was evaluated by loss- and gain-of-function techniques, as well as qRT-PCR, western blot, and Alizarin Red staining. In addition, the microRNAs (miRNAs) sponge potential of lnc-NTF3-5 was assessed through RNA immunoprecipitation, dual luciferase reporter assay, and in vivo ectopic bone formation.Lnc-NTF3-5, RUNX2, OSX, and ALP increased alone with the differentiation. Inhibition of lnc-NTF3-5 decreased the expression of RUNX2, OSX, and ALP both at mRNA and protein levels. Alizarin red staining showed similar trend. In contrast, overexpression of lnc-NTF3-5 presented totally opposite effects. Besides, overexpression of lnc-NTF3-5 could decrease the expression of microRNA-93-3p (miR-93-3p). Enhance miR-93-3p could also inhibit the expression level of lnc-NTF3-5. RNA immunoprecipitation demonstrated that lnc-NTF3-5 is directly bound to miR-93-3p and dual luciferase reporter assay proved that miR-93-3p targets 3' UTR of RUNX2 to regulate its expression. Ultimately, in vivo bone formation study showed that lnc-NTF3-5 and miR-93-3p inhibitor co-transfection group displayed the strongest bone formation.The novel pathway lnc-NTF3-5/miR-93-3p/RUNX2 could regulate osteogenic differentiation of MSMSCs and might serve as a therapeutic target for bone regeneration in the posterior maxilla.

To explore the core mechanism of long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) in the regulation of multidrug resistance of pancreatic cancer cells.mRNA levels of GAS5, miR-181c-5p and Hippo pathway related genes were detected by quantitative real-time PCR (qRT-PCR). Protein levels of MDR-1, MST1, YAP and TAZ were measured by western blot. Cell viability was detected by MTT assay. The combination between GAS5 and miR-181c-5p was confirmed by RNA pull-down and RNA immunoprecipitation (RIP) assay. We also established pancreatic cancer-bearing mice model and analyzed tumor volumes.Our data showed GAS5 expression was significantly down-regulated, miR-181c-5p expression was significantly up-regulated in pancreatic cancer cells. Besides, Overexpresson of GAS5 obviously inhibited cell viability, while GAS5 knockdown showed the opposite outcome. Additionally, we also found that GAS5 negatively regulated miR-181c-5p, and miR-181c-5p dramatically promoted pancreatic cancer cell chemoresistance through inactivating the Hippo signaling. GAS5 regulated chemoresistance and Hippo pathway of pancreatic cancer cells via miR-181c-5p/Hippo. Finally, we confirmed that overexpression of GAS5 inhibited tumor growth in pancreatic cancer-bearing mice model.GAS5 regualtes Hippo signaling pathway via miR-181c-5p to antagonize the development of multidrug resistance in pancreatic cancer cells.