RNA Binding Assays Research Articles

RETRACTED: LncRNA PVT1 Facilitates Tumorigenesis and Progression of Glioma via Regulation of MiR-128-3p/GREM1 Axis and BMP Signaling Pathway

The current research was aimed at probing into the role of long noncoding RNA (lncRNA) PVT1 in the pathogenesis of glioma and the regulatory mechanism of PVT1/miR-128-3p/GREM1 network in glioma via regulation of the bone morphogenetic protein (BMP) signaling pathway. Microarray analysis was used for preliminary screening for candidate lncRNAs and mRNAs in glioma tissues. Real-time quantitative polymerase chain reaction, Western blot, MTT assay, flow cytometry, migration and invasion assays, and xenograft tumor model were utilized to examine the influence of the lncRNA PVT1/miR-128-3p/GREM1 network on the biological functions of glioma cells. Luciferase assay and RNA-binding protein immunoprecipitation assay were used to validate the miR-128-3p-target relationships with lncRNA PVT1 or GREM1. In addition, the impact of GREM1 on BMP signaling pathway downstream proteins BMP2 and BMP4 was detected via Western blot. LncRNA PVT1 was highly expressed in human glioma tissues and significantly associated with WHO grade (I-II vs III-IV; p < 0.05). There existed a regulatory relationship between lncRNA PVT1 and miR-128-3p as well as that between miR-128-3p and GREM1. MiR-128-3p was downregulated, whereas GREM1 was upregulated in glioma tissues in comparison with para-carcinoma tissues. Overexpression of GREM1 promoted the proliferation and metastatic potential of glioma cells, whereas miR-128-3p mimics inhibited the glioma cell activity through targeting GREM1. Furthermore, lncRNA PVT1 acted as a sponge of miR-128-3p and, thus, influenced the BMP signaling pathway downstream proteins BMP2 and BMP4 through regulating GREM1. LncRNA PVT1 modulated GREM1 and BMP downstream signaling proteins through sponging miR-128-3p, thereby promoting tumorigenesis and progression of glioma.

The impact of Cymbopogon martinii essential oil on Cutibacterium (formerly Propionibacterium) acnes strains and its interaction with keratinocytes.

The human skin microbiota is mainly composed of bacteria belonging to the genera Staphylococcus, Cutibacterium, Micrococcus and Corynebacterium, but on the skin of the face and back, ca. 50% of the total microbiota is represented by the bacterium Cutibacterium acnes. The aim of this research was to evaluate the impact of C.martini EO and its major compound, geraniol, on C.acnes. The minimum inhibitory concentration against C.acnes strains, phenotypic changes and responses of the proteome was determined. In addition, was assessed the effect of compounds in RNA-binding assay, on C.acnes-exposed keratinocytes and on the C.acnes type distribution on shoulder skin. The range of the MIC was 0.7 to 1.6mg/ml for the three main C.acnes types. There were no cytotoxic effects of compounds in the absence or presence of C.acnes; after 7days of exposure to C.martini EO, we could not detect a major shift of the C.acnes types on shoulder skin that was found to be dominated by C.acnes strains of types II and IA2. Our work gives novel insight into the skin microbiota-interacting properties of C.martini EO.

Upregulation of long noncoding RNA MEG3 inhibits the osteogenic differentiation of periodontal ligament cells.

This study aims to discuss long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) function of regulating osteogenesis in human periodontal ligament cells (hPDLCs). First, use of a mineralizing solution induced osteogenic differentiation of hPDLCs to establish a differentiated cell model. Through microarray analysis, we selected a lncRNA MEG3 with marked changes between differentiated and undifferentiated cells. The quantitative polymerase chain reaction was used to detect the MEG3 content and an enzyme-linked immunosorbent assay was used to detect changes in related proteins. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and apoptosis was measured by flow cytometry. Alizarin red staining was also used to evaluate cells' osteogenic level. Finally,RNA-binding protein immunoprecipitation assays were conducted to further clarify the endogenous relationship between MEG3 and bone morphogenetic protein 2 ( BMP2) in hPDLCs. MEG3 was downregulated in osteogenic differentiation hPDLCs induced by mineralizing solution. Overexpression of MEG3 inhibited cell viability and increased cell apoptosis. MEG3 overexpression can reverse osteogenic differentiation induced by mineralizing solution. MEG3 can suppress BMP2 through interaction with heterogeneous nuclear ribonucleoprotein I. Upregulation of MEG3 inhibits the osteogenic differentiation of periodontal ligament cells by downregulating BMP2 expression.

LncRNA NBR2 inhibits epithelial-mesenchymal transition by regulating Notch1 signaling in osteosarcoma cells.

Long noncoding RNAs (lncRNAs) have been identified to have increasingly important roles in tumorigenesis, and they may serve as novel biomarkers for cancer therapy. Recent studies have demonstrated that lncRNA NBR2 (neighbor of BRCA1 gene 2), a novel identified lncRNA, is decreased in several cancers; however, the role of NBR2 in the development of osteosarcoma has not been elucidated. In our study, we found that NBR2 expression was downregulated in osteosarcoma tissues, and osteosarcoma cases with lower NBR2 expression exhibited a shorter overall survival time compared with those with higher NBR2 expression. NBR2 overexpression inhibited osteosarcoma cell proliferation, invasion, and migration but did not increase apoptosis. Furthermore, RNA-binding protein immunoprecipitation assays confirmed that NBR2 directly binds to Notch1 protein. Furthermore, overexpression of Notch1 in NBR2-overexpressing osteosarcoma cells reversed the effects of NBR2 on cell proliferation, invasion, migration, and epithelial-mesenchymal transition. The in vivo results showed that NBR2 overexpression inhibited tumor growth in nude mice that were inoculated with osteosarcoma cells. NBR2 overexpression also suppressed the messenger RNA (mRNA) expression of Notch1, N-cadherin, and vimentin and increased the mRNA expression of E-cadherin in the tumor tissues. These data indicated that NBR2 served as a tumor suppressor gene in osteosarcoma and inhibited osteosarcoma cell proliferation, invasion, and migration. The current study provides a novel insight and treatment strategy for osteosarcoma.

Long noncoding RNA H19 competitively binds miR-93-5p to regulate STAT3 expression in breast cancer.

The long noncoding RNA H19 is overexpressed in many cancers and acts as an oncogene. Here, we explored the role of H19 in breast cancer cells, including the effect of H19 on proliferation, migration, and invasion of breast cancer cells. We also investigated the relation of H19 to microRNA miR-93-5p and signal transducers and activators of transcription 3 (STAT3), the target gene of miR-93-5p. Ectopic expression of H19 in MCF-7 cells and knockdown of H19 in MDA-MB-231 cells showed that overexpression of H19 promoted proliferation, migration, and invasion, whereas knockdown of H19 reduced proliferation, migration, and invasion in vitro. Dual-luciferase reporter assays and RNA-binding protein immunoprecipitation assays proved that H19 was a target of miR-93-5p. In addition, H19 antagonized the downregulation of miR-93-5p on its target STAT3 and antagonized miR-93-5p-mediated cell proliferation. Our study revealed a new network in the expression of STAT3 involving H19 and miR-93-5p, which may contribute to a better understanding of breast cancer pathogenesis and provide new insights into the treatment of this disease.

Long noncoding RNA MALAT1 acts as a competing endogenous RNA to regulate Amadori-glycated albumin-induced MCP-1 expression in retinal microglia by a microRNA-124-dependent mechanism.

To determine whether the long noncoding RNA MALAT1 may be involved in the inflammatory effect of Amadori-glycated albumin (AGA) in retinal microglia via a microRNA-124 (miR-124)-dependent mechanism. Diabetes mellitus was induced by streptozotocin (STZ) injection. The expression of monocyte chemotactic protein-1 (MCP-1) in the retinas of rats was determined using quantitative reverse transcription-PCR (qRT-PCR) analyses and enzyme-linked immunosorbent assay (ELISA). Both qRT-PCR and ELISA were used to detect the levels of MCP-1 mRNA and soluble MCP-1 protein in the primary rat retinal microglia treated with AGA. The regulation of a putative target of miR-124 was validated by luciferase reporter assays. MALAT1 knockdown ameliorated diabetic retinopathy (DR) and inhibited MCP-1 release in the retinas of STZ-induced diabetic rats. The cultured retinal microglial cells treated with AGA-released MCP-1 in a dose- and time-dependent manner. In addition, AGA consistently induced MALAT1 expression in the retinal microglial cells. Next, we demonstrated that the expression of MCP-1 is controlled by miR-124, which binds to the 3'-UTR of MCP-1 in microglial cells. Luciferase reporter assays and RNA-binding protein immunoprecipitation assays showed that MALAT1 targets miR-124. Finally, we demonstrated that MALAT1 acts as a competing endogenous RNA by directly binding to miR-124 to regulate AGA-induced MCP-1 expression in microglial cells. MALAT1-miR-124-MCP-1 signaling pathway may be involved in AGA-induced MCP-1 expression in microglial cells, which may provide a new approach for the treatment of DR.

Dicer1 Deficiency in the Idiopathic Pulmonary Fibrosis Fibroblastic Focus Promotes Fibrosis by Suppressing MicroRNA Biogenesis.

The lung extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) mediates progression of fibrosis by decreasing fibroblast expression of miR-29 (microRNA-29), a master negative regulator of ECM production. The molecular mechanism is undefined. IPF-ECM is stiffer than normal. Stiffness drives fibroblast ECM production in a YAP (yes-associated protein)-dependent manner, and YAP is a known regulator of miR-29. Therefore, we tested the hypothesis that negative regulation of miR-29 by IPF-ECM was mediated by mechanotransduction of stiffness. To determine how IPF-ECM negatively regulates miR-29. We decellularized lung ECM using detergents and prepared polyacrylamide hydrogels of defined stiffness by varying acrylamide concentrations. Mechanistic studies were guided by immunohistochemistry of IPF lung and used cell culture, RNA-binding protein assays, and xenograft models. Contrary to our hypothesis, we excluded fibroblast mechanotransduction of ECM stiffness as the primary mechanism deregulating miR-29. Instead, systematic examination of miR-29 biogenesis revealed a microRNA processing defect that impeded processing of miR-29 into its mature bioactive forms. Immunohistochemical analysis of the microRNA processing machinery in IPF lung specimens revealed decreased Dicer1 expression in the procollagen-rich myofibroblastic core of fibroblastic foci compared with the focus perimeter and adjacent alveolar walls. Mechanistically, IPF-ECM increased association of the Dicer1 transcript with RNA binding protein AUF1 (AU-binding factor 1), and Dicer1 knockdown conferred primary human lung fibroblasts with cell-autonomous fibrogenicity in zebrafish and mouse lung xenograft models. Our data identify suppression of fibroblast Dicer1 expression in the myofibroblast-rich IPF fibroblastic focus core as a central step in the mechanism by which the ECM sustains fibrosis progression in IPF.

The RNA-binding Protein MEX3B Mediates Resistance to Cancer Immunotherapy by Downregulating HLA-A Expression.

Purpose: Cancer immunotherapy has shown promising clinical outcomes in many patients. However, some patients still fail to respond, and new strategies are needed to overcome resistance. The purpose of this study was to identify novel genes and understand the mechanisms that confer resistance to cancer immunotherapy.Experimental Design: To identify genes mediating resistance to T-cell killing, we performed an open reading frame (ORF) screen of a kinome library to study whether overexpression of a gene in patient-derived melanoma cells could inhibit their susceptibility to killing by autologous tumor-infiltrating lymphocytes (TIL).Results: The RNA-binding protein MEX3B was identified as a top candidate that decreased the susceptibility of melanoma cells to killing by TILs. Further analyses of anti-PD-1-treated melanoma patient tumor samples suggested that higher MEX3B expression is associated with resistance to PD-1 blockade. In addition, significantly decreased levels of IFNγ were secreted from TILs incubated with MEX3B-overexpressing tumor cells. Interestingly, this phenotype was rescued upon overexpression of exogenous HLA-A2. Consistent with this, we observed decreased HLA-A expression in MEX3B-overexpressing tumor cells. Finally, luciferase reporter assays and RNA-binding protein immunoprecipitation assays suggest that this is due to MEX3B binding to the 3' untranslated region (UTR) of HLA-A to destabilize the mRNA.Conclusions: MEX3B mediates resistance to cancer immunotherapy by binding to the 3' UTR of HLA-A to destabilize the HLA-A mRNA and thus downregulate HLA-A expression on the surface of tumor cells, thereby making the tumor cells unable to be recognized and killed by T cells. Clin Cancer Res; 24(14); 3366-76. ©2018 AACRSee related commentary by Kalbasi and Ribas, p. 3239.

Abstract 4445: Post-transcriptional inhibition of PTPN23 by SND1: Potential mechanism for hepatocellular carcinoma

Abstract Oncoprotein SND1 regulates gene expression at a post-transcriptional level in multiple cancers including hepatocellular carcinoma (HCC). Staphylococcal nuclease domains of SND1 function as an RNAse and tudor domain facilitates protein-oligonucleotide interaction. In the present study, we aimed to identify RNA interactome of SND1 to obtain better insights into gene regulation by SND1. RNA interactome was identified by immunoprecipitation of RNA using anti-SND1 antibody from human HCC cells followed by RNA-sequencing (RIP-Seq). With an adjusted p value of &amp;lt;0.01 and &amp;gt;2-fold enrichment over control, 282 mRNAs were identified to significantly associate with SND1 protein. We focused on the tumor suppressor PTPN23 because its regulation by SND1 and its role in HCC are not known. PTPN23 levels were downregulated in human HCC cells versus normal hepatocytes and in human HCC tissues versus normal adjacent liver as revealed by immunohistochemistry. In human HCC cells, knocking down SND1 increased and overexpression of SND1 decreased PTPN23 protein. RNA binding and degradation assays revealed that SND1 specifically binds to and degrades 3'-UTR of PTPN23 mRNA. Tetracycline-inducible PTPN23 overexpression in human HCC cells resulted in significant inhibition in proliferation, migration and invasion and in vivo tumorigenesis. PTPN23 induction caused inhibition in activation of c-Met, EGFR, Src and FAK, suggesting that as a phosphatase PTPN23 inhibits activation of these oncogenic kinases. The present study unravels a novel function of SND1 in targeting PTPN23 and identifies PTPN23 as a unique tumor suppressor for HCC. PTPN23 might function as a homeostatic regulator of multiple kinases restraining their activation. Citation Format: Nidhi H. Jariwala, Rachel G. Mendoza, Garcia Dawn, Lai Zhao, Mark A. Subler, Jolene J. Windle, Paul B. Fisher, Chen Yidong, Devanand Sarkar. Post-transcriptional inhibition of PTPN23 by SND1: Potential mechanism for hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4445.

Novel insights into nucleoamino acids: biomolecular recognition and aggregation studies of a thymine-conjugated L-phenyl alanine.

This article deals with the synthesis in solid phase and characterization of a nucleoamino amide, based on a phenylalaninamide moiety which was N-conjugated to a thymine nucleobase. In analogy to the natural nucleobase-amino acid conjugates, endowed with a wide range of biological properties, the nucleoamino amide interacts with single-stranded nucleic acids as verified in DNA- and RNA-binding assays conducted by CD and UV spectroscopies. These technologies were used to show also that this conjugate binds serum proteins altering significantly their secondary structure, as evidenced by CD and UV using BSA as a model. The biomolecular recognition seems to rely on the ability of the novel compound to bind aromatic and heteroaromatic moieties in protein and nucleic acids, not hindered by its propensity to self-assemble in aqueous solution, behavior suggested by dynamic light scattering (DLS) and CD spectroscopy in concentration- and temperature-dependent experiments. Finally, the high stability in human serum concurs to define the picture of the nucleoamino amide: this enzymatically stable drug candidate could interfere with protein and single-stranded nucleic acid-driven biological processes, particularly those associated with mRNA poly(A) tail, and its self-assembling nature, in analogy to other L-Phe-based systems, discloses new scenarios in drug delivery technology.

Defensive effect of microRNA-200b/c against amyloid-beta peptide-induced toxicity in Alzheimer's disease models.

MiRNA molecules are important post-transcriptional regulators of gene expression in the brain function. Altered miRNA profiles could represent a defensive response against the pathogenesis of neurodegenerative disorders, such as Alzheimer's disease (AD). Endogenous miRNAs have lower toxic effects than other gene silencing methods, thus enhancing the expression of defensive miRNA could be an effective therapy. However, little is known about the potential of targeting miRNAs for the treatment of AD. Here, we examined the function of the miR-200 family (miR-200a, -141, -429, -200b, -200c), identified using miRNA microarray analysis of cortical tissue from Tg2576 transgenic mice. In murine primary neurons, we found that upregulation of miR-200b or -200c was induced by the addition of amyloid beta (Aβ). Neurons transfected with miR-200b or -200c reduced secretion of Aβ in conditioned medium. Moreover, mice infused with miR-200b/c into the brain were relieved of memory impairments induced by intracerebroventricular injection of oligomeric Aβ, and demonstrated proper spatial learning in the Barnes maze. To gain further understanding of the relationship between miR-200b/c and Aβ, we identified target mRNAs via an RNA-binding protein immunoprecipitation-microarray assay. Western blot analysis showed that expression of ribosomal protein S6 kinase B1 (S6K1), a candidate target, was inhibited by miR-200c. S6K1, a downstream effector of mammalian target of rapamycin (mTOR), serves as a negative feedback mediator that phosphorylates insulin receptor substrate 1 at serine residues (IRS-1pSer). S6K1-dependent IRS-1pSer suppresses insulin signaling leading to insulin resistance, which is frequently observed in AD brains. Notably, miR-200b/c transfection of SH-SY5Y cells reduced the levels of IRS-1pSer. This finding indicates that miR-200b/c has the potential to alleviate insulin resistance via modulation of S6K1. Taken together, miR-200b/c may contribute to reduce Aβ secretion and Aβ-induced cognitive impairment by promoting insulin signaling.

Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5' splice site.

Phenylketonuria (PKU), one of the most common inherited diseases of amino acid metabolism, is caused by mutations in the phenylalanine hydroxylase (PAH) gene. Recently, PAH exon 11 was identified as a vulnerable exon due to a weak 3’ splice site, with different exonic mutations affecting exon 11 splicing through disruption of exonic splicing regulatory elements. In this study, we report a novel intron 11 regulatory element, which is involved in exon 11 splicing, as revealed by the investigated pathogenic effect of variants c.1199+17G>A and c.1199+20G>C, identified in PKU patients. Both mutations cause exon 11 skipping in a minigene system. RNA binding assays indicate that binding of U1snRNP70 to this intronic region is disrupted, concomitant with a slightly increased binding of inhibitors hnRNPA1/2. We have investigated the effect of deletions and point mutations, as well as overexpression of adapted U1snRNA to show that this splicing regulatory motif is important for regulation of correct splicing at the natural 5’ splice site. The results indicate that U1snRNP binding downstream of the natural 5’ splice site determines efficient exon 11 splicing, thus providing a basis for development of therapeutic strategies to correct PAH exon 11 splicing mutations. In this work, we expand the functional effects of non-canonical intronic U1 snRNP binding by showing that it may enhance exon definition and that, consequently, intronic mutations may cause exon skipping by a novel mechanism, where they disrupt stimulatory U1 snRNP binding close to the 5’ splice site. Notably, our results provide further understanding of the reported therapeutic effect of exon specific U1 snRNA for splicing mutations in disease.

An evolutionarily conserved P-subfamily pentatricopeptide repeat protein is required to splice the plastid ndhA transcript in the moss Physcomitrella patens and Arabidopsis thaliana.

Pentatricopeptide repeat (PPR) proteins are known to play important roles in post-transcriptional regulation in plant organelles. However, the function of the majority of PPR proteins remains unknown. To examine their functions, Physcomitrella patens PpPPR_66 knockout (KO) mutants were generated and characterized. The KO mosses exhibited a wild-type-like growth phenotype but showed aberrant chlorophyll fluorescence due to defects in chloroplast NADH dehydrogenase-like (NDH) activity. Immunoblot analysis suggested that disruption of PpPPR_66 led to a complete loss of the chloroplast NDH complex. To examine whether the loss of PpPPR_66 affects the expression of plastid ndh genes, the transcript levels of 11 plastid ndh genes were analyzed by reverse transcription PCR. This analysis indicated that splicing of the ndhA transcript was specifically impaired while mRNA accumulation levels as well as the processing patterns of other plastid ndh genes were not affected in the KO mutants. Complemented PpPPR_66 KO lines transformed with the PpPPR_66 full-length cDNA rescued splicing of the ndhA transcript. Arabidopsis thaliana T-DNA tagged lines of a PPR_66 homolog (At2g35130) showed deficient splicing of the ndhA transcript. This indicates that the two proteins are functionally conserved between bryophytes and vascular plants. An invitro RNA-binding assay demonstrated that the recombinant PpPPR_66 bound preferentially to the region encompassing a part of exon 1 to a 5' part of the ndhA group II intron. Taken together, these results indicate that PpPPR_66 acts as a specific factor to splice ndhA pre-mRNA.

Studying the mechanism of PLAGL2 overexpression and its carcinogenic characteristics based on 3'-untranslated region in colorectal cancer

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.

Long noncoding RNA HOTAIR mediates the estrogen-induced metastasis of endometrial cancer cells via the miR-646/NPM1 axis.

LncRNA homeobox (HOX) transcript antisense intergenic RNA (HOTAIR) has been confirmed to be involved in the tumorigenic progression of endometrial carcinoma (EC). However, the molecular mechanisms of HOTAIR in EC are not fully elucidated. The expression of HOTAIR and miR-646 in human EC tissues was determined by qRT-PCR. The effect of miR-646 on EC cells was assessed by the cell viability, migration, and invasion using CCK-8 assays and transwell assays. RNA-binding protein immunoprecipitation assays and RNA pull-down assays were performed to explore the interaction between HOTAIR and miR-646. The regulation of miR-646 on nucleophosmin 1 (NPM1) was tested using luciferase reporter assays. MiR-646 expression was significantly decreased both in human EC tissues ( n = 23) and cell lines (Ishikawa and HEC-1-A) compared with the control. Moreover, miR-646 expression was negatively related to HOTAIR in human EC tissues ( n = 23). Our results also showed that miR-646 overexpression considerably attenuated the E2-promoted viability, migration, and invasion of Ishikawa and HEC-1-A cells in vitro. In addition, HOTAIR was confirmed to regulate the viability, migration, and invasion of EC cells through negative regulating miR-646. More importantly, we also demonstrated that NPM1 was the target of miR-646, and HOTAIR promoted NPM1 expression through interacting with miR-646 in EC cells. Taken together, our findings presented that HOTAIR could regulate NPM1 via interacting with miR-646, thereby governing the viability, migration, and invasion of EC cells.

Repression of PDK1- and LncRNA HOTAIR-Mediated EZH2 Gene Expression Contributes to the Enhancement of Atractylenolide 1 and Erlotinib in the Inhibition of Human Lung Cancer Cells

Background/Aims: We previously showed that the major bioactive compound of Atractylodes macrocephula Koidz atractylenolide 1 (ATL-1) inhibited human lung cancer cell growth by suppressing the gene expression of 3-Phosphoinositide dependent protein kinase-1 (PDK1 or PDPK1). However, the potentially associated molecules and downstream effectors of PDK1 underlying this inhibition, particularly the mechanism for enhancing the anti-tumor effects of epidermal growth factor receptor-tyrosine-kinase inhibitors (EGFR-TKIs), remain unknown. Methods: Cell viability and cell cycle distribution were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. Western blot analyses were performed to examine the protein expressions of PDK1 and of zeste homolog 2 (EZH2). The levels of long non-coding RNA (lncRNA) and HOX transcript antisense RNA (HOTAIR) were examined via qRT-PCR. RNA-binding protein immunoprecipitation assays were used to analyze HOTAIR interaction with EZH2. The promoter activity of the EZH2 gene was determined using Secrete-Pair Dual Luminescence Assay Kit. Exogenous expressions of PDK1, HOTAIR, and EZH2 were conducted via transient transfection assays. A xenografted tumor model was used to further evaluate the effect of ATL-1 in the presence or absence of erlotinib in vivo. Results: We showed that the combination of ATL-1 and EGFR-TKI erlotinib further inhibited growth and induced cell arrest of the human lung cancer cells, determined by both MTT and flow cytometry assays. ATL-1 inhibited the protein expression and the promoter activity of EZH2, which was reversed in cells with PDK1 overexpression. Interestingly, ATL-1 inhibited the expression levels of HOTAIR. While silencing HOTAIR inhibited the expressions of PDK1 and EZH2, overexpression of HOTAIR reduced the ATL-1-reduced PDK1 and EZH2 protein expressions and EZH2 promoter activity. In addition, ATL-1 reduced the HOTAIR binding to the EZH2 protein. Moreover, we found that exogenously expressed EZH2 antagonized the effect of ATL-1 on cell growth inhibition. Consistent with the in vitro results, ATL-1 inhibited tumor growth and the expression levels of HOTAIR, protein expressions of EZH2 and PDK1 in vivo. Importantly, there was synergy of the combination of ATL-1 and erlotinib in this process. Conclusion: Here, we provide the first evidence that ATL-1 inhibits lung cancer cell growth through inhibiting not only the PDK1 but also the lncRNA HOTAIR, which results in the reduction of one downstream effector EZH2 expression. The novel interplay between the HOTAIR and EZH2, as well as repressions of the PDK1 and HOTAIR coordinate the overall effects of ATL-1. Importantly, the combination of ATL-1 and EGFR-TKI erlotinib exhibits synergy. Thus, targeting the PDK1- and HOTAIR-mediated downstream molecule EZH2 by the combination of ATL-1 and erlotinib potentially facilitates the development of an additional novel strategy to combat lung cancer.

Structural insights into the assembly and polyA signal recognition mechanism of the human CPSF complex.

3' polyadenylation is a key step in eukaryotic mRNA biogenesis. In mammalian cells, this process is dependent on the recognition of the hexanucleotide AAUAAA motif in the pre-mRNA polyadenylation signal by the cleavage and polyadenylation specificity factor (CPSF) complex. A core CPSF complex comprising CPSF160, WDR33, CPSF30 and Fip1 is sufficient for AAUAAA motif recognition, yet the molecular interactions underpinning its assembly and mechanism of PAS recognition are not understood. Based on cross-linking-coupled mass spectrometry, crystal structure of the CPSF160-WDR33 subcomplex and biochemical assays, we define the molecular architecture of the core human CPSF complex, identifying specific domains involved in inter-subunit interactions. In addition to zinc finger domains in CPSF30, we identify using quantitative RNA-binding assays an N-terminal lysine/arginine-rich motif in WDR33 as a critical determinant of specific AAUAAA motif recognition. Together, these results shed light on the function of CPSF in mediating PAS-dependent RNA cleavage and polyadenylation.

Identification of Splicing Factors Involved in DMD Exon Skipping Events Using an In Vitro RNA Binding Assay.

Mutation-induced exon skipping in the DMD gene can modulate the severity of the phenotype in patients with Duchenne or Becker Muscular Dystrophy. These alternative splicing events are most likely the result of changes in recruitment of splicing factors at cis-acting elements in the mutated DMD pre-mRNA. The identification of proteins involved can be achieved by an affinity purification procedure. Here, we provide a detailed protocol for the in vitro RNA binding assay that we routinely apply to explore molecular mechanisms underlying splicing defects in the DMD gene. In vitro transcribed RNA probes containing either the wild type or mutated sequence are oxidized and bound to adipic acid dihydrazide-agarose beads. Incubation with a nuclear extract allows the binding of nuclear proteins to the RNA probes. The unbound proteins are washed off and then the specifically RNA-bound proteins are released from the beads by an RNase treatment. After separation by SDS-PAGE, proteins that display differential binding affinities for the wild type and mutant RNA probes are identified by mass spectrometry.

Proteomic analyses of nucleus laminaris identified candidate targets of the fragile X mental retardation protein.

The avian nucleus laminaris (NL) is a brainstem nucleus necessary for binaural processing, analogous in structure and function to the mammalian medial superior olive. In chickens (Gallus gallus), NL is a well-studied model system for activity-dependent neural plasticity. Its neurons have bipolar extension of dendrites, which receive segregated inputs from two ears and display rapid and compartment-specific reorganization in response to unilateral changes in auditory input. More recently, fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates local protein translation, has been shown to be enriched in NL dendrites, suggesting its potential role in the structural dynamics of these dendrites. To explore the molecular role of FMRP in this nucleus, we performed proteomic analysis of NL, using micro laser capture and liquid chromatography tandem mass spectrometry. We identified 657 proteins, greatly represented in pathways involved in mitochondria, translation and metabolism, consistent with high levels of activity of NL neurons. Of these, 94 are potential FMRP targets, by comparative analysis with previously proposed FMRP targets in mammals. These proteins are enriched in pathways involved in cellular growth, cellular trafficking and transmembrane transport. Immunocytochemistry verified the dendritic localization of several proteins in NL. Furthermore, we confirmed the direct interaction of FMRP with one candidate, RhoC, by in vitro RNA binding assays. In summary, we provide a database of highly expressed proteins in NL and in particular a list of potential FMRP targets, with the goal of facilitating molecular characterization of FMRP signaling in future studies.

Abstract 3098: Nanotherapeutics for combination drug and gene therapy in treating glioblastoma multiforme

Abstract Gliomas represent approximately 80% of all malignant brain tumors, and glioblastoma multiforme (GBM), the most aggressive type, accounts for nearly half of all gliomas. Despite treatment strategies including surgery, radiation, and chemotherapy, the 5-year survival rate for brain cancer is only 35%. New therapeutic strategies are necessary to improve the outcomes of this disease. Chemotherapy with DNA alkylating agents is commonly used in treatment for GBM. Research has shown that better therapeutic response of GBM tumors to alkylating agents and increased survival rate is indicative in patients with epigenetic silencing of O6-methylguanine-DNA methyltransferase (MGMT), a gene responsible for DNA repair. Therefore, we propose a treatment strategy combining drug and gene therapy to target and silence MGMT to sensitize cells to treatment with temozolomide (TMZ) or lomustine (CCNU), both DNA alkylating agents. We previously developed cationic, amphiphilic copolymer poly(lactide-co-glycolide)-g-polyethylenimine (PgP) and demonstrated its utility for nucleic acid delivery. Here, we examine the ability of PgP as a drug and siRNA delivery carrier to overcome drug resistance and improve anticancer activity through combination drug and gene therapy for GBM treatment. PgP micelles were designed and synthesized for delivery of hydrophobic drugs in the PLGA core and negatively charged nucleic acids in the positively charged PEI shell through electrostatic interactions. RNA binding and polyplex stability assays were performed using agarose gel electrophoresis. Cytotoxicity of TMZ, CCNU, and/or PgP/siMGMT polyplexes was determined by MTT assay. Silencing of MGMT on the protein and mRNA level was determined using western blotting and qPCR, respectively. Our results demonstrated that PgP effectively forms stable complexes with siRNA and protects siRNAs from serum- and ribonuclease-mediated degradation, confirming the potential of the polyplex for in vivo delivery. We demonstrated that PgP/siMGMT polyplexes mediate knockdown of MGMT protein as well as a significant ~56% and ~68% knockdown of MGMT mRNA in T98G GBM cells compared to cells treated with PgP complexed with non-targeting siRNA (siNT) at a 60:1 and 80:1 nitrogen:phosphate (N:P) ratio, respectively. Further, co-treatment of PgP/siMGMT polyplexes with TMZ or CCNU enhanced anticancer activity in T98G GBM cells compared to treatment with the PgP/siMGMT polyplex, TMZ, or CCNU alone. Future studies will determine efficacy of drug-loaded and siRNA-complexed PgP for combination therapy in vitro as well as using a xenograft GBM model for local delivery. Successful combinatorial drug and gene therapy using PgP may overcome drug resistance and improve therapeutic outcomes for patients with glioblastoma. Citation Format: Angela A. Alexander-Bryant, Breanne Hourigan, Michael Lynn, Jeoung Soo Lee. Nanotherapeutics for combination drug and gene therapy in treating glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3098. doi:10.1158/1538-7445.AM2017-3098