Radiation-induced lattice relaxation in [Formula: see text]-Fe[Formula: see text]O[Formula: see text] nanorods.

We report radiation-induced lattice relaxation of the [Formula: see text]-Fe[Formula: see text]O[Formula: see text] and its associated alteration of particle morphology. The [Formula: see text]-Fe[Formula: see text]O[Formula: see text] was grown in solution by microwave hydrothermal synthesis technique in which more than half of the synthesized material was nanorods with axis along the (001) direction. Five sets of the synthesized [Formula: see text]-Fe[Formula: see text]O[Formula: see text] samples were irradiated using gamma-ray from [Formula: see text]Co cell with doses of 600 kGy, 700 kGy, 800 kGy, 900 kGy, and 1 MGy. The investigation of the pristine and gamma-irradiated samples was carried out using X-ray powder diffraction, transmission electron microscope, and electron paramagnetic resonance methods. Results showed that continuous alternation of radiation-induced lattice compression and expansion causes lattice relaxation. The morphology of the [Formula: see text]-Fe[Formula: see text]O[Formula: see text] nanorods was found to change with absorbed dose into buckyball-shaped particles in response to the alternation of the compression and expansion strain. The EPR results showed a correlation between distortion in the [Formula: see text]-[Formula: see text] octahedron structure and the relaxation of the lattice. The synthesis, growth, and relaxation are discussed in detail.

Piwil1 Regulates Glioma Stem Cell Maintenance and Glioblastoma Progression.

Piwi proteins are a subfamily of Argonaute proteins that maintain germ cells in eukaryotes. However, the role of their human homologs in cancer stem cells, and more broadly in cancer, is poorly understood. Here, we report that Piwi-like family members are overexpressed in glioblastoma (GBM), with Piwil1 (Hiwi) most frequently overexpressed (88%). Piwil1 is enriched in glioma stem-like cells (GSCs) to maintain self-renewal. Silencing Piwil1 in GSCs leads to global changes in gene expression resulting in cell-cycle arrest, senescence, or apoptosis. Piwil1 knockdown increases expression of the transcriptional co-regulator BTG2 and the E3-ubiquitin ligase FBXW7, leading to reduced c-Myc expression, as well as loss of expression of stem cell factors Olig2 and Nestin. Piwil1 regulates mRNA stability of BTG2, FBXW7, and CDKN1B. In animal models of GBM, Piwil1 knockdown suppresses tumor growth and promotes mouse survival. These findings support a role of Piwil1 in GSC maintenance and glioblastoma progression.

Myeloid-Derived Suppressor Cell Subsets Drive Glioblastoma Growth in a Sex-Specific Manner.

Myeloid-derived suppressor cells (MDSC) that block antitumor immunity are elevated in glioblastoma (GBM) patient blood and tumors. However, the distinct contributions of monocytic (mMDSC) versus granulocytic (gMDSC) subsets have yet to be determined. In mouse models of GBM, we observed that mMDSCs were enriched in the male tumors, whereas gMDSCs were elevated in the blood of females. Depletion of gMDSCs extended survival only in female mice. Using gene-expression signatures coupled with network medicine analysis, we demonstrated in preclinical models that mMDSCs could be targeted with antiproliferative agents in males, whereas gMDSC function could be inhibited by IL1ß blockade in females. Analysis of patient data confirmed that proliferating mMDSCs were predominant in male tumors and that a high gMDSC/IL1ß gene signature correlated with poor prognosis in female patients. These findings demonstrate that MDSC subsets differentially drive immune suppression in a sex-specific manner and can be leveraged for therapeutic intervention in GBM. SIGNIFICANCE: Sexual dimorphism at the level of MDSC subset prevalence, localization, and gene-expression profile constitutes a therapeutic opportunity. Our results indicate that chemotherapy can be used to target mMDSCs in males, whereas IL1 pathway inhibitors can provide benefit to females via inhibition of gMDSCs.See related commentary by Gabrilovich et al., p. 1100.This article is highlighted in the In This Issue feature, p. 1079.

Transcriptome dynamics of long noncoding RNAs and transcription factors demarcate human neonatal, adult, and human mesenchymal stem cell-derived engineered cartilage.

The engineering of a native-like articular cartilage (AC) is a long-standing objective that could serve the clinical needs of millions of patients suffering from osteoarthritis and cartilage injury. An incomplete understanding of the developmental stages of AC has contributed to limited success in this endeavor. Using next generation RNA sequencing, we have transcriptionally characterized two critical stages of AC development in humans-that is, immature neonatal and mature adult, as well as tissue-engineered cartilage derived from culture expanded human mesenchymal stem cells. We identified key transcription factors (TFs) and long noncoding RNAs (lncRNAs) as candidate drivers of the distinct phenotypes of these tissues. AGTR2, SCGB3A1, TFCP2L1, RORC, and TBX4 stand out as key TFs, whose expression may be capable of reprogramming engineered cartilage into a more expandable and neonatal-like cartilage primed for maturation into biomechanically competent cartilage. We also identified that the transcriptional profiles of many annotated but poorly studied lncRNAs were dramatically different between these cartilages, indicating that lncRNAs may also be playing significant roles in cartilage biology. Key neonatal-specific lncRNAs identified include AC092818.1, AC099560.1, and KC877982. Collectively, our results suggest that tissue-engineered cartilage can be optimized for future clinical applications by the specific expression of TFs and lncRNAs.

A FAM83A Positive Feed-back Loop Drives Survival and Tumorigenicity of Pancreatic Ductal Adenocarcinomas.

Pancreatic ductal adenocarcinomas (PDAC) are deadly on account of the delay in diagnosis and dearth of effective treatment options for advanced disease. The insurmountable hurdle of targeting oncogene KRAS, the most prevalent genetic mutation in PDAC, has delayed the availability of targeted therapy for PDAC patients. An alternate approach is to target other tumour-exclusive effector proteins important in RAS signalling. The Family with Sequence Similarity 83 (FAM83) proteins are oncogenic, tumour-exclusive and function similarly to RAS, by driving the activation of PI3K and MAPK signalling. In this study we show that FAM83A expression is significantly elevated in human and murine pancreatic cancers and is essential for the growth and tumorigenesis of pancreatic cancer cells. Elevated FAM83A expression maintains essential MEK/ERK survival signalling, preventing cell death in pancreatic cancer cells. Moreover, we identified a positive feed-forward loop mediated by the MEK/ERK-activated AP-1 transcription factors, JUNB and FOSB, which is responsible for the elevated expression of oncogenic FAM83A. Our data indicates that targeting the MEK/ERK-FAM83A feed-forward loop opens up additional avenues for clinical therapy that bypass targeting of oncogenic KRAS in aggressive pancreatic cancers.

Exon1 and -116 C/G Promoter Polymorphism on the X-Box DNA Binding Protein- 1 Gene is not Associated with Breast Cancer among Jordanian Women.

Background: Human X -box binding protein 1 (XBP1), a critical gene in the endoplasmic reticulum stress response, is located on chromosome 22q12, which has been linked with the pathogenesis of many diseases, particularly cancers such as breast cancer (BC). Single nucleotide polymorphisms (SNPs) in the XBP1 gene can alter structure and function of the gene. In this study, polymorphism in the promoter region and exon1 of the gene XBP1 and its association with BC in Jordanian women was investigated. Methods: Polymorphism in the promoter and exon1 of XBP1 was analyzed in 100 subjects (controls: n=40; BC patients=60). −116 C/G SNP was genotyped by Polymerase Chain Reaction (PCR)-sequence specific primer technique. The odd ratios (ORs) at 95% confidence intervals (CIs) were computed to assess the strength of this association. Results: The three genotypes of the SNP (GG, GC, CC) and their allelic frequencies have nonsignificant differences between patients and control group. It was noticed that the frequencies of the mutant allele (G) were (75.8% versus 24.2%)) in the patients and control groups, respectively, while those of the normal allele (C) were (67.5% versus 32.5%). XBP1 (-116 G→C) G allele did not show significant association with BC risk (confidence interval = 0.3534- 1.2395, odds ratio = 0.6619, P= 0.197). Moreover, there were no significant mutations in the XBP1 exon1 neither in BC subjects nor control subjects. Conclusions: This is the first study to evaluate the effect of polymorphism in the promoter and exon1 of XBP1 gene in the pathogenesis of BC in Jordanian women. The results do not support a role for polymorphism in development of BC and further studies with a larger sample size and detailed data should be performed in other populations.

Analysis of -5p and -3p Strands of miR-145 and miR-140 During Mesenchymal Stem Cell Chondrogenic Differentiation.

The chondrogenic differentiation of mesenchymal stem cells (MSCs) is mediated by transcription factors and small noncoding RNAs such as microRNAs (miRNAs). Each miRNA is initially transcribed as a long transcript, which matures to produce -5p and -3p strands. It is widely believed that the mature and functional miRNA from any given pre-miRNA, usually the -5p strand, is functional, while the opposing -3p strand is degraded. However, recent cartilage literature started to show functional -3p strands for a few miRNAs. This study aimed at examining both -5p and -3p strands of two key miRNAs miR-140 and miR-145, known to be involved in the chondrogenic differentiation of MSCs. The level (copy number) of both -5p and -3p strands of miR-145 and miR-140 along the time line of MSC chondrogenic differentiation was determined by polymerase chain reaction. The gene expression profiles of several genes related to MSC chondrogenesis were compared with these miRNA profiles along the same timeline. While miR-145-3p is declining in step with miR-145-5p in pellet cultures during the process, the -3p strand is only 1-2% of the total miR-145 products. In contrast, the mature -3p and -5p products of miR-140 are found to increase with near-equal molar expression throughout chondrogenic differentiation. Numerous genes are expressed by cartilage progenitor cells during development. One such target gene, Sox9, is a regulatory target of the dominant miR-145-5p, consistent with the data. Further experimental validations are warranted to confirm that ACAN, FOXO1, and RUNX3 as direct targets of miR-145-5p in the context of MSC chondrogenesis. Similarly, TRSP1 and ACAN are worth further validation as direct targets of miR-145-3p. For miR-140, SOX4 shall be further validated as a direct target of miR-140-5p, while KLF4, PTHLH, and WNT5A can be validated as direct targets of miR-140-3p.

In vivo combined treatment of rats with ivermectin and aged garlic extract attenuates ivermectin-induced cytogenotoxicity in bone marrow cells.

Ivermectin (IVM) is widely used in human and veterinary medicine for the control of parasitic infections. Researches revealed new avenues of medicinal applications of IVM as an antiviral and an anticancer agent. Very little is known about the genotoxic potential of IVM and the available literature is contradictory. The objective of this study was to evaluate the possible genetic damage caused by IVM. Male Sprague Dawley rats were intraperitoneally given IVM at doses between 0.2 mg and 3.2 mg/kg body weight (b. w). Percentages of mitotic and aberrant bone marrow cells were followed. The results indicated that IVM by itself, at doses higher than the recommended dose, induced significant levels of cytogenetic toxicity. To this end, we decided to investigate the potential use of combination of varying doses of aged garlic extract (AGE); 300, 600 and 1200 mg/kg b w and the minimum detectable toxic (MDT) dose of IVM; 0.4 mg/kg. A powerful capacity of AGE to reduce IVM cytogenetic effects was demonstrated. Overall, the data prove the safety of IVM at the recommended dose and provide a strong scientific evidence for superior protection of AGE against possible cytogenotoxic side effects of IVM, confirming the existence of a meaningful therapeutic window.

The DNMT1-associated lincRNA DACOR1 reprograms genome-wide DNA methylation in colon cancer.

BACKGROUND: DNA methylation is a key epigenetic mark in mammalian organisms that plays key roles in chromatin organization and gene expression. Although DNA methylation in gene promoters is generally associated with gene repression, recent studies demonstrate that DNA methylation in gene bodies and intergenic regions of the genome may result in distinct modes of gene regulation. Furthermore, the molecular mechanisms underlying the establishment and maintenance of DNA methylation in human health and disease remain to be fully elucidated. We recently demonstrated that a subset of long non-coding RNAs (lncRNAs) associates with the major DNA methyltransferase DNMT1 in human colon cancer cells, and the dysregulation of such lncRNAs contribute to aberrant DNA methylation patterns. RESULTS: In the current study, we assessed the impact of a key DNMT1-associated lncRNA, DACOR1, on genome-wide DNA methylation using reduced representation bisulfite sequencing (RRBS). Our findings demonstrated that induction of DACOR1 in colon cancer cells restores DNA methylation at thousands of CpG sites throughout the genome including promoters, gene bodies, and intergenic regions. Importantly, these sites overlap with regions of the genome that become hypomethylated in colon tumors. Furthermore, induction of DACOR1 results in repression of FOS and JUN and, consequently, reduced AP-1 transcription factor activity. CONCLUSION: Collectively, our results demonstrate a key role of lncRNAs in regulating DNA methylation in human cells, and the dysregulation of such lncRNAs could emerge as a key mechanism by which DNA methylation patterns become altered in human tumors.

Colon Cancer-Upregulated Long Non-Coding RNA lincDUSP Regulates Cell Cycle Genes and Potentiates Resistance to Apoptosis.

Long non-coding RNAs (lncRNAs) are frequently dysregulated in many human cancers. We sought to identify candidate oncogenic lncRNAs in human colon tumors by utilizing RNA sequencing data from 22 colon tumors and 22 adjacent normal colon samples from The Cancer Genome Atlas (TCGA). The analysis led to the identification of ~200 differentially expressed lncRNAs. Validation in an independent cohort of normal colon and patient-derived colon cancer cell lines identified a novel lncRNA, lincDUSP, as a potential candidate oncogene. Knockdown of lincDUSP in patient-derived colon tumor cell lines resulted in significantly decreased cell proliferation and clonogenic potential, and increased susceptibility to apoptosis. The knockdown of lincDUSP affects the expression of ~800 genes, and NCI pathway analysis showed enrichment of DNA damage response and cell cycle control pathways. Further, identification of lincDUSP chromatin occupancy sites by ChIRP-Seq demonstrated association with genes involved in the replication-associated DNA damage response and cell cycle control. Consistent with these findings, lincDUSP knockdown in colon tumor cell lines increased both the accumulation of cells in early S-phase and γH2AX foci formation, indicating increased DNA damage response induction. Taken together, these results demonstrate a key role of lincDUSP in the regulation of important pathways in colon cancer.

Transcriptome-Wide Analyses of Human Neonatal Articular Cartilage and Human Mesenchymal Stem Cell-Derived Cartilage Provide a New Molecular Target for Evaluating Engineered Cartilage.

Cellular differentiation comprises a progressive, multistep program that drives cells to fabricate a tissue with specific and site distinctive structural and functional properties. Cartilage constitutes one of the potential differentiation lineages that mesenchymal stem cells (MSCs) can follow under the guidance of specific bioactive agents. Single agents such as transforming growth factor beta (TGF-ß) and bone morphogenetic protein 2 in unchanging culture conditions have been historically used to induce in vitro chondrogenic differentiation of MSCs. Despite the expression of traditional chondrogenic biomarkers such as type II collagen and aggrecan, the resulting tissue represents a transient cartilage rather than an in vivo articular cartilage (AC), differing significantly in structure, chemical composition, cellular phenotypes, and mechanical properties. Moreover, there have been no comprehensive, multicomponent parameters to define high-quality and functional engineered hyaline AC. To address these issues, we have taken an innovative approach based on the molecular interrogation of human neonatal articular cartilage (hNAC), dissected from the knees of 1-month-old cadaveric specimens. Subsequently, we compared hNAC-specific transcriptional regulatory elements and differentially expressed genes with adult human bone marrow (hBM) MSC-derived three-dimensional cartilage structures formed in vitro. Using microarray analysis, the transcriptome of hNAC was found to be globally distinct from the transient, cartilage-like tissue formed by hBM-MSCs in vitro. Specifically, over 500 genes that are highly expressed in hNAC were not expressed at any time point during in vitro human MSC chondrogenesis. The analysis also showed that the differences were less variant during the initial stages (first 7 days) of the in vitro chondrogenic differentiation program. These observations suggest that the endochondral fate of hBM-MSC-derived cartilage may be rerouted at earlier stages of the TGF-ß-stimulated chondrogenic differentiation program. Based on these analyses, several key molecular differences (transcription factors and coded cartilage-related proteins) were identified in hNAC that will be useful as molecular inductors and identifiers of the in vivo AC phenotype. Our findings provide a new gold standard of a molecularly defined AC phenotype that will serve as a platform to generate novel approaches for AC tissue engineering.

Review: Regulation of the cancer epigenome by long non-coding RNAs.

Long non-coding RNAs have emerged as highly versatile players in the regulation of gene expression in development and human disease, particularly cancer. Hundreds of lncRNAs become dysregulated across tumor types, and multiple lncRNAs have demonstrated functions as tumor-suppressors or oncogenes. Furthermore, studies have demonstrated that dysregulation of lncRNAs results in alterations of the epigenome in cancer cells, potentially providing a novel mechanism for the massive epigenomic alterations observed in many tumors. Here, we highlight and provide some illustrious examples of lncRNAs in various epigenetic regulatory processes, including coordination of chromatin dynamics, regulation of DNA methylation, modulation of other non-coding RNAs and mRNA stability, and control of epigenetic substrate availability through altered tumor metabolism. In light of all these known and emerging functions in epigenetic regulation of tumorigenesis and cancer progression, lncRNAs represent attractive targets for future therapeutic strategies in cancer.

Regulation of paxillin-p130-PI3K-AKT signaling axis by Src and PTPRT impacts colon tumorigenesis.

Protein tyrosine phosphatase receptor T (PTPRT) is frequently mutated in a variety of human cancers including colorectal cancer. Here we report that PTPRT knockout increases the size of mouse colon tumors in the Apcmin+/- genetic background, suggesting that inactivation of PTPRT promotes tumor progression. We previously demonstrated that PTPRT dephosphorylates paxillin at tyrosine-Y88 residue. Consistently, phosphorylation of Y88 paxillin (pY88) is up-regulated in colon tumors derived from Apcmin+/- Ptprt-/- mice. An important downstream effector of pY88 paxillin is the oncogene Akt. Here, we show that pY88 paxillin impacts the Akt pathway by regulating the interaction between p130cas and the p85 regulatory subunit of PI3-Kinase. Additionally, while pY88 paxillin is a substrate of the tumor suppressor phosphatase PTPRT, the corresponding kinase has not been previously identified. In this study, we demonstrate that the oncogenic kinase Src directly phosphorylates paxillin at Y88. Moreover, colorectal cancer cells that express high levels of pY88 paxillin are sensitive to dasatinib treatment, suggesting that pY88 paxillin may serve as a predictive biomarker for Src family kinase inhibitors.

S-Nitrosoglutathione Attenuates Airway Hyperresponsiveness in Murine Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is characterized by lifelong obstructive lung disease and profound, refractory bronchospasm. It is observed among survivors of premature birth who have been treated with prolonged supplemental oxygen. Therapeutic options are limited. Using a neonatal mouse model of BPD, we show that hyperoxia increases activity and expression of a mediator of endogenous bronchoconstriction, S-nitrosoglutathione (GSNO) reductase. MicroRNA-342-3p, predicted in silico and shown in this study in vitro to suppress expression of GSNO reductase, was decreased in hyperoxia-exposed pups. Both pretreatment with aerosolized GSNO and inhibition of GSNO reductase attenuated airway hyperresponsiveness in vivo among juvenile and adult mice exposed to neonatal hyperoxia. Our data suggest that neonatal hyperoxia exposure causes detrimental effects on airway hyperreactivity through microRNA-342-3p-mediated upregulation of GSNO reductase expression. Furthermore, our data demonstrate that this adverse effect can be overcome by supplementing its substrate, GSNO, or by inhibiting the enzyme itself. Rates of BPD have not improved over the past two decades; nor have new therapies been developed. GSNO-based therapies are a novel treatment of the respiratory problems that patients with BPD experience.

Transcriptome-wide identification of mRNAs and lincRNAs associated with trastuzumab-resistance in HER2-positive breast cancer.

Approximately, 25-30% of early-stage breast tumors are classified at the molecular level as HER2-positive, which is an aggressive subtype of breast cancer. Amplification of the HER2 gene in these tumors results in a substantial increase in HER2 mRNA levels, and consequently, HER2 protein levels. HER2, a transmembrane receptor tyrosine kinase (RTK), is targeted therapeutically by a monoclonal antibody, trastuzumab (Tz), which has dramatically improved the prognosis of HER2-driven breast cancers. However, ~30% of patients develop resistance to trastuzumab and recur; and nearly all patients with advanced disease develop resistance over time and succumb to the disease. Mechanisms of trastuzumab resistance (TzR) are not well understood, although some studies suggest that growth factor signaling through other receptors may be responsible. However, these studies were based on cell culture models of the disease, and thus, it is not known which pathways are driving the resistance in vivo. Using an integrative transcriptomic approach of RNA isolated from trastuzumab-sensitive and trastuzumab-resistant HER2+ tumors, and isogenic cell culture models, we identified a small set of mRNAs and lincRNAs that are associated with trastuzumab-resistance (TzR). Functional analysis of a top candidate gene, S100P, demonstrated that inhibition of S100P results in reversing TzR. Mechanistically, S100P activates the RAS/MEK/MAPK pathway to compensate for HER2 inhibition by trastuzumab. Finally, we demonstrated that the upregulation of S100P appears to be driven by epigenomic changes at the enhancer level. Our current findings should pave the path toward new therapies for breast cancer patients.

DNMT1-associated long non-coding RNAs regulate global gene expression and DNA methylation in colon cancer.

The cancer epigenome exhibits global loss of DNA methylation, which contributes to genomic instability and aberrant gene expression by mechanisms that are yet to be fully elucidated. We previously discovered over 3300 long non-coding (lnc)RNAs in human cells and demonstrated that specific lncRNAs regulate gene expression via interactions with chromatin-modifying complexes. Here, we tested whether lncRNAs could also associate with DNA methyltransferases to regulate DNA methylation and gene expression. Using RIP-seq, we identified a subset of lncRNAs that interact with the DNA methyltransferase DNMT1 in a colon cancer cell line, HCT116. One lncRNA, TCONS_00023265, which we named DACOR1 (DNMT1-associated Colon Cancer Repressed lncRNA 1), shows high, tissue-specific expression in the normal colon (including colon crypts) but was repressed in a panel of colon tumors and patient-derived colon cancer cell lines. We identified the genomic occupancy sites of DACOR1, which we found to significantly overlap with known differentially methylated regions (DMRs) in colon tumors. Induction of DACOR1 in colon cancer cell lines significantly reduced their ability to form colonies in vitro, suggesting a growth suppressor function. Consistent with the observed phenotype, induction of DACOR1 led to the activation of tumor-suppressor pathways and attenuation of cancer-associated metabolic pathways. Notably, DACOR1 induction resulted in down-regulation of Cystathionine ß-synthase, which is known to lead to increased levels of S-adenosyl methionine-the key methyl donor for DNA methylation. Collectively, our results demonstrate that deregulation of DNMT1-associated lncRNAs contributes to aberrant DNA methylation and gene expression during colon tumorigenesis.

Integrative transcriptome-wide analyses reveal critical HER2-regulated mRNAs and lincRNAs in HER2+ breast cancer.

Breast cancer is a major health problem affecting millions of women worldwide. Over 200,000 new cases are diagnosed annually in the USA, with approximately 40,000 of these cases resulting in death. HER2-positive (HER2+) breast tumors, representing 20-30 % of early-stage breast cancer diagnoses, are characterized by the amplification of the HER2 gene. However, the critical genes and pathways that become affected by HER2 amplification in humans are yet to be specifically identified. Furthermore, it is yet to be determined if HER2 amplification also affects the expression of long intervening non-coding (linc)RNAs, which are involved in the epigenetic regulation of gene expression. We examined changes in gene expression by next generation RNA sequencing in human tumors pre- and post- HER2 inhibition by trastuzumab in vivo, and changes in gene expression in response to HER2 knock down in cell culture models. We integrated our results with gene expression analysis of HER2+ tumors vs matched normal tissue from The Cancer Genome Atlas. The integrative analyses of these datasets led to the identification of a small set of mRNAs, and the associated biological pathways that become deregulated by HER2 amplification. Furthermore, our analyses identified three lincRNAs that become deregulated in response to HER2 amplification both in vitro and in vivo. Our results should provide the foundation for functional studies of these candidate mRNAs and lincRNAs to further our understanding of how HER2 amplification results in tumorigenesis. Also, the identified lincRNAs could potentially open the door for future RNA-based biomarkers and therapeutics in HER2+ breast cancer.

Diverse functions and mechanisms of mammalian long noncoding RNAs.

Long noncoding RNAs are becoming increasingly appreciated as major players in gene regulation. They have been reported to play diverse roles in many biological processes. Here, we discuss their discovery, features, and known functions in cells. While not comprehensive, this chapter should serve to illustrate the power and promise of studying long noncoding RNAs.

The functional characterization of long noncoding RNA SPRY4-IT1 in human melanoma cells.

Expression of the long noncoding RNA (lncRNA) SPRY4-IT1 is low in normal human melanocytes but high in melanoma cells. siRNA knockdown of SPRY4-IT1 blocks melanoma cell invasion and proliferation, and increases apoptosis. To investigate its function further, we affinity purified SPRY4-IT1 from melanoma cells and used mass spectrometry to identify the protein lipin 2, an enzyme that converts phosphatidate to diacylglycerol (DAG), as a major binding partner. SPRY4-IT1 knockdown increases the accumulation of lipin2 protein and upregulate the expression of diacylglycerol O-acyltransferase 2 (DGAT2) an enzyme involved in the conversion of DAG to triacylglycerol (TAG). When SPRY4-IT1 knockdown and control melanoma cells were subjected to shotgun lipidomics, an MS-based assay that permits the quantification of changes in the cellular lipid profile, we found that SPRY4-IT1 knockdown induced significant changes in a number of lipid species, including increased acyl carnitine, fatty acyl chains, and triacylglycerol (TAG). Together, these results suggest the possibility that SPRY4-IT1 knockdown may induce apoptosis via lipin 2-mediated alterations in lipid metabolism leading to cellular lipotoxicity.

Identification of mRNAs and lincRNAs associated with lung cancer progression using next-generation RNA sequencing from laser micro-dissected archival FFPE tissue specimens.

OBJECTIVES: Adenocarcinoma in situ (AIS) is an intermediate step in the progression of normal lung tissue to invasive adenocarcinoma. However, molecular mechanisms underlying this progression remain to be fully elucidated due to challenges in obtaining fresh clinical samples for downstream analyses. Formalin fixation and paraffin embedding (FFPE) is a tissue preservation system widely used for long-term storage. Until recently, challenges in working with FFPE precluded using new RNA sequencing technologies (RNA-seq), which would help clarify key pathways in cancer progression. Also, isolation techniques including laser-capture micro-dissection provide the ability to select histopathologically distinct tissues, allowing researchers to study transcriptional variations between tightly juxtaposed cell and tissue types. MATERIALS AND METHODS: Utilizing these technologies and new alignment tools we examined differential expression of long intergenic non-coding RNAs (lincRNAs) and mRNAs across normal, AIS and invasive adenocarcinoma samples from six patients to identify possible markers of lung cancer progression. RESULTS: RNA extracted and sequenced from these 18 samples generated an average of 198 million reads per sample. After alignment and filtering, uniquely aligned reads represented an average 35% of the total reads. We detected differential expression of a number of lincRNAs and mRNAs when comparing normal to AIS, or AIS to invasive adenocarcinoma. Of these, 5 lincRNAs and 31 mRNAs were consistently up- or down-regulated from normal to AIS and more so to invasive carcinoma. We validated the up-regulation of two mRNAs and one lincRNA by RT-qPCR as proof of principle. CONCLUSION: Our findings indicate a potential role of not only mRNAs, but also lincRNAs in the progression to invasive adenocarcinoma. We anticipate that these findings will lay the groundwork for future experimental studies of candidate RNAs from FFPE to identify their functional roles in lung cancer.