MALAT1 Inhibits Proliferation of HPV16-Positive Cervical Cancer by Sponging miR-485-5p to Promote Expression of MAT2A

DNA and cell biology

2021 Oct 04

Tie, W;Ge, F;
PMID: 34610246 | DOI: 10.1089/dna.2020.6205

Cervical cancer is the leading cause of morbidity and mortality in women throughout the world, human papillomavirus 16 (HPV16) is the main type of HPV causing invasive cervical cancer. However, the underlying mechanism of the high carcinogenicity of HPV16 remains unclear. In the current study, we documented that metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long noncoding RNA, is upregulated in HPV16-positive cervical cancer tissue and cell lines. The results of immunohistochemistry and immunofluorescence showed that MALAT1 was mainly localized in the cytoplasm. To clarify the biological functions of MALAT1 in cervical cancer cells, we performed gain- and loss-of-function experiments to explore the underlying molecular mechanism. Functionally, the proliferation of cervical cancer was detected by Cell Counting Kit-8 (CCK-8) and colony formation assay in MALAT1 overexpression or knockdown cells, our data showed that MALAT1 promotes the proliferation of cervical cancer cells. Mechanistically, our results suggested that MALAT1 upregulates Methionine adenosyltransferase 2A (MAT2A) by sponging miR-485-5p. Moreover, the gain-of-function assay validated the function of MAT2A in HPV16-positive cervical cancer proliferation. Taken together, our results demonstrated that MALAT1 acts as a competitive endogenous RNA (ceRNA) to regulate MAT2A by sponging miR-485-5p in HPV16-positive cervical cancer, suggesting that MALAT1 may act as a potential therapeutic target for HPV16-positive cervical cancer.

Recovery of Latent HIV-1 from Brain Tissue by Adoptive Cell Transfer in Virally Suppressed Humanized Mice

Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

2021 Sep 15

Su, H;Sravanam, S;Sillman, B;Waight, E;Makarov, E;Mathews, S;Poluektova, LY;Gorantla, S;Gendelman, HE;Dash, PK;
PMID: 34528173 | DOI: 10.1007/s11481-021-10011-w

Defining the latent human immunodeficiency virus type 1 (HIV-1) burden in the human brain during progressive infection is limited by sample access. Human hematopoietic stem cells (hu-HSCs)-reconstituted humanized mice provide an opportunity for this study. The model mimics, in measure, HIV-1 pathophysiology, transmission, treatment, and elimination in an infected human host. However, to date, brain HIV-1 latency in hu-HSC mice during suppressive antiretroviral therapy (ART) was not studied. To address this need, hu-HSC mice were administered long acting (LA) ART 14 days after HIV-1 infection was established. Animals were maintained under suppressive ART for 3 months, at which time HIV-1 infection was detected at low levels in brain tissue by droplet digital polymerase chain reaction (ddPCR) test on DNA. Notably, adoptive transfer of cells acquired from the hu-HSC mouse brains and placed into naive hu-HSC mice demonstrated viral recovery. These proof-of-concept results demonstrate replication-competent HIV-1 reservoir can be established in hu-HSC mouse brains that persists during long-term ART treatment. Hu-HSC mice-based mouse viral outgrowth assay (hu-MVOA) serves as a sensitive tool to interrogate latent HIV-1 brain reservoirs.

The noncoding genome and hearing loss

Human genetics

2021 Sep 07

Avraham, KB;Khalaily, L;Noy, Y;Kamal, L;Koffler-Brill, T;Taiber, S;
PMID: 34491412 | DOI: 10.1007/s00439-021-02359-z

The age of sequencing has provided unprecedented insights into the human genome. The coding region of the genome comprises nearly 20,000 genes, of which approximately 4000 are associated with human disease. Beyond the protein-coding genome, which accounts for only 3% of the genome, lies a vast pool of regulatory elements in the form of promoters, enhancers, RNA species, and other intricate elements. These features undoubtably influence human health and disease, and as a result, a great deal of effort is currently being invested in deciphering their identity and mechanism. While a paucity of material has caused a lag in identifying these elements in the inner ear, the emergence of technologies for dealing with a minimal number of cells now has the field working overtime to catch up. Studies on microRNAs (miRNAs), long non-coding RNAs (lncRNAs), methylation, histone modifications, and more are ongoing. A number of microRNAs and other noncoding elements are known to be associated with hearing impairment and there is promise that regulatory elements will serve as future tools and targets of therapeutics and diagnostics. This review covers the current state of the field and considers future directions for the noncoding genome and implications for hearing loss.

Signaling through retinoic acid receptors is essential for mammalian uterine receptivity and decidualization

JCI insight

2021 Jul 22

Yin, Y;Haller, ME;Chadchan, SB;Kommagani, R;Ma, L;
PMID: 34292881 | DOI: 10.1172/jci.insight.150254

Retinoic Acid (RA) signaling has long been speculated to regulate embryo implantation, because many enzymes and proteins responsible for maintaining RA homeostasis and transducing RA signals are tightly regulated in the endometrium during this critical period. However, due to lack of genetic data, it was unclear whether RA signaling is truly required for implantation, and which specific RA signaling cascades are at play. Herein we utilize a genetic murine model that expresses a dominant negative form of RA receptor specifically in female reproductive organs to show that functional RA signaling is fundamental to female fertility, particularly implantation and decidualization. Reduction in RA signaling activity severely affects the ability of the uterus to achieve receptive status and decidualize, partially through dampening follistatin expression and downstream activin B/BMP2 signaling. To confirm translational relevance of these findings to humans, human endometrial stromal cells (hESCs) were treated with pan-RAR antagonist to show that in vitro decidualization is impaired. RNAi perturbation of individual RAR transcripts in hESCs revealed that RARα in particular is essential for proper decidualization. These data provide direct functional evidence that uterine RAR-mediated RA signaling is crucial for mammalian embryo implantation, and its disruption leads to failure of uterine receptivity and decidualization resulting in severely compromised fertility.

LINC01348 suppresses hepatocellular carcinoma metastasis through inhibition of SF3B3-mediated EZH2 pre-mRNA splicing

Oncogene

2021 Jun 17

Lin, YH;Wu, MH;Liu, YC;Lyu, PC;Yeh, CT;Lin, KH;
PMID: 34140643 | DOI: 10.1038/s41388-021-01905-3

Long non-coding RNAs (lncRNA) play crucial roles in hepatocellular carcinoma (HCC) progression. However, the specific functions of lncRNAs in alternative splicing (AS) and the metastatic cascade in liver cancer remain largely unclear. In this study, we identified a novel lncRNA, LINC01348, which was significantly downregulated in HCC and correlated with survival functions in HCC patients. Ectopic expression of LINC01348 induced marked inhibition of cell growth, and metastasis in vitro and in vivo. Conversely, these phenotypes were reversed upon knockdown of LINC01348. Mechanistically, LINC01348 complexed with splicing factor 3b subunit 3 (SF3B3) acted as a modulator of EZH2 pre-mRNA AS, and induced alterations in JNK/c-Jun activity and expression of Snail. Notably, C-terminal truncated HBx (Ct-HBx) negatively regulated LINC01348 through c-Jun signaling. Our data collectively highlight those novel regulatory associations involving LINC01348/SF3B3/EZH2/JNK/c-Jun/Snail are an important determinant of metastasis in HCC cells and support the potential utility of targeting LINC01348 as a therapeutic strategy for HCC.

LncRNA H19 aggravates intervertebral disc degeneration by promoting the autophagy and apoptosis of nucleus pulposus cells via the miR-139/CXCR4/NF-κB axis

Stem cells and development

2021 May 20

Sun, Z;Tang, X;Wang, H;Sun, H;Chu, P;Sun, L;Tian, J;
PMID: 34015968 | DOI: 10.1089/scd.2021.0009

The etiology of lumbocrural pain is closely related to intervertebral disc degeneration (IDD). Long noncoding RNAs (LncRNAs) serve crucial roles in IDD progression. This study investigated the effect of lncRNA H19 on autophagy and apoptosis of nucleus pulposus cells (NPCs) in IDD. The rat model of IDD was established. Normal NPCs and degenerative NPCs (DNPCs) were cultured in vitro. H19 expression in IDD rat was detected. DNPCs were treated with si-H19 to evaluate autophagy and apoptosis of DNPCs. The binding relationships between H19 and miR-139-3p, and miR-139-3p and CXCR4 were verified. DNPCs were co-transfected si-H19 and miR-139-3p inhibitor. The phosphorylation of NF-κB pathway related p65 in DNPCs was detected. LncRNA H19 was upregulated in IDD rats. Downregulation of H19 inhibited autophagy and apoptosis of DNPCs. LncRNA H19 sponged miR-139-3p to inhibit CXCR4 expression. si-H19 and miR-139-3p inhibitor co-treatment induced autophagy and apoptosis, and enhanced CXCR4 expression. si-H19 decreased p-p65 phosphorylation, while si-H19 and miR-139-3p inhibitor co-treatment partially elevated p-p65 phosphorylation. In conclusion, lncRNA H19 facilitated the autophagy and apoptosis of DNPCs via the miR-139-3p/CXCR4/NF-κB axis, thereby aggravating IDD. This study may offer new insights for the management of IDD.

LncRNA PCIR Is an Oncogenic Driver via Strengthen the Binding of TAB3 and PABPC4 in Triple Negative Breast Cancer

Frontiers in oncology

2021 May 03

Guo, W;Li, J;Huang, H;Fu, F;Lin, Y;Wang, C;
PMID: 34012913 | DOI: 10.3389/fonc.2021.630300

Long non-coding RNAs (LncRNA) as the key regulators in all stages of tumorigenesis and metastasis. However, the underlying mechanisms are largely unknown. Here, we report a lncRNA RP11-214F16.8, which renamed Lnc-PCIR, is upregulated and higher RNA level of Lnc-PCIR was positively correlated to the poor survival of patients with triple negative breast cancer (TNBC) tissues. Lnc-PCIR overexpression significantly promoted cell proliferation, migration, and invasion in vitro and in vivo. RNA pulldown, RNA immunoprecipitation (RIP) and RNA transcriptome sequencing technology (RNA-seq) was performed to identify the associated proteins and related signaling pathways. Mechanistically, higher Lnc-PCIR level of blocks PABPC4 proteasome-dependent ubiquitination degradation; stable and highly expressed PABPC4 can further increase the stability of TAB3 mRNA, meanwhile, overexpression of Lnc-PCIR can disrupt the binding status of TAB3 and TAB2 which lead to activate the TNF-α/NF-κB pathway in TNBC cells. Our findings suggest that Lnc-PCIR promotes tumor growth and metastasis via up-regulating the mRNA/protein level of TAB3 and PABPC4, activating TNF-α/NF-κB signaling pathway in TNBC.

The noncoding MIR100HG RNA enhances the autocrine function of transforming growth factor β signaling

Oncogene

2021 May 01

Papoutsoglou, P;Rodrigues-Junior, DM;Morén, A;Bergman, A;Pontén, F;Coulouarn, C;Caja, L;Heldin, CH;Moustakas, A;
PMID: 33941855 | DOI: 10.1038/s41388-021-01803-8

Activation of the transforming growth factor β (TGFβ) pathway modulates the expression of genes involved in cell growth arrest, motility, and embryogenesis. An expression screen for long noncoding RNAs indicated that TGFβ induced mir-100-let-7a-2-mir-125b-1 cluster host gene (MIR100HG) expression in diverse cancer types, thus confirming an earlier demonstration of TGFβ-mediated transcriptional induction of MIR100HG in pancreatic adenocarcinoma. MIR100HG depletion attenuated TGFβ signaling, expression of TGFβ-target genes, and TGFβ-mediated cell cycle arrest. Moreover, MIR100HG silencing inhibited both normal and cancer cell motility and enhanced the cytotoxicity of cytostatic drugs. MIR100HG overexpression had an inverse impact on TGFβ signaling responses. Screening for downstream effectors of MIR100HG identified the ligand TGFβ1. MIR100HG and TGFB1 mRNA formed ribonucleoprotein complexes with the RNA-binding protein HuR, promoting TGFβ1 cytokine secretion. In addition, TGFβ regulated let-7a-2-3p, miR-125b-5p, and miR-125b-1-3p expression, all encoded by MIR100HG intron-3. Certain intron-3 miRNAs may be involved in TGFβ/SMAD-mediated responses (let-7a-2-3p) and others (miR-100, miR-125b) in resistance to cytotoxic drugs mediated by MIR100HG. In support of a model whereby TGFβ induces MIR100HG, which then enhances TGFβ1 secretion, analysis of human carcinomas showed that MIR100HG expression correlated with expression of TGFB1 and its downstream extracellular target TGFBI. Thus, MIR100HG controls the magnitude of TGFβ signaling via TGFβ1 autoinduction and secretion in carcinomas.

Detection of Epstein-Barr virus encoded RNA in fixed cells and tissues using CRISPR/Cas-mediated RCasFISH

Analytical biochemistry

2021 Apr 26

Chen, K;Wang, M;Zhang, R;Li, J;
PMID: 33915117 | DOI: 10.1016/j.ab.2021.114211

Identification of Epstein-Barr virus (EBV)-infected cells is critical for the diagnosis and clinical management of EBV-associated diseases. EBV-encoded RNA (EBER) located in the nucleus is a reliable marker due to its high levels of expression and inherent stability in tissue specimens. EBER in situ hybridization has long been the gold standard for detecting tumor-associated latent EBV infection and is valuable in determining the primary site and radiation fields of EBV-related malignancies. However, reliable detection is somewhat restricted by diffused signal and time-consuming procedure of this method, especially when proteins and RNA needed to be labeled simultaneously. Here, we optimized and validated our CRISPR-dCas9 mediated in situ RNA imaging tool-RCasFISH that enabled us to detect EBER rapidly and was compatible with IHC methods in fixed cells and tissue sections. Our approach could provide an attractive alternative for the molecular diagnosis of latent EBV infection.

Emerging role of lncRNA ELDR in development and cancer

The FEBS journal

2021 Apr 15

Sur, S;Ray, RB;
PMID: 33860640 | DOI: 10.1111/febs.15876

Whole-genome sequencing and transcriptome analysis revealed more than 90% of the human genome transcribes noncoding RNAs including lncRNAs. From the beginning of the 21st century, lncRNAs have gained widespread attention as a new layer of regulation in biological processes. lncRNAs are > 200 nucleotides in size, transcribed by RNA polymerase II, and share many similarities with mRNAs. lncRNA interacts with DNA, RNA, protein, and miRNAs, thereby regulating many biological processes. In this review, we have focused mainly on LINC01156 [also known as the EGFR long non-coding downstream RNA (ELDR) or Fabl] and its biological importance. ELDR is a newly identified lncRNA and first reported in a mouse model, but it has a human homolog. The human ELDR gene is closely localized downstream of epidermal growth factor receptor (EGFR) gene at chromosome 7 on the opposite strand. ELDR is highly expressed in neuronal stem cells and associated with neuronal differentiation and mouse brain development. ELDR is upregulated in head and neck cancer, suggesting its role as an oncogene and its importance in prognosis and therapy. Publicly available RNA-seq data further support its oncogenic potential in different cancers. Here, we summarize all the aspects of ELDR in development and cancer, highlighting its future perspectives in the context of mechanism.

CALINCA—A Novel Pipeline for the Identification of lncRNAs in Podocyte Disease

Cells

2021 Mar 20

Talyan, S;Filipów, S;Ignarski, M;Smieszek, M;Chen, H;Kühne, L;Butt, L;Göbel, H;Hoyer-Allo, K;Koehler, F;Altmüller, J;Brinkkötter, P;Schermer, B;Benzing, T;Kann, M;Müller, R;Dieterich, C;
| DOI: 10.3390/cells10030692

Diseases of the renal filtration unit—the glomerulus—are the most common cause of chronic kidney disease. Podocytes are the pivotal cell type for the function of this filter and focal-segmental glomerulosclerosis (FSGS) is a classic example of a podocytopathy leading to proteinuria and glomerular scarring. Currently, no targeted treatment of FSGS is available. This lack of therapeutic strategies is explained by a limited understanding of the defects in podocyte cell biology leading to FSGS. To date, most studies in the field have focused on protein-coding genes and their gene products. However, more than 80% of all transcripts produced by mammalian cells are actually non-coding. Here, long non-coding RNAs (lncRNAs) are a relatively novel class of transcripts and have not been systematically studied in FSGS to date. The appropriate tools to facilitate lncRNA research for the renal scientific community are urgently required due to a row of challenges compared to classical analysis pipelines optimized for coding RNA expression analysis. Here, we present the bioinformatic pipeline CALINCA as a solution for this problem. CALINCA automatically analyzes datasets from murine FSGS models and quantifies both annotated and de novo assembled lncRNAs. In addition, the tool provides in-depth information on podocyte specificity of these lncRNAs, as well as evolutionary conservation and expression in human datasets making this pipeline a crucial basis to lncRNA studies in FSGS.

The shape of pleomorphic virions determines resistance to cell-entry pressure

Nature microbiology

2021 Mar 18

Li, T;Li, Z;Deans, EE;Mittler, E;Liu, M;Chandran, K;Ivanovic, T;
PMID: 33737748 | DOI: 10.1038/s41564-021-00877-0

Many enveloped animal viruses produce a variety of particle shapes, ranging from small spherical to long filamentous types. Characterization of how the shape of the virion affects infectivity has been difficult because the shape is only partially genetically encoded, and most pleomorphic virus structures have no selective advantage in vitro. Here, we apply virus fractionation using low-force sedimentation, as well as antibody neutralization coupled with RNAScope, single-particle membrane fusion experiments and stochastic simulations to evaluate the effects of differently shaped influenza A viruses and influenza viruses pseudotyped with Ebola glycoprotein on the infection of cells. Our results reveal that the shape of the virus particles determines the probability of both virus attachment and membrane fusion when viral glycoprotein activity is compromised. The larger contact interface between a cell and a larger particle offers a greater probability that several active glycoproteins are adjacent to each other and can cooperate to induce membrane merger. Particles with a length of tens of micrometres can fuse even when 95% of the glycoproteins are inactivated. We hypothesize that non-genetically encoded variable particle shapes enable pleomorphic viruses to overcome selective pressure and may enable adaptation to infection of cells by emerging viruses such as Ebola. Our results suggest that therapeutics targeting filamentous virus particles could overcome antiviral drug resistance and immune evasion in pleomorphic viruses.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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