miRNA and mRNA expression in the hippocampal region during postnatal developments in rats

Hjernestrukturene entorhinal cortex og hippocampus er viktige for minnefunksjon og navigering. Innenfor hver av disse strukturene finnes underregioner som har forskjellige celletyper og forbindelser i hjernen. Noen av celletypene og forbindelsene dannes før fødsel, mens andre kommer til etter fødsel. I tillegg spesialiseres alle celletypene etter fødsel. Alle disse begivenhetene kommer av en blanding av gener og miljøpåvirkning. Hovedmålet med dette arbeidet var å karakterisere genuttrykket i underregionene mens disse spesialiseres etter fødsel hos rotter, og finne potensiell molekylær basis for forskjellene som sees mellom underregionene. Artikkel 1 tar for seg to underregioner i hippocampus, den dorsale og ventrale delen. Flere egenskaper viser forskjeller langs denne aksen, inkludert elektrofysiologi, genuttrykk, og forbindelser til andre deler av hjernen. I tillegg er dorsal hippocampus viktig for minne og navigering, mens ventral hippocampus er mer involvert i følelser og angst-relatert oppførsel. Vi undersøkte når denne forskjellen var til stede etter fødsel basert på anatomi og genuttrykk. Anatomisk sett så vi ingen forskyvninger mellom den dorsale og ventrale delen, og forskjellene i hjerneforbindelser var til stede allerede den første uken etter fødsel. Vi fant også forskjeller i genuttrykk mellom dorsal og ventral hippocampus allerede ved fødsel, og mange av genene var fortsatt forskjellig uttrykt hos voksne dyr. Vi konkluderte derfor at forskjellene mellom dorsal og ventral hippocampus allerede er til stede den første uka etter fødsel. I artikkel 2 undersøkte vi forskjellene i uttrykket av mikroRNA og proteinkodende gener mellom lag II og de dypere lagene i medial entorhinal cortex ved fire tidspunkt etter fødsel. Lag II i entorhinal cortex inneholder en høy prosentandel av stellat nevroner, som har unikt utseende og elektrofysiologi, og påvirkes særlig i Alzheimer’s sykdom. Vi fant at forskjellene i uttrykk av både mikroRNA og protein-kodende gener var større mellom alder enn mellom lag. MikroRNA er små RNA molekyler som regulerer proteinsyntesen fra protein-kodende gener, og som har viktige funksjoner i hjerneutvikling og funksjon. For å finne hvilke gener som var regulert av mikroRNA molekyler med forskjellig uttrykk, fant vi gener som hadde konservert bindingssted for mikroRNA samt hadde motsatt uttrykksprofil. Når genene er kjent, kan en også finne ut potensielle funksjoner for mikroRNA. Vi fant at mikroRNA sannsynligvis bidrar til cellespesialisering i medial entorhinal cortex. Flere av mikroRNAene som hadde forskjeller i uttrykk mellom lag er også involvert i Alzheimer’s sykdom, som åpner for muligheten at disse bidrar til de molekylære sykdomsmekanismene. Vi sammenlignet også mikroRNA uttrykk mellom stellat nevroner og resten av cellene i medial entorhinal cortex. Et av mikroRNAene som var oppregulert i lag II, miR-143, var også høyere uttrykt i stellat nevronene. Analysen vår viste at miR-143 mest sannsynlig regulerer Lmo4 genet, som er viktig for navigeringsminnet samt utvikling av entorhinal cortex på fosterstadiet. I tillegg til forskjellene som finnes mellom lag i entorhinal cortex, ser man også forskjeller i celletyper, elektrofysiologi, og hjerneforbindelser mellom den laterale og den mediale delen av strukturen. Det finnes også forskjeller i minnefunksjon, da medial entorhinal cortex er mer involvert i navigering, mens lateral entorhinal cortex er involvert i minne for fysiske objekter og lukter. I artikkel 3 karakteriserte vi forskjellene i uttrykket av protein-kodende og ikkekodende gener mellom de to regionene ved fire forskjellige tidspunkt etter fødsel. Forskjellene mellom lag II og de dypere lagene i hver region ble også undersøkt. Vi fant at forskjellene i genuttrykk mellom de mediale og laterale delene var størst i lag II sammenliknet med de dypere lagene, og at mange av disse genene kodet for neuropeptidreseptorer, som er viktige for minnefunksjon. Forskjellene mellom lag II og de dypere lagene besto i strukturen som omgir cellene, blodkardannelse, nevronspesialisering og funksjon, samt myelindannelse. Basert på ulike genkategorier fant vi kandidatgener som kan forklare forskjellene i funksjon mellom ulike lag og mellom medial og lateral entorhinal cortex, inkludert gener involvert i nevronenes elektriske egenskaper, minnefunksjon, og sykdomssensitivitet. Dette arbeidet representer det første molekylære overblikket av forskjeller i underregioner av hippocampus og entorhinal cortex under utvikling etter fødsel. Dataene er gjort tilgjengelige for videre studier av den molekylære bakgrunnen for utvikling av minne- og navigeringsfunksjonene i disse regionene

MicroRNAs contribute to postnatal development of laminar differences and neuronal subtypes in the rat medial entorhinal cortex

The medial entorhinal cortex (MEC) is important in spatial navigation and memory formation and its layers have distinct neuronal subtypes, connectivity, spatial properties, and disease susceptibility. As little is known about the molecular basis for the development of these laminar differences, we analyzed microRNA (miRNA) and messenger RNA (mRNA) expression differences between rat MEC layer II and layers III–VI during postnatal development. We identified layer and age-specific regulation of gene expression by miRNAs, which included processes related to neuron specialization and locomotor behavior. Further analyses by retrograde labeling and expression profiling of layer II stellate neurons and in situ hybridization revealed that the miRNA most up-regulated in layer II, miR-143, was enriched in stellate neurons, whereas the miRNA most up-regulated in deep layers, miR-219-5p, was expressed in ependymal cells, oligodendrocytes and glia. Bioinformatics analyses of predicted mRNA targets with negatively correlated expression patterns to miR-143 found that miR-143 likely regulates the Lmo4 gene, which is known to influence hippocampal-based spatial learning

Basal level of autophagy and MAP1LC3B-II as potential biomarkers for DHA-induced cytotoxicity in colorectal cancer cells

The omega‐3 fatty acid docosahexaenoic acid (DHA) is known as an anticancer agent. Colorectal cancer (CRC) cells exhibit different sensitivity toward DHA, but the mechanisms involved are still unclear. Gene expression profiling of 10 CRC cell lines demonstrated a correlation between the level of DHA sensitivity and different biological stress responses, such as endoplasmic reticulum (ER) stress, oxidative stress, and autophagy. The basal level of autophagy and MAP1LC3B‐II protein correlated with DHA sensitivity in the cell lines studied. DHA induced oxidative stress, ER stress, and autophagy in DHA‐sensitive DLD‐1 cells, while the less sensitive LS411N cells were affected to a much lesser extent. Co‐treatment with DHA and the autophagy inducer rapamycin reduced DHA sensitivity in DLD‐1 and HCT‐8 cells, while co‐treatment with DHA and the autophagy inhibitors chloroquine and 3‐methyladenine increased the DHA sensitivity in LS411N and LS513 cells. Differentially expressed genes correlating with DHA sensitivity and the level of autophagy demonstrated an overlap in biological pathways involved. Results indicate the basal level of autophagy and MAP1LC3B‐II protein as potential biomarkers for DHA sensitivity in CRC cells

Joint changes in RNA, RNA polymerase II, and promoter activity through the cell cycle identify non-coding RNAs involved in proliferation

Proper regulation of the cell cycle is necessary for normal growth and development of all organisms. Conversely, altered cell cycle regulation often underlies proliferative diseases such as cancer. Long non-coding RNAs (lncRNAs) are recognized as important regulators of gene expression and are often found dysregulated in diseases, including cancers. However, identifying lncRNAs with cell cycle functions is challenging due to their often low and cell-type specific expression. We present a highly effective method that analyses changes in promoter activity, transcription, and RNA levels for identifying genes enriched for cell cycle functions. Specifically, by combining RNA sequencing with ChIP sequencing through the cell cycle of synchronized human keratinocytes, we identified 1009 genes with cell cycle-dependent expression and correlated changes in RNA polymerase II occupancy or promoter activity as measured by histone 3 lysine 4 trimethylation (H3K4me3). These genes were highly enriched for genes with known cell cycle functions and included 57 lncRNAs. We selected four of these lncRNAs—SNHG26, EMSLR, ZFAS1, and EPB41L4A-AS1—for further experimental validation and found that knockdown of each of the four lncRNAs affected cell cycle phase distributions and reduced proliferation in multiple cell lines. These results show that many genes with cell cycle functions have concomitant cell-cycle dependent changes in promoter activity, transcription, and RNA levels and support that our multi-omics method is well suited for identifying lncRNAs involved in the cell cycle

MicroRNA profiling of psoriatic skin identifies 11 miRNAs associated with disease severity

MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as central regulators of gene expression and powerful biomarkers of disease. Much is yet unknown about their role in psoriasis pathology. To globally characterize the miRNAome of psoriatic skin, skin biopsies were collected from psoriatic cases (n = 75) and non-psoriatic controls (n = 46) and RNA sequenced. Count data were meta-analysed with a previously published dataset (cases, n = 24, controls, n = 20), increasing the number of psoriatic cases fourfold from previously published studies. Differential gene expression analyses were performed comparing lesional psoriatic (PP), non-lesional psoriatic (PN) and control (NN) skin. Further, functional enrichment and cell-specific analyses were performed. Across all contrasts, we identified 439 significantly differentially expressed miRNAs (DEMs), of which 85 were novel for psoriasis and 11 were related to disease severity. Meta-analyses identified 20 DEMs between PN and NN, suggesting an inherent change in the constitution of all skin in psoriasis. By integrating the miRNA transcriptome with mRNA target interactions, we identified several functionally enriched terms, including “thyroid hormone signalling,” “insulin resistance” and various infectious diseases. Cell-specific expression analyses revealed that the upregulated DEMs were enriched in epithelial and immune cells. This study provides the most comprehensive overview of the miRNAome in psoriatic skin to date and identifies a miRNA signature related to psoriasis severity. Our results may represent molecular links between psoriasis and related comorbidities and have outlined potential directions for future functional studies to identify biomarkers and treatment targets

Establishment of a patient-derived xenograft model of colorectal cancer in CIEA NOG mice and exploring smartfish liquid diet as a source of omega-3 fatty acids

Cancer patient-derived xenografts (PDXs) better preserve tumor characteristics and microenvironment than traditional cancer cell line derived xenografts and are becoming a valuable model in translational cancer research and personalized medicine. We have established a PDX model for colorectal cancer (CRC) in CIEA NOG mice with a 50% engraftment rate. Tumor fragments from patients with CRC (n = 5) were engrafted in four mice per tumor (n = 20). Mice with established PDXs received a liquid diet enriched with fish oil or placebo, and fatty acid profiling was performed to measure fatty acid content in whole blood. Moreover, a biobank consisting of tissue and blood samples from patients was established. Histology, immunohistochemistry and in situ hybridization procedures were used for staining of tumor and xenograft tissue slides. Results demonstrate that key histological characteristics of the patients’ tumors were retained in the established PDXs, and the liquid diets were consumed as intended by the mice. Some of the older mice developed lymphomas that originated from human Ki67+, CD45+, and EBV+ lymphoid cells. We present a detailed description of the process and methodology, as well as possible issues that may arise, to refine the method and improve PDX engraftment rate for future studies. The established PDX model for CRC can be used for exploring different cancer treatment regimes, and liquid diets enriched with fish oil may be successfully delivered to the mice through the drinking flasks

Alkyladenine DNA glycosylase associates with transcription elongation to coordinate DNA repair with gene expression

Base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG) is essential for removal of aberrantly methylated DNA bases. Genome instability and accumulation of aberrant bases accompany multiple diseases, including cancer and neurological disorders. While BER is well studied on naked DNA, it remains unclear how BER efficiently operates on chromatin. Here, we show that AAG binds to chromatin and forms complex with RNA polymerase (pol) II. This occurs through direct interaction with Elongator and results in transcriptional co-regulation. Importantly, at co-regulated genes, aberrantly methylated bases accumulate towards the 3′end in regions enriched for BER enzymes AAG and APE1, Elongator and active RNA pol II. Active transcription and functional Elongator are further crucial to ensure efficient BER, by promoting AAG and APE1 chromatin recruitment. Our findings provide insights into genome stability maintenance in actively transcribing chromatin and reveal roles of aberrantly methylated bases in regulation of gene expression