5 research outputs found
Transkriptionelle Charakterisierung und funktionelle Analyse von langen nicht- kodierenden RNA/Protein-kodierenden Genpaaren des menschlichen Genoms
Many protein-coding gene (PCG) promoters in the human genome initiate
transcription in two directions, thereby expressing an mRNA and an upstream
non-coding RNA (ncRNA). Diverse species of these promoter-associated ncRNAs
are abundantly detected in genome-wide transcriptome studies but the functions
of these non-coding transcripts remain mostly elusive. In this thesis, a set
of 1,107 long ncRNA/PCG pairs that are expressed from bidirectional promoters
is defined. These bidirectional promoters exhibit a high degree of sequence
conservation and mediate linked expression of paired genes. This is determined
by expression quantification and reporter assays of selected candidates.
Expression of these long ncRNA/PCG pairs is detected to frequently occur from
promoters of cancer-related proteins. One of the bidirectional promoters
mediates simultaneous expression of the tumor suppressor gene RB1 and ncRNA-
RB1 as detected by assaying the effects of retinoblastoma-associated point
mutations in a bidirectional reporter assay. The linked expression of both
genes is further shown by mutation of core promoter elements residing in both
promoter directions. Changes of single or few base-pairs, is found to affect
transcription initiation in both promoter directions equally. To determine the
functionality of paired genes and their involvement in common biological
pathways, ncRNA-RB1 and RB1 mRNA were individually depleted in a cell culture
system. This revealed that both genes are not regulating each otherâs
expression and that ncRNA-RB1 conveys regulatory effects that are different
but also to a certain degree overlapping to the RB1 controlled transcriptional
program. NcRNA-RB1 positively regulates the expression of calreticulin (CALR),
an endoplasmic reticulum-sessile chaperone that can translocate to the surface
of tumor cells after chemotherapy, thereby serving as an âeat-me-signalâ to
phagocytes. Knock-down of the nuclear-retained ncRNA-RB1 in tumor cells
reduces the expression of the CALR gene on chromatin, impairs translocation of
the CALR protein to the cell surface upon treatment with anthracylines, and
consequently inhibits uptake of the cells by macrophages. In conclusion, co-
transcription of ncRNA-RB1 from the bidirectional RB1 promoter provides a
positive link between the regulation of two the tumor suppressors RB1 and
CALR. Loss of expression of either gene product of the ncRNA-RB1/RB1 pair
entails the abolition of additional tumor-inhibitory mechanisms.Viele Promotoren Protein-kodierender Gene im menschlichen Genom initiieren die
Transkription in zwei Richtungen und exprimieren dabei eine Boten-RNA (mRNA)
sowie eine nicht-kodierende RNA (ncRNA), welche upstream des Promoters liegt.
Verschiedene Varianten solcher Promoter-assoziierten, ncRNAs wurden kĂźrzlich
in Genom-weiten Transkriptionsstudien detektiert. Dennoch sind ihre Funktionen
bisher weitgehend ungeklärt. In dieser Doktorarbeit, wird ein Set bestehend
aus 1107 Paaren langer ncRNAs (>200 bp) und Protein-kodierender Gene
definiert, die von bidirektionellen Promotoren exprimiert werden.
Entsprechende Promotoren weisen ein hohes MaĂ an Sequenzkonservierung auf und
initiieren gleichzeitig die Expression von Genpaaren. Durch Quantifizierung
der Expression und Verwendung von Reporter Assays fßr ausgewählte
Kandidatengenpaare wurde dieses Verhalten nachgewiesen. Viele dieser
bidirektionellen Promotoren exprimieren Gene, die im Zusammenhang zur
Entstehung von Tumoren stehen. Einer dieser Promotoren vermittelt die
gleichzeitige Expression des Tumorsuppressors RB1 und der ncRNA-RB1. Dieses
wird mittels eines Reporter-Assays gezeigt, welcher die Auswirkungen von
Retinoblastoma-assoziierten Punktmutationen auf die Bidirektionionalität des
Promotors nachvollzieht. Weiterhin wird die gekoppelte Expression der Genpaare
durch Mutation von Core-Promoterelementen gezeigt, welche sich in beide
Richtungen des bidirektionellen ncRNA-RB1/RB1 Promoters befinden. Dabei
beeinflusste die artifizielle Veränderung einzelner oder einiger weniger
Basenpaare die Transkriptionsinitiation in beide Promoterrichtungen. Um die
Funktionalität beider Gene eines Genpaares sowie ihre Beteiligung in
gemeinsamen biologischen Stoffwechselwegen aufzuklären, wurden die ncRNA-RB1
und die RB1 mRNA einzeln inaktiviert. Dieser Versuch zeigte, dass beide Gene
nicht gegenseitig ihre Expression beeinflussen und die ncRNA-RB1
regulatorische Effekte besitzt, die unterschiedlich von, andererseits aber
auch Ăźberlappend mit der transkriptionellen Regulation durch RB1 sind.
Unabhängig von RB1 beeinflusst die ncRNA-RB1 die Expression von Calreticulin
(CALR), eines Chaperons des endoplasmatischen Retikulums, positiv. Nach
Behandlung mit spezifischen Chemotherapeutika kann CALR zur Zelloberfläche von
Tumorzellen translozieren und dort als Fress-Signal fĂźr phagozytierende Zellen
dienen. Der Knock-down der nukleären ncRNA-RB1 in Tumorzellen reduziert die
Transkription des CALR-Gens und verhindert nachfolgend die Translokation des
CALR-Proteins zur Zelloberfläche als Auswirkung der Behandlung mit
Anthracyclinen. Die Konsequenz daraus ist eine verhinderte Aufnahme der ncRNA-
RB1 knock-down Zellen durch Makrophagen. Als Ergebnis stellt die gleichzeitige
Transkription von ncRNA-RB1 und RB1 von einem gemeinsamen bidirektionalen
Promoter eine VerknĂźpfung zwischen der Regulation der zwei Tumorsuppressoren
RB1 und CALR her. Der Verlust der Expression jedes Gens des Paares ncRNA-
RB1/RB1 fßhrt zur Beeinträchtigung Tumor unterdrßckender Mechanismen in der
Zelle
Validation of the position of the femoral tunnels in anatomic double-bundle ACL reconstruction with 3-D CT scan
This study compares the positioning of femoral AM and PL tunnels obtained with specific ancillary instruments during anatomic double-bundle ACL reconstruction with the native ACL footprint using three-dimensional computed tomography (3-D CT). In 35 consecutive patients, anatomic double-bundle ACL reconstruction was performed with specific ancillary instruments. Three-dimensional CT reconstruction of both knees was performed using the volume rendering technique. In the controls (contralateral knee, with intact ACL), the angle between the longitudinal axis of the footprint and the axis of the femur, the "footprint angle" (FA) was measured. On the involved side, using the axis passing through the tunnel centers, FA was also measured. In both the groups, footprint's length and width, and distances to cartilage margins were measured. FA was 28.1A degrees A A +/- A 5.0A degrees in the controls and 32.9A degrees A A +/- A 15.8A degrees on the involved side (n.s.). There was no statistical difference between the two groups for the other morphometric parameters: footprint's length and width, and distances to cartilage margins. Using specific ancillary instruments the morphometric parameters of the reconstructed femoral ACL footprint were similar to the native ACL
Bioabsorbable interference screw versus bioabsorbable cross pins: influence of femoral graft fixation on the clinical outcome after ACL reconstruction
Argonaute2 Mediates Compensatory Expansion of the Pancreatic β Cell
Pancreatic β cells adapt to compensate for increased metabolic demand during insulin resistance. Although the microRNA pathway has an essential role in β cell proliferation, the extent of its contribution is unclear. Here, we report that miR-184 is silenced in the pancreatic islets of insulin-resistant mouse models and type 2 diabetic human subjects. Reduction of miR-184 promotes the expression of its target Argonaute2 (Ago2), a component of the microRNA-induced silencing complex. Moreover, restoration of miR-184 in leptin-deficient ob/ob mice decreased Ago2 and prevented compensatory β cell expansion. Loss of Ago2 during insulin resistance blocked β cell growth and relieved the regulation of miR-375-targeted genes, including the growth suppressor Cadm1. Lastly, administration of a ketogenic diet to ob/ob mice rescued insulin sensitivity and miR-184 expression and restored Ago2 and β cell mass. This study identifies the targeting of Ago2 by miR-184 as an essential component of the compensatory response to regulate proliferation according to insulin sensitivity