123 research outputs found
THE NON-CODING RNA, PRINS AFFECTS AIM2 INFLAMMASOME ACTIVATION IN KERATINOCYTES
The non-coding RNA, PRINS was previously described by our research group as a differentially expressed transcript in psoriatic uninvolved and healthy skin. The expression level of PRINS in cultured keratinocytes is altered after exposure to various stress factors and silencing of it decreases the viability of keratinocytes during stress stimulations suggesting its role in stress response of the cells. A potential stress signal in psoriatic skin may be the extracellular DNA, which activates the AIM2 inflammasome. The activated inflammasome cleaves the precursor proIL-1β form into mature, functioning IL-1β. The role of the AIM2 inflammasome and the IL-1β cytokine in psoriasis has been described recently.
The aim of our study was to investigate if the PRINS non coding RNA affects the expression and activation of the inflammasome members and IL-1β in normal human epidermal keratinocytes (NHEK) after exposure to extracellular DNA.
The expression of PRINS was transiently silenced by a vector based RNA interference method in cultured NHEK cells. Silenced and non-silenced NHEK cells were primed with the cytokines TNF-α and IFN-γ and transfected with the synthetic DNA analogue poly(dA:dT). The expression of PRINS and inflammasome members was detected by real-time RT-PCR and the secreted IL-1β was measured by ELISA.
Poly(dA:dT) treatment caused a moderate increase in PRINS expression and IL-1β secretion as well, whereas priming with a combination of TNF-α and IFN-γ before poly(dA:dT) transfection resulted in a highly elevated PRINS expression and higher secreted IL-1β levels. The silencing of PRINS decreased the amount of secreted IL-1β, but did not affect the expression of the proIL-1β or AIM2.
Our results suggest that the PRINS non-coding RNA regulates the IL-1β production of NHEK cells, but not through the regulation of proIL-1β expression, rather contributing to inflammasome-activation
PPIG, SFRS-18 és LUC7L3 Splicing regulárok vizsgálata szinkronizált, immortalizált sejtvonalakban és pikkelysömörben
Az mRNS Ă©rĂ©s (splicing) folyamatának vizsgálata egĂ©szen Ăşjfajta megközelĂtĂ©snek számĂt a pikkelysömör kutatásban: a splicing mechanizmus e betegsĂ©gben tapasztalt eltĂ©rĂ©seirĹ‘l mindössze nĂ©hány közlemĂ©ny jelent meg napjainkig.
Egy nemrĂ©g elvĂ©gzett microarray vizsgálatban több olyan gĂ©nt is azonosĂtottunk, amelyek T-limfokin kezelĂ©s hatására eltĂ©rĹ‘ kifejezĹ‘dĂ©s változással reagálnak, majd bioinformatikai mĂłdszerekkel tovább elemeztĂĽk azokat. A gĂ©nek között három olyat is találtunk (serine/ arginine-rich splicing factor 18 (SFRS18), peptydilpropyl isomerase G (PPIG), luc-7 like 3 (LUC7L3)) amelyek funkciĂłja az mRNS Ă©rĂ©s szabályozásához kötĹ‘dik.
A PPIG, SFRS18 Ă©s LUC7L3 gĂ©nek kifejezĹ‘dĂ©sĂ©t szinkronizált, immortalizált sejtvonalak segĂtsĂ©gĂ©vel követtĂĽk. KĂ©t fĂĽggetlen sejtvonal (HaCaT Ă©s HPV-KER) esetĂ©ben is azt tapasztaltuk, hogy a splicing regulátorok kifejezĹ‘dĂ©si mintázata jelentĹ‘s hasonlĂłságot mutat. EzekbĹ‘l az eredmĂ©nyekbĹ‘l arra következtettĂĽnk, hogy a gĂ©nek upstream reguláciĂłja közös transzkripciĂłs faktorok Ăştján valĂłsulhat meg.
A splicing gĂ©nek fehĂ©rjeszintű expressziĂłját is meghatároztuk Western blot technika segĂtsĂ©gĂ©vel. ElĹ‘kĂsĂ©rleteinkben azt találtuk, hogy az SFRS18 Ă©s a PPIG expressziĂłja fehĂ©rje
szinten is hasonlóságot mutat a szinkronizált, immortalizált HPV-KER sejtekben.
Az in vitro analĂzisek mellett egĂ©szsĂ©ges Ă©s pikkelysömörös betegekbĹ‘l származĂł tĂĽnetes Ă©s tĂĽnetmentes mintákon is megvizsgáltuk, mikĂ©nt alakul a három fehĂ©rje expressziĂłja. KĂsĂ©rleteink során megmutattuk, hogy a PPIG fehĂ©rje expressziĂłs mintázata Ă©s kifejezĹ‘dĂ©sĂ©nek mennyisĂ©ge is jelentĹ‘sen megváltozik: a tĂĽnetmentes bĹ‘rben a PPIG festĹ‘dĂ©se fokozottnak mutatkozott Ă©s eloszlása is eltĂ©r az egĂ©szsĂ©ges bĹ‘rĂ©tĹ‘l.
EredmĂ©nyeink alapján Ăşgy gondoljuk, hogy a splicing szabályozás eltĂ©rĂ©seinek fontos szerepe lehet a pikkelysömör tĂĽneteinek kifejlĹ‘dĂ©sĂ©ben. A splicing folyamatának további tanulmányozása nagyban segĂtheti a betegsĂ©g komplex molekuláris hátterĂ©nek megĂ©rtĂ©sĂ©t
Regulatory Networks Contributing to Psoriasis Susceptibility
The non-involved, healthy-looking skin of psoriatic pa-
tients displays inherent characteristics that make it pro-
ne to develop typical psoriatic symptoms. Our primary
aim was to identify genes and proteins that are diffe-
rentially regulated in the non-involved psoriatic and the
normal epidermis, and to discover regulatory networks
responsible for these differences. A cDNA microarray ex-
periment was performed to compare the gene expression
proiles of 4 healthy and 4 psoriatic non-involved epider-
mis samples in response to T-cell lymphokine induction
in organotypic cultures. We identiied 61 annotated genes
and another 11 expressed transcripts that were differen-
tially regulated in the psoriatic tissues. Bioinformatics
analysis suggested that the regulation of cell morpholo-
gy, development and cell death is abnormal, and that the
metabolism of small molecules and lipids is differentially
regulated in psoriatic epidermis. Our results indicate
that one of the early steps of psoriasis pathogenesis may
be the abnormal regulation of IL-23A and IL-1B genes in
psoriatic keratinocytes. Key words: non-involved psoriatic
epidermis; T-cell lymphokines; gene expression analysis;
regulatory networks; IL-23A
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