- These lncRNAs were assigned, depending on their position relative to annotated genes, to “MSC- related long intergenic non-coding RNAs” named “Mlinc”, and to “MSC-related long overlapping antisense RNAs”. - Of these, 34712 were found to be intergenic and were thus referred to as “Mlinc” RNAs, and 38751 were found to overlap with coding regions but in anti-sense orientation and thus referred to as “Mloanc” RNAs (with criteria described as in “Methods” section and Fig. - Mlinc.28428.2, MlincV MlincV4.89912.1 and MlincV4.64225.1. - In particular, for Mlinc.28428.2, the transcript observed with long-reads sequencing cor- responded to the prediction made with short reads. - It was also supported by Mlinc.28428.1, a variant that differs by the absence of the second exon. - Similar characteristics were observed for Mlinc.128022, which also produced two variants with a different organisation of 5 exons. - The two other candidates, Mlinc.89912.1 and Mlinc.64225.1, are mono-exonic. - Mlinc.89912.1 occurs at the close proximity of FGF5 3’end, in reverse orientation. - For Mlinc.64225.1, the long-read sequence is longer than the ab initio short read prediction.. - Except for Mlinc.64225, and in accordance to the start of long reads, we observed CAGE enrichments at the 5’. - The results permitted the classification of our Mlincs according to observed specificity, from the most promising to the least restricted profile: Mlinc.28428.2 is expressed in Ad and BM derived MSCs. - Mlinc.128022.2 is expressed in Ad and BM-. - a List of tissues for the cell specific expression exploration (samples with ID numbers are listed in Additional file 8) b Relative expression of Mlinc.28428.2, Mlinc.128022.2, and Mlinc.89912.1 across ENCODE’s ribodepleted RNAseq data, made by k-mer quantification, normalised by k-mer per million. - Mlinc.89912.1 is principally expressed in BM-MSCs and less in UC and Ad- MSCs, but shows expression in epithelial and endothelial cells. - Finally, Mlinc.64225.1 differs from other Mlincs as it is also strongly expressed in keratinocytes, hematopoietic stem cells and epithelial cells (Figure in Additional file 12).. - We designed two primer pairs for both Mlinc.128022 variants to validate the existence of first splice, and two pairs for Mlinc.28428 variants, one overlapping the second exon and another corresponding to a splice between first and third exons.. - We confirmed most of the expression profiles obtained by k-mers quantifica- tion using RT-qPCR, notably the specificity of expression dependency on the MSC tissular origin: over expres- sion of Mlinc.28428 and 128022 in BM and Ad-MSCs.. - Nevertheless, few exceptions such as Mlinc.89912.1, pre- sented an enrichment in UC-MSCs not found with k-mers quantification. - To this end, we explored assumptions on the function of Mlinc.28428.2, Mlinc.128022.2 and Mlinc.89912.1 candidates using different published meth- ods. - We noted a predicted interaction between Mlinc.28428.2 and Beta- catenin (CTNNB1) as part of apoptosis-linked modules, 5’-3’ Exoribonuclease 1, component of the CCR4-NOT complex, mRNA Decapping Enzyme 1B as part of the. - Mlinc.128022 could interact with important genes like THY1 (CD90), NRF1 (mitochon- dria metabolism) with no module clearly highlighted.. - Mlinc.89912 could interact with tubulins, UBB (ubiqui- tin B), SMG6 nonsense mediated mRNA decay factor and ribosomes subunits (RPSX) proteins, RPL24 for nonsense mediated decay (NMD), PINK1 (mitophagy) and finally MGMT as part of the MGMT mediated DNA damage reversal module.. - Mlinc.28428.2 is down-regulated when JPX, SERTAD4- AS1, BOLA3-AS1, and SNRPD3 are KD and over- expressed with the KD of PTCHD3P1, ERVK3.1 and MEG3, among other lncRNAs without reported function (Fig. - All these features converge on the hypothesis of a link between the function of Mlinc.28428, stress response, senescence and cellular maintenance. - Mlinc.128022.2 is down-regulated with the KD of FOXN3-AS1, A1BG-AS1, CD27-AS1, and FLVCR1-AS1 (Fig. - Moreover, the reported over-expression of FOXN3 during the early stages of MSC osteodiffer- entiation [48], and the down-regulation of CD27-AS1 in MSCs of donors with bone fracture [49], allow us to hypothesise a possible function of Mlinc.128022 in bone remodelling and osteogenesis. - For each Mlinc (Mlinc.28428 (a) Mlinc.128022 (b) and Mlinc.89912 (c) respectively) 3 steps of prediction were performed. - Expression of differentially expressed annotated genes between positive (in turquoise) and negative (in pink) cells for Mlinc.28428, Mlinc.128022 and Mlinc.89912 respectively. - Finally, Mlinc.89912.1 is down-regulated after the KD of NEAT1-1 and PCAT6, and over-expressed when. - The manifest relations between cell pro- liferation and CDKN2B-AS MFI2 [58], MFI.AS1 [59], PCAT6 [60] and NEAT a combination with the DNA damage repair response, [63, 64] reinforce the prediction of a role of Mlinc.89912 in these mechanisms.. - Mlinc.28428 and Mlinc.128022 are enriched in at least cytoplasm (Fig. - 5a- b), whereas Mlinc.89912 is enriched in free nucleus fraction suggesting interactions with nuclear component (Fig. - were Mlinc.28428-positives, 4944 were Mlinc.128022- positives, and 404 were Mlinc.89912-positives. - We found that Mlinc.28428-positive cells under-express H19 and PI16 (Fig. - Despite the low number of differentially expressed genes in Mlinc.28428-positive cells, their functional behaviour and their known targets suggest a pathway linked to stress response and senescence establishment that reinforce our previous assumptions on Mlinc.28428 function.. - Mlinc.128022-positive cells are enriched in FTH1, TPM2, FTL and CD24 and present a lower expression in HMGN2, HMGB1, ODC1, PTTG1, BIRC5, EIF5A, MKI67, UBE2S, FGF5, HAS2-AS1 (Fig. - The Mlinc.128022-positive cells have an increased expression of ferritin (light and heavy chains), major actor in iron metabolism in osteoblastic cell line [69], that is also involved in osteogenic differentiation [70]. - Two genes, enriched in Mlinc.128022-positive cells, are positively linked to the osteogenic differentiation potential of MSCs: the tropomyosin 2 (TPM2), downregulated when human. - In addition ODC1, under- represented in Mlinc.128022-positive cells, inhibited the MSCs osteogenic differentiation [74, 75]. - These data suggest that the expression profile of Mlinc.128022 positive cells indicate a subpopulation of undifferentiated osteogenic progenitors, probably in senescence or quiescence.. - Mlinc.89912-positive cells are enriched in FGF5 and HIST1H4C (Fig. - It reinforces our assumptions that Mlinc.89912 has a role in cell pro- liferation and DNA damage repair. - stress and senescence for Mlinc.28428.2, osteodifferenti- ation for Mlinc.128022.2 and cell cycle/proliferation for Mlinc.89912. - 6, we observed a statistically rele- vant increase of Mlinc.28428 expression in MSCs under replicative stress and in MSCs with CRISPR-Cas9 deple- tion of genes with important role against senescence.. - Mlinc.28428 expres- sion increased with tunicamycin treatment, late passage. - a Expression of Mlinc.28428.1 in the context of oxydative, replicative, or KO-driven, stress and senescence (PRJNA396193, PRJNA433339). - b Expression of Mlinc.128022 in osteodifferentiation conditions (PRJNA515466) or osteodifferentiation potential (PRJNA379707). - c Expression of Mlinc.89912 in the context of proliferation (PRJNA328824 and PRJNA498109).. - Interestingly, YAP KO, significantly increases the expression of Mlinc.28428.2. - This would lead us to conclude that Mlinc.28428 is overexpressed in senescence and stress conditions, suggesting a role in one or both of these phenomena.. - The change in Mlinc.128022 expression is strictly linked to osteodifferentiation conditions. - Mlinc.128022 expres- sion shows a relevant increase in MSCs exposed to fungal metabolite Cytochalasin D (CytoD). - studied osteogenic MSCs differenti- ation [82], with a similar increase of Mlinc.128022 being observed after ten days.. - We then quantified the expression of Mlinc.89912 in a study that compares proliferating MSCs versus con- fluent MSCs [83, 84]. - However Mlinc.89912 expression was reduced when IWR-1, an inhibitor of beta-catenin nuclear translocation, that reduced the proliferation of MSCs, was added to the medium. - The functional domains of these genes are sum- marised in Table 1 and confirm the potential functional role suggested from FANTOM data: stress-related path- ways for Mlinc.28428, MSCs differentiation with a pre- sumed orientation in osteo-progenitors for Mlinc.128022. - and a more restricted role in proliferation and DNA repair for Mlinc.89912.. - Mlinc.28428 Apoptosis, mRNA decay, PPARA activity, intracellular transport, response to hypoxia and cell cycle. - Mlinc.128022 THY1, NRF1 Chromatin, cytoplasm FOXN3-AS1,. - Mlinc.89912 MGMT-mediated DNA damage reversal, Nonsense Mediated Decay, Tubulin metabolism. - Mlinc.28428 has concomitant expression with genes related to the stress response pathway. - Mlinc.28428 could be a good target for treatment to study the senescence process, age pathologies or stress response. - Mlinc.128022 poten- tially interacts with THY1 (CD90) and has co-occurences with genes linked to osteoprogenitors and cell differen- tiation. - Finally, Mlinc.89912 poten- tially interacts with damage repair and RNA decay, and tubulin metabolism, all linked to cell proliferation and cell cycle. - Moreover, the subcompartment enrichment corre- sponds to this prediction: Mlinc.89912.1 is the only can- didate to have possible interactions with DNA-repair sys- tem, a hypothesis corresponding to its observed enrich- ment in the nucleus. - 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https://www.frontiersin.org/. - http://www.nature.com/. - https://www.nature.com/. - http://www.sciencedirect.com/. - www.ncbi.nlm.nih.gov/pmc/articles/PMC4039077/.. - https://www.nature.. - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5788881/.. - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4982273/.. - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2973839/.. - http://www.sciencedirect.com/science/article/. - https://www.nature.com/articles/. - http://www.nature.. - www.ncbi.nlm.nih.gov/pmc/articles/PMC4937194/.. - https://www.cell.com/. - www.ncbi.nlm.nih.gov/pmc/articles/PMC1297577/.
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