RESUMEN
Healing of large calvarial bone defects remains challenging. An RNA-guided Split dCas12a system is previously harnessed to activate long non-coding RNA H19 (lncRNA H19, referred to as H19 thereafter) in bone marrow-derived mesenchymal stem cells (BMSCs). H19 activation in BMSCs induces chondrogenic differentiation, switches bone healing pathways, and improves calvarial bone repair. Since adipose-derived stem cells (ASCs) can be harvested more easily in large quantity, here it is aimed to use ASCs as an alternative cell source. However, H19 activation alone using the Split dCas12a system in ASCs failed to elicit evident chondrogenesis. Therefore, split dCas12a activators are designed more to co-activate other chondroinductive transcription factors (Sox5, Sox6, and Sox9) to synergistically potentiate differentiation. It is found that co-activation of H19/Sox5/Sox6 in ASCs elicited more potent chondrogenic differentiation than activation of Sox5/Sox6/Sox9 or H19 alone. Co-activating H19/Sox5/Sox6 in ASCs significantly augmented in vitro cartilage formation and in vivo calvarial bone healing. These data altogether implicated the potentials of the Split dCas12a system to trigger multiplexed gene activation in ASCs for differentiation pathway reprogramming and tissue regeneration.
Asunto(s)
Diferenciación Celular , Condrogénesis , ARN Largo no Codificante , Factores de Transcripción SOXD , Cráneo , Factores de Transcripción SOXD/metabolismo , Factores de Transcripción SOXD/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Animales , Humanos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Tejido Adiposo/citología , Células Madre/metabolismo , Células Madre/citología , Osteogénesis/genéticaRESUMEN
We investigated the influence of 17ß-estradiol (17ß-E2) on cartilage extracellular matrix (ECM) homeostasis in postmenopausal women. We focused on the roles of estrogen receptors (ESR) and SOX6 in 17ß-E2-mediated stimulation of ECM metabolism during chondrocyte (CH) degeneration. We compared the expression of anabolic genes (collagen II and aggrecan) and catabolic genes (MMPs and TIMPs) in IL-1ß-induced CH degeneration in vitro, with and without 17ß-E2 supplementation. We separately silenced the SOX6, ESR1, and ESR2 genes in CHs to determine their impact on 17ß-E2 treatment. Additionally, we used Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) and luciferase assays to investigate protein-DNA interactions within ESR2 and SOX6-promoter complexes. After three days of IL-1ß treatment, ESR1/2, SOX6, collagen II, aggrecan, and TIMP1/3 were decreased, while MMP3/9/13 were increased. The addition of 17ß-E2 partially reversed these effects, but silencing SOX6, ESR1, or ESR2 weakened the protective effects of 17ß-E2. Silencing ESR2, but not ESR1, abolished the upregulation of SOX6 induced by 17ß-E2. ESR2 was found to bind the SOX6 promoter and regulate SOX6 expression. 17ß-E2 upregulates SOX6 through ESR2 mediation, and the synergistic effect of 17ß-E2 and ESR2 on SOX6 balances ECM metabolism in CHs.
Asunto(s)
Condrocitos , Estradiol , Receptor beta de Estrógeno , Matriz Extracelular , Interleucina-1beta , Factores de Transcripción SOXD , Condrocitos/metabolismo , Condrocitos/efectos de los fármacos , Estradiol/farmacología , Humanos , Receptor beta de Estrógeno/metabolismo , Receptor beta de Estrógeno/genética , Femenino , Matriz Extracelular/metabolismo , Matriz Extracelular/efectos de los fármacos , Factores de Transcripción SOXD/metabolismo , Factores de Transcripción SOXD/genética , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacología , Receptor alfa de Estrógeno/metabolismo , Receptor alfa de Estrógeno/genética , Regiones Promotoras Genéticas/genética , Células CultivadasRESUMEN
The identification of regulatory networks contributing to fetal/adult gene expression switches is a major challenge in developmental biology and key to understand the aberrant proliferation of cancer cells, which often reactivate fetal oncogenes. One key example is represented by the developmental gene LIN28B, whose aberrant reactivation in adult tissues promotes tumor initiation and progression. Despite the prominent role of LIN28B in development and cancer, the mechanisms of its transcriptional regulation are largely unknown. Here, by using quantitative RT-PCR and single cell RNA sequencing data, we show that in erythropoiesis the expression of the transcription factor SOX6 matched a sharp decline of LIN28B mRNA during human embryo/fetal to adult globin switching. SOX6 overexpression repressed LIN28B not only in a panel of fetal-like erythroid cells (K562, HEL and HUDEP1; ≈92% p < 0.0001, 54% p = 0.0009 and ≈60% p < 0.0001 reduction, respectively), but also in hepatoblastoma HepG2 and neuroblastoma SH-SY5H cells (≈99% p < 0.0001 and ≈59% p < 0.0001 reduction, respectively). SOX6-mediated repression caused downregulation of the LIN28B/Let-7 targets, including MYC and IGF2BP1, and rapidly blocks cell proliferation. Mechanistically, Lin28B repression is accompanied by SOX6 physical binding within its locus, suggesting a direct mechanism of LIN28B downregulation that might contribute to the fetal/adult erythropoietic transition and restrict cancer proliferation.
Asunto(s)
Proteínas de Unión al ARN , Factores de Transcripción SOXD , Humanos , Factores de Transcripción SOXD/genética , Factores de Transcripción SOXD/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Línea Celular Tumoral , Regulación del Desarrollo de la Expresión Génica , Eritropoyesis/genética , MicroARNs/genética , MicroARNs/metabolismo , Células Hep G2 , Células K562 , Regulación Neoplásica de la Expresión Génica , Células Eritroides/metabolismoRESUMEN
SET (SuVar3-9, Enhancer of Zeste, Trithorax) domain bifurcated histone lysine methyltransferase 1, setdb1, is the predominant histone lysine methyltransferase catalyzing H3K9me3. Prior studies have illustrated that setdb1 and H3K9me3 critically regulate sex differentiation and gametogenesis. However, the molecular details by which setdb1 is involved in these processes in fish have been poorly reported. Here, we cloned and characterized the setdb1 ORF (open reading frame) sequence from Chinese tongue sole (Cynoglossus semilaevis). The setdb1 ORF sequence was 3,669 bp, encoding a 1,222-amino-acid protein. Phylogenetic analysis showed that setdb1 was structurally conserved. qRT-PCR revealed that setdb1 had a high expression level in the testes at 12 mpf (months post fertilization). Single-cell RNA-seq data at 24 mpf indicated that setdb1 was generally expressed in spermatogenic cells at each stage except for sperm and was centrally expressed in oogonia. H3K9me3 modification was observed in gonads with the immunofluorescence technique. Furthermore, the overexpression experiment suggested that sox5 was a candidate target of setdb1. sox5 was abundantly expressed in male and pseudomale gonads at 24 mpf. Single-cell RNA-seq data showed that sox5 was mainly expressed in spermatogonia and its expression gradually declined with differentiation. Taken together, our findings imply that setdb1 regulates sox5 transcription in gonads, which provides molecular clues into histone modification-mediated orchestration of sex differentiation and gametogenesis.
Asunto(s)
Proteínas de Peces , Lenguado , Código de Histonas , N-Metiltransferasa de Histona-Lisina , Factores de Transcripción SOXD , Animales , Masculino , Lenguado/genética , Gónadas/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Filogenia , Semen/metabolismo , Factores de Transcripción SOXD/metabolismo , Proteínas de Peces/metabolismoRESUMEN
OBJECTIVE: MicroRNA-23b-3p has been demonstrated to act as a safeguard against several autoimmune diseases. However, its role in Sjögren's syndrome (SS) remains unclear. METHODS: In order to investigate its role in SS, we administered agomiR-23b-3p or agomiR-NC to non-obese diabetic (NOD) mice via tail vein weekly for 6 weeks. The study examined the saliva flow rate, histological changes in submandibular glands, and levels of autoantibodies. Additionally, the levels of several cytokines, cell apoptosis, and NF-κB signaling were evaluated. The protective effect of miR-23b-3p was confirmed in a cell model. RESULTS: The results demonstrated that miR-23b-3p overexpression improved salivary flow rates, inhibited lymphocyte infiltration, reduced cytokine levels, and suppressed cell apoptosis in NOD mice. Moreover, NF-κB signaling was inactivated following miR-23b-3p overexpression. In a cellular model of SS, overexpression of miR-23b-3p protected submandibular gland epithelial cells exposed to IFN-γ against apoptosis and inflammation by targeting SOX6. CONCLUSIONS: The study concludes that miR-23b-3p alleviates SS by targeting SOX6 and inhibiting the NF-κB signaling pathway. The miR-23b-3p/SOX6 axis represents a promising avenue for the development of novel therapeutic strategies for SS.
Asunto(s)
Apoptosis , Ratones Endogámicos NOD , MicroARNs , FN-kappa B , Factores de Transcripción SOXD , Transducción de Señal , Síndrome de Sjögren , Animales , Femenino , Humanos , Ratones , Apoptosis/genética , MicroARNs/genética , FN-kappa B/metabolismo , Síndrome de Sjögren/genética , Síndrome de Sjögren/inmunología , Factores de Transcripción SOXD/genética , Factores de Transcripción SOXD/metabolismo , Glándula Submandibular/patología , Glándula Submandibular/metabolismoRESUMEN
BACKGROUND: Psoriasis is a complex and recurrent chronic inflammatory skin disease, and the abnormal proliferation of keratinocytes plays a crucial role in the pathogenesis of psoriasis. Long non-coding RNAs (lncRNAs) play an indispensable role in regulating cellular functions. This research aims to explore the potential impact of lncRNA MIR181A2HG on the regulation of keratinocyte proliferation. METHODS: The expression level of MIR181A2HG and the mRNA level of KRT6, KRT16, and SOX6 were assessed using qRT-PCR. The viability and proliferation of keratinocytes were evaluated using CCK-8 and EdU assays. Cell cycle analysis was performed using flow cytometry. Dual-luciferase reporter assays were applied to test the interaction among MIR181A2HG/miR-223-3p/SOX6. Protein level was detected by Western blotting analysis. RESULTS: The findings indicated that psoriasis lesions tissue exhibited lower levels of MIR181A2HG expression compared to normal tissue. The overexpression of MIR181A2HG resulted in the inhibition of HaCaT keratinocytes proliferation. The knockdown of MIR181A2HG promoted cell proliferation. The dual-luciferase reporter assay and rescue experiments provided evidence of the interaction among MIR181A2HG, SOX6, and miR-223-3p. CONCLUSIONS: The lncRNA MIR181A2HG functions as a miR-223-3p sponge targeting SOX6 to regulate the proliferation of keratinocytes, which suggested that MIR181A2HG/miR-223-3p/SOX6 might be potential diagnostic and therapeutic targets for psoriasis.
Asunto(s)
Proliferación Celular , Queratinocitos , MicroARNs , Psoriasis , ARN Largo no Codificante , Factores de Transcripción SOXD , Humanos , MicroARNs/metabolismo , MicroARNs/genética , Queratinocitos/metabolismo , Proliferación Celular/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Factores de Transcripción SOXD/metabolismo , Factores de Transcripción SOXD/genética , Psoriasis/genética , Psoriasis/metabolismo , Psoriasis/patología , Células HaCaTRESUMEN
SRY-box transcription factor 6 (SOX6) is a member of the SOX gene family and inhibits the proliferation of cervical cancer cells by inducing cell cycle arrest. However, the final cell fate and significance of these cell-cycle-arrested cervical cancer cells induced by SOX6 remains unclear. Here, we report that SOX6 inhibits the proliferation of cervical cancer cells by inducing cellular senescence, which is mainly mediated by promoting transforming growth factor beta 2 (TGFB2) gene expression and subsequently activating the TGFß2-Smad2/3-p53-p21WAF1/CIP1-Rb pathway. SOX6 promotes TGFB2 gene expression through the MAP4K4-MAPK (JNK/ERK/p38)-ATF2 and WT1-ATF2 pathways, which is dependent on its high-mobility group (HMG) domain. In addition, the SOX6-induced senescent cervical cancer cells are resistant to cisplatin treatment. ABT-263 (navitoclax) and ABT-199 (venetoclax), two classic senolytics, can specifically eliminate the SOX6-induced senescent cervical cancer cells, and thus significantly improve the chemosensitivity of cisplatin-resistant cervical cancer cells. This study uncovers that the MAP4K4/WT1-ATF2-TGFß2 axis mediates SOX6-induced cellular senescence, which is a promising therapeutic target in improving the chemosensitivity of cervical cancer.