Expression and clinical significance of interferon regulatory factor 4 and soluble suppression of tumorigenesis 2 in conjunctival epithelial cells and tears of patients with dry eye / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the expression and clinical significance of interferon regulatory factor 4(IRF4)and soluble suppression of tumorigenesis 2(sST2)in conjunctival epithelial cells and tears of patients with dry eye.METHODS: A total of 94 patients with dry eye who admitted to our hospital from January 2019 to December 2021 were selected as the dry eye group, and 97 physical examiners who underwent ophthalmic examination were selected as the control group at the same time. The conjunctival epithelial cells and tears of the subjects were collected, and the clinical indicators, including tear film break-up time(BUT), corneal fluorescein staining(CFS)score, and Schirmer Ⅰ test(SⅠt)were recorded. The levels of IRF4 and sST2 in conjunctival epithelial cells were detected by quantitative real-time polymerase chain reaction(qRT-PCR), and the levels of IRF4 and sST2 in tears were detected by enzyme-linked immunosorbent assay(ELISA). Pearson method was used to analyze the correlation between IRF4 and sST2 levels in conjunctival epithelial cells and tears and clinical indicators of dry eye patients.RESULTS: The levels of IRF4 and sST2 in conjunctival epithelial cells and tears in dry eye group before treatment were significantly higher than those in control group(P<0.001). The levels of IRF4 and sST2 in conjunctival epithelial cells and tears of dry eye patients at 4wk after treatment were significantly lower than those before treatment(P<0.001). The BUT and SⅠt of dry eye patients increased significantly at 4wk after treatment, and the CFS score decreased significantly(P<0.001). The levels of IRF4 and sST2 in conjunctival epithelial cells and tears of dry eye patients before treatment were positively correlated with CFS score before treatment and negatively correlated with BUT and SⅠt before treatment(P<0.001).CONCLUSION: The levels of IRF4 and sST2 in conjunctival epithelial cells and tears of patients with dry eye are increased, and are significantly correlated with BUT, SⅠt and CFS scores, which has potential to become a new therapeutic target for dry eye.

Application of CRISPR-Cas9 to interrogate novel gene functions in cutaneous melanoma

Melanoma accounts for 3% of skin neoplasms and is the leading cause of death from skin disorders worldwide. The high mortality rate associated with this disease stems from the high capacity of melanoma patients to develop metastases and treatment relapse with inhibitors of the MAPK signaling pathway (such as BRAF inhibitors), commonly used in melanoma therapy. Thus, the investigation of genes involved in the mechanisms of melanoma development is essential for new and more effective therapeutic strategies. Hence, we describe in this thesis two projects involving the genes SIN3B and IRF4 as possible biomarkers for cutaneous melanoma. Initially, through bioinformatics analyses performed by our group, an upregulation of SIN3B was found in metastatic melanomas. This result together with the understanding of SIN3B role in regulating gene expression and oncogenic transformation, prompted us to describe in this thesis some mechanisms by which SIN3B may influence melanoma development. We then sought to characterize the gene function using SIN3B-deleted cells, generated by the CRISPR-Cas9 methodology. Initially, we observed increased SIN3B expression in BRAF-mutant metastatic melanomas, where we noted that the long splicing variant of the gene (NM_001297595.1) was effectively prevalent in melanomas. Subsequently, we designed gRNAs between the exons 2 and 3 of the human SIN3B gene and engineered three knockout clones and three control clones (containing empty lentiCRISPRv2 plasmid) from different melanoma cell lines (SKMEL28, A2058, and A375). Through functional analyses, it was observed that the absence of the gene did not interfere in the proliferation of tumor cells; however, it led to a decrease in invasive properties. These results were verified by Boyden chamber assays and transcriptome analysis (total RNA sequencing of deleted cells), where a decrease in migration and motility pathways was observed. Additionally, a screening of synthetically lethal genes with SIN3B was performed with a genome wide CRISPR library. These results showed that USP7 and STK11 genes, which belong to the FoxO signaling pathway, were essential in SIN3B-depleted melanoma cells. Finally, through a collaborative project with the Wellcome Trust Sanger Institute, previous large-scale sequencing analyses demonstrated that deletion of the IRF4 gene was lethal for melanoma cells. Accordingly, we performed IRF4 silencing in vitro and noticed that the lack of IRF4 promotes cell death and apoptosis, independently of MYC and MITF, known in the literature to be downstream targets of this gene. Therefore, these data suggest that IRF4 plays a vital role in melanoma cell survival. Taken together, both works herein described in this thesis demonstrate how CRISPR-Cas9 can be applied to study the functions and mechanisms of genes involved in melanoma progression, collectively helping in the development of more effective therapeutic strategies for this tumor

O melanoma representa 3% dos tipos de neoplasias cutâneas e é a maior causa das mortes por distúrbios de pele no mundo. A alta taxa de mortalidade associada à essa doença advém da alta capacidade de pacientes com melanoma desenvolverem metástases, e apresentarem recidiva após tratamento com inibidores da via de sinalização MAPK (como da proteína BRAF), comumente utilizados no tratamento de pacientes metastáticos. Assim, a investigação de genes envolvidos nos mecanismos de desenvolvimento do melanoma é primordial para novas estratégias terapêuticas mais efetivas. Dessa forma, descrevemos no presente trabalho dois projetos envolvendo os genes SIN3B e IRF4 como possíveis biomarcadores para melanoma cutâneo. Em análises prévias de bioinformática realizados pelo nosso grupo, SIN3B foi identificado tendo maior expressão em melanomas metastáticos. Além disso, diversos estudos mostraram que o gene está envolvido na regulação da expressão gênica e transformação oncogênica. Dessa forma, descrevemos nessa tese alguns mecanismos pelos quais SIN3B pode influenciar no desenvolvimento do melanoma, através da caracterização funcional de células SIN3B-deletadas pela metodologia CRISPR-Cas9. Inicialmente, observamos aumento na expressão de SIN3B em melanomas metastáticos BRAF-mutados, onde notamos que a variante de splicing longa do gene (NM_001297595.1), era efetivamente prevalente em melanomas. Assim, desenhamos sequências de RNA guias entre os éxons 2 e 3 do gene SIN3B humano e, obtivemos três clones knockout e outros três clones controle (contendo plasmídeo vazio) em diferentes linhagens de melanoma (SKMEL28, A2058 e A375), para caracterização funcional. Observou-se que a ausência do gene não interferiu na proliferação das células tumorais, contudo, acarretou na diminuição de processos invasivos. Esses resultados foram averiguados através de ensaios em câmara de Boyden e análises de transcriptoma (sequenciamento de RNA total das células deletadas), onde notou-se diminuição das vias de migração e motilidade. Adicionalmente, um rastreamento de genes sinteticamente letais com SIN3B foi realizado com uma biblioteca de CRISPR capaz de silenciar todo o genoma. Esses resultados mostraram que os genes USP7 e STK11, ambos pertencentes à via de sinalização de FoxO, são essenciais nas células SIN3B deletadas. Por fim, através de um projeto colaborativo com o Wellcome Trust Sanger Institute, análises prévias de sequenciamento de larga escala demonstraram que a deleção do gene IRF4 era letal para células de melanoma. Dessa forma, realizamos o silenciamento de IRF4 in vitro e notamos que a ausência do gene promove morte celular e apoptose, independentemente de MYC e MITF, conhecidos na literatura por serem alvos downstream do gene. Portanto, esses dados sugerem que IRF4 tem um papel importante na sobrevivência de células de melanoma. Em conjunto, ambos trabalhos descritos nessa tese, demonstram como a metodologia CRISPR-Cas9 pode auxiliar no entendimento de processos importantes para a malignidade do melanoma e contribuir para estratégias terapêuticas mais efetivas para esse tumor

IRF4 and IRF8 expression are associated with clinical phenotype and clinico-hematological response to hydroxyurea in essential thrombocythemia / 医学前沿

The morbidity and mortality of myeloproliferative neoplasms (MPNs) are primarily caused by arterial and venous complications, progression to myelofibrosis, and transformation to acute leukemia. However, identifying molecular-based biomarkers for risk stratification of patients with MPNs remains a challenge. We have previously shown that interferon regulatory factor-8 (IRF8) and IRF4 serve as tumor suppressors in myeloid cells. In this study, we evaluated the expression of IRF4 and IRF8 and the JAK2V617F mutant allele burden in patients with MPNs. Patients with decreased IRF4 expression were correlated with a more developed MPN phenotype in myelofibrosis (MF) and secondary AML (sAML) transformed from MPNs versus essential thrombocythemia (ET). Negative correlations between the JAK2V617F allele burden and the expression of IRF8 (P < 0.05) and IRF4 (P < 0.001) and between white blood cell (WBC) count and IRF4 expression (P < 0.05) were found in ET patients. IRF8 expression was negatively correlated with the JAK2V617F allele burden (P < 0.05) in polycythemia vera patients. Complete response (CR), partial response (PR), and no response (NR) were observed in 67.5%,10%, and 22.5% of ET patients treated with hydroxyurea (HU), respectively, in 12 months. At 3 months, patients in the CR group showed high IRF4 and IRF8 expression compared with patients in the PR and NR groups. In the 12-month therapy period, low IRF4 and IRF8 expression were independently associated with the unfavorable response to HU and high WBC count. Our data indicate that the expression of IRF4 and IRF8 was associated with the MPN phenotype, which may serve as biomarkers for the response to HU in ET.

LBH589/lenalidomide regulates, IRF4 and promotes apoptosis of multiple myeloma cells / 重庆医学

Objective We aimed to investigate the expression of IRF4 and apoptosis of the histone deacetylase inhibitor LBH589 against MM cells in vitro,and study the mechanism of apoptosis when LBH589 alone or/and combined with lenalidomide in RPMI8226 cell so as to provide a new strategy for the treatment of multiple myeloma.Methods The cell viability on the growth of RPMI8226 cell were assessed by CCK8 assay.Apoptosis were measured by flow cytometry,The Grandpad software analyzes the statistical significance and evaluates the synergistic effect of the drug.The expression level of the related transcription factor and apoptotic gene protein were determined by western blot.The cell viability on the growth of RPMI8226-R cell were assessed by CCK8.Results LBH589 combined with lenalidomide have significant effect on inhibit cell proliferation and induce apoptosis in a dose-dependent manner.Apoptosis induced by LBH589/lenalidomide alone or combination was shown to involved PARP activation,decreased Bcl-2 and Bcl-xl expression.significantly down regulated transcriptional related factors of IRF4 and c-MYC expression compared with either agent alone.Conclusion LBH589 and lenalidomide alone or combination decrease the expression of transcription factor IRF4 and c-MYC,and have a significant synergistic effect,and highly expression of IRF in RPMI8226-R reduce proliferation inhibition.

Collagen I-induced dendritic cells activation is regulated by TNF-α production through down-regulation of IRF4.

Previously we have shown that collagen I enhances the maturation and function of dendritic cells (DCs). Inflammatory mediators such as tumour necrosis factor (TNF)-α, interleukin (IL)-1β and lipopolysaccharide (LPS) are also known to activate DCs. Here we investigated the involvement of TNF-α on the collagen I-induced DCs activation. TNF-a neutralization inhibited collagen I-induced IL-12 secretions by DCs. Additionally, we observed suppression of collagen I-induced costimulatory molecules expression along with down-regulation of genes involved in DCs activation pathway. Furthermore, TNF-α inhibition upon collagen Istimulation up-regulated the expression of interferon regulatory transcription factor IRF4, when compared to collagen I only treated cells. Collectively, our data demonstrate that collagen I induce TNF-α production, which is crucial for the activation and function of DCs, through down-regulation of IRF4, and implicates the importance in development of anti- TNF-α therapeutics for several inflammatory diseases.

Expression of IRF-4 and IBP in Skin Lesions of Patients with Psoriasis Vulgaris / 华中科技大学学报（医学）（英德文版）

The expression of the interferon regulatory factor 4 (IRF-4) and the IRF-4-binding protein (IBP) in psoriatic skin lesions was investigated.The expression of IRF-4 and IBP in skin lesions of 20 patients with psoriasis vulgaris were immunohistochemically dectected.Normal skin from 10 healthy people was used as normal control.The study showed that expression of IRF-4 was increased significantly in keratinocytes and inflammatory cells in the lesions of psoriasis vulgaris than that in the normal control.The detection revealed that IBP expression in keratinocytes,lymphocytes,hair follicles,and sebaceous glands in normal skin was significantly lower than that in the lesions of psoriasis vulgaris (P＜0.05).Both IRF-4 and IBP might be involved in the pathogenesis of psoriasis vulgaris.

The Association between Cytogenetic Abnormalities and Clinical Outcomes Based on Prognostic Factors of the Children Cancer Group (CCG) in Pediatric Patients with Acute Leukemia: Two Institutional Retrospective Studies / 대한혈액학회지

BACKGROUND: We investigated the incidence of cytogenetic abnormalities as well as the correlation of the cytogenetic abnormalities and clinical outcomes based on the prognostic factors of the Children Cancer Group (CCG) in children with acute leukemia. METHODS: We retrospectively reviewed the cytogenetic studies and clinical data from 99 children that were diagnosed with acute leukemia and treated with CCG regimens in two institutions. A conventional cytogenetic analysis was performed. RESULTS: The incidence of cytogenetic abnormalities was 51 (51.5%) in 99 patients, and 27 (39.7%) in acute lymphoblastic leukemia (ALL) patients and 24 (77.4%) in acute myelogenous leukemia (AML) patients. The most frequent cytogenetic abnormality was hyperdiploidy and t(8:21) in the ALL and AML patients, respectively. The overall survival rate (OS)/disease free survival rate (DFS) of the ALL patients was 74.0%/73.9%. The OS/DFS of the standard risk group (88.8%/85.2%) was significantly higher than that of the high-risk group (49.4%/39.3%) in the ALL patients (P=0.0005/P<0.0001). There was no significant difference in the survival rates according to the type of cytogenetic abnormalities among the ALL patients for the standard/high risk groups, based on the CCG prognostic factors. The OS/DFS of the AML patients were 43.4% and 41.7%, respectively, without significant differences of the survival rates according to the type of chromosomal abnormalities. CONCLUSION: There were significant differences of OS/DFS based on the risk groups in ALL patients when evaluated with the CCG prognostic factors (standard/high) and chromosomal abnormalities (good/ poor), respectively. However, there was no significant correlation between type of cytogenetic abnormalities and clinical outcomes based on the CCG prognostic factors in children with ALL as well as with AML.

Prognostic Significance of Biomarkers of Diffuse Large B-cell Lymphomas / 대한혈액학회지

BACKGROUND: Biomarkers of lymphomas identified by immunostaining of lymphoma tissues were recently found to have a prognostic value for diffuse large B-cell lymphoma (DLBL). Thus, it seems likely that the prognostic prediction of lymphomas might be improved by incorporating biological markers into well known prognostic systems. METHODS: To determine the clinical significance of the biological markers expressed in DLBL, 26 patients, with de novo DLBL, were retrospectively studied at the Chungnam National University Hospital. Archival specimens from the patients were stained with antibodies for the bcl-2, bcl-6, Ki-67, CD 10, IRF-4, Granzyme-B, MHC-II and p16 antigens. Two immunophenotypic patterns of DLBL were identified by the pattern of differentiation; the germinal center (GC, CD10+/-/Bcl-6+/IRF-4-)-like subgroup and the post germinal center (pGC, CD10+/-/bcl-6+/-/IRF4+)-like subgroup. RESULTS: The median age of the subjects was 56 years, ranging form 37 to 69. After a median follow up duration of 48 months, the median survival time was 44 month, ranging from 1~100 months. The five-year overall survival rate Using the Kaplan-Meier method was 32%. The only biomarker affecting the survival was bcl-2 (P=0.009). The survival of the GC-like subgroup was superior to that of the pGC-like subgroup, but without statistical significance (P=0.064). Among 18 patients with IPI scores 0~2, those expressing bcl-2 (P=0.002) and the pGC-like subgroup had a worse prognosis compared to the GC-like subgroup (P=0.049). CONCLUSION: The prognostic assessment of DLBL patients might be improved by the addition of immunohistochemical profiles, especially for bcl-2, to the traditional IPI system.