Gene activation guided by nascent RNA-bound transcription factors.

Technologies for gene activation are valuable tools for the study of gene functions and have a wide range of potential applications in bioengineering and medicine. In contrast to existing methods based on recruiting transcriptional modulators via DNA-binding proteins, we developed a strategy termed Narta (nascent RNA-guided transcriptional activation) to achieve gene activation by recruiting artificial transcription factors (aTFs) to transcription sites through nascent RNAs of the target gene. Using Narta, we demonstrate robust activation of a broad range of exogenous and endogenous genes in various cell types, including zebrafish embryos, mouse and human cells. Importantly, the activation is reversible, tunable and specific. Moreover, Narta provides better activation potency of some expressed genes than CRISPRa and, when used in combination with CRISPRa, has an enhancing effect on gene activation. Quantitative imaging illustrated that nascent RNA-directed aTFs could induce the high-density assembly of coactivators at transcription sites, which may explain the larger transcriptional burst size induced by Narta. Overall, our work expands the gene activation toolbox for biomedical research.

Clinical efficacy of chemotherapy in colorectal cancer patients over 80 years old.

PURPOSE: Colorectal cancer (CRC) is a common and aggressive gastrointestinal cancer, and the prognostic impact associated with chemotherapy in super elderly (over 80 years old) patients remains poorly defined. We aimed to define the effect of chemotherapy on the prognosis of patients with CRC over 80 years old. PATIENTS AND METHODS: A retrospective study including CRC patients over 80 years old was conducted. The patients were screened from the Surveillance Epidemiology and End Results (SEER) database from 2010 to 2015. Overall survival (OS) and cancer-specific survival (CSS) were applied as the primary and secondary outcome. Cox proportional hazards regression models were used to evaluate factors associated with OS and CSS. Survival curves of OS and CSS were estimated by Kaplan-Meier method and compared by log-rank test. RESULTS: In total, 14,748 CRC patients over 80 years old were included in this study. The median patient age was 85 (IQR: 82-87). All patients were divided into surgical group and non-surgical group. The OS and CSS of the surgical group were significantly better than those of the non-surgical group (P < 0.001). Chemotherapy can improve OS and CSS for patients with stage III and IV (P < 0.001) in surgical group. For the super elderly patients with CRC, chemotherapy significantly improved OS and CSS in all TNM stages in non-surgical group. CONCLUSION: For super elderly patients with colorectal cancer, tumor treatment should not be abandoned because of their age. It is necessary to carry out clinical trials in super elderly patients.

Circ_SEC61A1 contributes to the progression of multiple myeloma cells via regulating miR-660-5p/CDK6 axis.

BACKGROUND: Circular RNAs (circRNAs) have been reported to play critical roles in the malignant progression of diverse human cancers, including multiple myeloma (MM). This study aimed to explore the functional role and underlying mechanism of circ_SEC61A1 in MM progression. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed to detect the expression levels of circ_SEC61A1, microRNA (miR)-660-5p and cyclin-dependent kinase 6 (CDK6) mRNA. The localization of circ_SEC61A1 in MM cells was tested by the subcellular fractionation location assay. Actinomycin D assay was conducted to determine the characteristics of circ_SEC61A1. Cell proliferation was evaluated by colony formation assay, 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Western blot assay was exploited to examine the expression of proteins. Cell migration and invasion were tested via transwell assay, and cell apoptosis was measured by flow cytometry analysis. Dual-luciferase reporter assay was utilized to confirm the interaction between miR-660-5p and circ_SEC61A1 or CDK6. RESULTS: Circ_SEC61A1 level was increased in MM tissues and cells. Circ_SEC61A1 was a stable circRNA and mainly located in cytoplasm. Circ_SEC61A1 silence restrained the proliferation, metastasis and expedited the apoptosis in MM cells. CDK6 was identified as the target of miR-660-5p, and circ_SEC61A1 sponged miR-660-5p to positively regulate CDK6 expression. The inhibitory impacts of circ_SEC61A1 knockdown on the progression of MM cells were mitigated by miR-660-5p inhibition. MiR-660-5p overexpression blocked the malignant phenotypes of MM cells by targeting CDK6. CONCLUSION: Our study manifested that circ_SEC61A1 could accelerate MM progression at least partially through modulating miR-660-5p/CDK6 axis.

Phase separation of RNA-binding protein promotes polymerase binding and transcription.

An RNA-involved phase-separation model has been proposed for transcription control. However, the molecular links that connect RNA to the transcription machinery remain missing. Here we find that RNA-binding proteins (RBPs) constitute half of the chromatin proteome in embryonic stem cells (ESCs), some being colocalized with RNA polymerase (Pol) II at promoters and enhancers. Biochemical analyses of representative RBPs show that the paraspeckle protein PSPC1 inhibits the RNA-induced premature release of Pol II, and makes use of RNA as multivalent molecules to enhance the formation of transcription condensates and subsequent phosphorylation and release of Pol II. This synergistic interplay enhances polymerase engagement and activity via the RNA-binding and phase-separation activities of PSPC1. In ESCs, auxin-induced acute degradation of PSPC1 leads to genome-wide defects in Pol II binding and nascent transcription. We propose that promoter-associated RNAs and their binding proteins synergize the phase separation of polymerase condensates to promote active transcription.

Thermal Analysis on Active Heat Dissipation Design with Embedded Flow Channels for Flexible Electronic Devices.

Heat generation is a major issue in all electronics, as heat reduces product life, reliability, and performance, especially in flexible electronics with low thermal-conductivity polymeric substrates. In this sense, the active heat dissipation design with flow channels holds great promise. Here, a theoretical model, validated by finite element analysis and experiments, based on the method of the separation of variables, is developed to study the thermal behavior of the active heat dissipation design with an embedded flow channel. The influences of temperature and flow velocity of the fluid on heat dissipation performance were systematically investigated. The influence of channel spacing on heat dissipation performance was also studied by finite element analysis. The study shows that performance can be improved by decreasing the fluid temperature or increasing the flow velocity and channel density. These results can help guide the design of active heat dissipation with embedded flow channels to reduce adverse effects due to excessive heating, thus enhancing the performance and longevity of electronic products.

Localization of RNAs in the nucleus: cis- and trans- regulation.

The subcellular localization of RNAs correlates with their function and how they are regulated. Most protein-coding mRNAs are exported into the cytoplasm for protein synthesis, while some mRNA species, long noncoding RNAs, and some regulatory element-associated unstable transcripts tend to be retained in the nucleus, where they function as a regulatory unit and/or are regulated by nuclear surveillance pathways. While the mechanisms regulating mRNA export and localization have been well summarized, the mechanisms governing nuclear retention of RNAs, especially of noncoding RNAs, are seldomly reviewed. In this review, we summarize recent advances in the mechanistic study of RNA nuclear retention, especially for noncoding RNAs, from the angle of cis-acting elements embedded in RNA transcripts and their interaction with trans-acting factors. We also try to illustrate the general principles of RNA nuclear retention and we discuss potential areas for future investigation.

Long non-coding RNA ANRIL and its target microRNAs (microRNA-34a, microRNA-125a and microRNA-186) relate to risk stratification and prognosis in multiple myeloma.

OBJECTIVE: This study aimed to investigate the correlations of long non-coding RNA ANRIL (lncRNA ANRIL), microRNA (miR)-34a, miR-125a and miR-186 with disease risk, clinical features and prognosis of multiple myeloma (MM). METHOD: Totally, 87 MM patients and 30 controls were recruited. LncRNA ANRIL and its target miRNAs (miR-34a, miR-125a and miR-186) in bone marrow derived plasma cells were detected by RT-qPCR. Treatment response was assessed and survivals were calculated in MM patients. RESULTS: LncRNA ANRIL expression was increased, while miR-34a, miR-125a and miR-186 expressions were reduced in MM patients compared with controls. Meanwhile, lncRNA ANRIL negatively correlated with miR-34a and miR-125a but not miR-186 in MM patients, while did not correlate with miR-34a, miR-125a or miR-186 in controls. In MM patients, lncRNA ANRIL high expression associated with higher beta-2-microglobulin (ß2-MG) and more advanced international staging system (ISS) stage; miR-125a high expression associated with lower ß2-MG, less advanced ISS stage and less t (14; 16) abnormality; miR186 high expression associated with increased albumin; while miR-34a did not associate with any clinical features. Furthermore, lncRNA ANRIL high expression associated with decreased complete response (CR), while miR-34a high and miR-125a high expression associated with increased CR and objective response rate. Additionally, lncRNA ANRIL high expression associated with shorter progression-free survival (PFS), while miR-34a high expression associated with prolonged overall survival (OS), and miR-125a high expression associated with longer PFS and OS. CONCLUSION: LncRNA ANRIL and its target miRNAs might serve as biomarkers for assisting with personalized treatment and prognosis improvement of MM.

Homotypic clustering of L1 and B1/Alu repeats compartmentalizes the 3D genome.

Organization of the genome into euchromatin and heterochromatin appears to be evolutionarily conserved and relatively stable during lineage differentiation. In an effort to unravel the basic principle underlying genome folding, here we focus on the genome itself and report a fundamental role for L1 (LINE1 or LINE-1) and B1/Alu retrotransposons, the most abundant subclasses of repetitive sequences, in chromatin compartmentalization. We find that homotypic clustering of L1 and B1/Alu demarcates the genome into grossly exclusive domains, and characterizes and predicts Hi-C compartments. Spatial segregation of L1-rich sequences in the nuclear and nucleolar peripheries and B1/Alu-rich sequences in the nuclear interior is conserved in mouse and human cells and occurs dynamically during the cell cycle. In addition, de novo establishment of L1 and B1 nuclear segregation is coincident with the formation of higher-order chromatin structures during early embryogenesis and appears to be critically regulated by L1 and B1 transcripts. Importantly, depletion of L1 transcripts in embryonic stem cells drastically weakens homotypic repeat contacts and compartmental strength, and disrupts the nuclear segregation of L1- or B1-rich chromosomal sequences at genome-wide and individual sites. Mechanistically, nuclear co-localization and liquid droplet formation of L1 repeat DNA and RNA with heterochromatin protein HP1α suggest a phase-separation mechanism by which L1 promotes heterochromatin compartmentalization. Taken together, we propose a genetically encoded model in which L1 and B1/Alu repeats blueprint chromatin macrostructure. Our model explains the robustness of genome folding into a common conserved core, on which dynamic gene regulation is overlaid across cells.

Expression and correlation of IL-2, IL-10 and TNF-α in patients with multiple myeloma-infected herpes zoster treated by bortezomib-containing regimen.

BACKGROUND: Multiple myeloma (MM) is a proliferative disease with complex pathogenesis. Most patients will have low body resistance and high inflammatory mediators. Bortezomib is an anti-tumor drug. There are few reports on the clinical efficacy and adverse reactions of bortezomib intervention. This research aimed to explore the effect of bortezomib on inflammation and immune lymphocytes of patients with MM-infected herpes zoster. OBJECTIVE: The aim of this study is to explore the effect of bortezomib on inflammation and immune lymphocytes, i.e. the expression and correlation of interleukin (IL)-2, IL-10 and tumor necrosis factor-α (TNF-α) in patients with MM-infected herpes zoster (HZ) receiving bortezomib-containing regimen. METHODS: From October 2017 to March 2020, 83 MM patients receiving bortezomib-containing regimen were analyzed retrospectively, patients were divided into infection group (28 cases, IG) and non-infection group (55 cases, NG) based on whether or not they are complicated with HZ Pre- and post-treatment. IL-2, IL-10, TNF-α and immune lymphocytes (CD3+, CD4+, CD8+) were tested by AimPlex multifactor flow detection technique, and the Eastern Cooperative Oncology Group (ECOG) performance status scores were compared before therapy. The independent risk factors of patients receiving bortezomib-containing regimen were analyzed via multivariate logistic regression. RESULTS: After therapy, serum IL-2 and TNF-α declined significantly in NG while changed insignificantly in IG. Compared with NG, serum CD3+ and CD4+ in IG increased after treatment, while CD8+ decreased significantly. Before therapy, ECOG score in IG was higher than that in NG. Correlation analysis showed that IL-2 and TNF-α were negatively correlated with CD3+ and CD4+, and positively correlated with CD8+ and ECOG score. IL-10 was the opposite. Multivariate logistic regression analysis identified the independence of declined CD3+, CD4+, CD8+ and IL-10, increased IL-2, TNF-α and ECOG score before treatment as risk factors for HZ. CONCLUSION: MM patients have a high incidence of HZ. Before treatment, lymphocytopenia, increased IL-2, TNF-α and decreased IL-10 are important risk factors for HZ.

LncRNA Platr22 promotes super-enhancer activity and stem cell pluripotency.

Super-enhancers (SEs) comprise large clusters of enhancers, which are co-occupied by multiple lineage-specific and master transcription factors, and play pivotal roles in regulating gene expression and cell fate determination. However, it is still largely unknown whether and how SEs are regulated by the noncoding portion of the genome. Here, through genome-wide analysis, we found that long noncoding RNA (lncRNA) genes preferentially lie next to SEs. In mouse embryonic stem cells (mESCs), depletion of SE-associated lncRNA transcripts dysregulated the activity of their nearby SEs. Specifically, we revealed a critical regulatory role of the lncRNA gene Platr22 in modulating the activity of a nearby SE and the expression of the nearby pluripotency regulator ZFP281. Through these regulatory events, Platr22 contributes to pluripotency maintenance and proper differentiation of mESCs. Mechanistically, Platr22 transcripts coat chromatin near the SE region and interact with DDX5 and hnRNP-L. DDX5 further recruits p300 and other factors related to active transcription. We propose that these factors assemble into a transcription hub, thus promoting an open and active epigenetic chromatin state. Our study highlights an unanticipated role for a class of lncRNAs in epigenetically controlling the activity and vulnerability to perturbation of nearby SEs for cell fate determination.

Long non­coding RNA ANRIL is a potential indicator of disease progression and poor prognosis in acute myeloid leukemia.

The present study explored the association of long non­coding RNA (lncRNA) antisense non­coding RNA in the INK4 locus (ANRIL) with the development of acute myeloid leukemia (AML) clinical features and prognosis of patients with AML. Bone marrow mononuclear cells (BMMCs) were obtained from 178 patients with de novo AML prior to initial therapy and from 30 healthy donors. The expression of lncRNA ANRIL in BMMCs was detected by reverse transcription­quantitative PCR. Complete remission (CR) was assessed after induction therapy. Event­free survival (EFS) and overall survival (OS) were evaluated during the follow­up. The levels of lncRNA ANRIL were increased in patients with AML compared with those in healthy donors and were capable of distinguishing patients with AML from healthy donors (area under the curve, 0.886; 95% CI, 0.820­0.952). Furthermore, lncRNA ANRIL was associated with an increased occurrence internal tandem duplications in the FMS­like tyrosine kinase 3, decreased occurrence inv(16) or t(16;6), intermediate­risk and poor­risk stratification while no association of lncRNA ANRIL was identified with French­American­British classification, cytogenetics, isolated biallelic CCAAT/enhancer­binding protein α mutation and nucleophosmin 1 mutation in patients with AML. Furthermore, lncRNA ANRIL was significantly associated with a lower CR rate. In addition, EFS and OS were shorter in patients with high expression of lncRNA ANRIL compared with those in patients with low expression of lncRNA ANRIL. Multivariate Cox regression analyses revealed that high expression of lncRNA ANRIL, poor­risk stratification and white blood cells (>10.0x109 cells/l) were independent prognostic factors for shorter EFS, while high expression of lncRNA ANRIL and poorer risk stratification were independent prognostic factors for shorter OS. The present results suggested that lncRNA ANRIL has clinical relevance as a biomarker for assisting diagnosis treatment decisions and prognosis prediction and the identification of potential drug target for AML.

Clinical value of bedside abdominal sonography performed by certified sonographer in emergency evaluation of blunt abdominal trauma.

PURPOSE: To investigate the accuracy and efficiency of bedside ultrasonography application performed by certified sonographer in emergency patients with blunt abdominal trauma. METHODS: The study was carried out from 2017 to 2019. Findings in operations or on computed tomography (CT) were used as references to evaluate the accuracy of bedside abdominal ultrasonography. The time needed for bedside abdominal ultrasonography or CT examination was collected separately to evaluate the efficiency of bedside abdominal ultrasonography application. RESULTS: Bedside abdominal ultrasonography was performed in 106 patients with blunt abdominal trauma, of which 71 critical patients received surgery. The overall diagnostic accordance rate was 88.68%. The diagnostic accordance rate for liver injury, spleen injury, kidney injury, gut perforation, retroperitoneal hematoma and multiple abdominal organ injury were 100%, 94.73%, 94.12%, 20.00%, 100% and 81.48%, respectively. Among the 71 critical patients, the diagnostic accordance rate was 94.37%, in which the diagnostic accordance rate for liver injury, spleen injury, kidney injury, gut perforation and multiple abdominal organ injury were 100%, 100%, 100%, 20.00% and 100%. The mean time for imaging examination of bedside abdominal ultrasonography was longer than that for CT scan (4.45 ± 1.63 vs. 2.38 ± 1.19) min; however, the mean waiting time before examination (7.37 ± 2.01 vs. 16.42 ± 6.37) min, the time to make a diagnostic report (6.42 ± 3.35 vs. 36.26 ± 13.33) min, and the overall time (17.24 ± 2.33 vs. 55.06 ± 6.96) min were shorter for bedside abdominal ultrasonography than for CT scan. CONCLUSION: Bedside ultrasonography application provides both efficiency and reliability for the assessment of blunt abdominal trauma. Especially for patients with free peritoneal effusion and critical patients, bedside ultrasonography has been proved obvious advantageous. However, for negative bedside ultrasonography patients with blunt abdominal trauma, we recommend further abdominal CT scan or serial ultrasonography scans subsequently.

Targeting lactate dehydrogenase A (LDHA) exerts antileukemic effects on T-cell acute lymphoblastic leukemia.

BACKGROUND: T-cell acute lymphoblastic leukemia (T-ALL) is an uncommon and aggressive subtype of acute lymphoblastic leukemia (ALL). In the serum of T-ALL patients, the activity of lactate dehydrogenase A (LDHA) is increased. We proposed that targeting LDHA may be a potential strategy to improve T-ALL outcomes. The current study was conducted to investigate the antileukemic effect of LDHA gene-targeting treatment on T-ALL and the underlying molecular mechanism. METHODS: Primary T-ALL cell lines Jurkat and DU528 were treated with the LDH inhibitor oxamate. MTT, colony formation, apoptosis, and cell cycle assays were performed to investigate the effects of oxamate on T-ALL cells. Quantitative real-time PCR (qPCR) and Western blotting analyses were applied to determine the related signaling pathways. A mitochondrial reactive oxygen species (ROS) assay was performed to evaluate ROS production after T-ALL cells were treated with oxamate. A T-ALL transgenic zebrafish model with LDHA gene knockdown was established using CRISPR/Cas9 gene-editing technology, and then TUNEL, Western blotting, and T-ALL tumor progression analyses were conducted to investigate the effects of LDHA gene knockdown on T-ALL transgenic zebrafish. RESULTS: Oxamate significantly inhibited proliferation and induced apoptosis of Jurkat and DU528 cells. It also arrested Jurkat and DU528 cells in G0/G1 phase and stimulated ROS production (all P < 0.001). Blocking LDHA significantly decreased the gene and protein expression of c-Myc, as well as the levels of phosphorylated serine/threonine kinase (AKT) and glycogen synthase kinase 3 beta (GSK-3ß) in the phosphatidylinositol 3'-kinase (PI3K) signaling pathway. LDHA gene knockdown delayed disease progression and down-regulated c-Myc mRNA and protein expression in T-ALL transgenic zebrafish. CONCLUSION: Targeting LDHA exerted an antileukemic effect on T-ALL, representing a potential strategy for T-ALL treatment.

Identification of cis-Elements for RNA Subcellular Localization Through REL-seq.

The subcellular localization of RNAs is regulated by cis-regulatory elements together with interacting trans factors. Here we describe a high-throughput sequencing-based method named REL-seq (RNA elements for subcellular localization by sequencing) to identify the cis-elements that contribute to RNA subcellular localization. By coupling REL-seq with random mutagenesis (mutREL-seq), we can further narrow down the cis-elements to key motifs at single-nucleotide resolution.

U1 snRNP regulates chromatin retention of noncoding RNAs.

Long noncoding RNAs (lncRNAs) and promoter- or enhancer-associated unstable transcripts locate preferentially to chromatin, where some regulate chromatin structure, transcription and RNA processing1-13. Although several RNA sequences responsible for nuclear localization have been identified-such as repeats in the lncRNA Xist and Alu-like elements in long RNAs14-16-how lncRNAs as a class are enriched at chromatin remains unknown. Here we describe a random, mutagenesis-coupled, high-throughput method that we name 'RNA elements for subcellular localization by sequencing' (mutREL-seq). Using this method, we discovered an RNA motif that recognizes the U1 small nuclear ribonucleoprotein (snRNP) and is essential for the localization of reporter RNAs to chromatin. Across the genome, chromatin-bound lncRNAs are enriched with 5' splice sites and depleted of 3' splice sites, and exhibit high levels of U1 snRNA binding compared with cytoplasm-localized messenger RNAs. Acute depletion of U1 snRNA or of the U1 snRNP protein component SNRNP70 markedly reduces the chromatin association of hundreds of lncRNAs and unstable transcripts, without altering the overall transcription rate in cells. In addition, rapid degradation of SNRNP70 reduces the localization of both nascent and polyadenylated lncRNA transcripts to chromatin, and disrupts the nuclear and genome-wide localization of the lncRNA Malat1. Moreover, U1 snRNP interacts with transcriptionally engaged RNA polymerase II. These results show that U1 snRNP acts widely to tether and mobilize lncRNAs to chromatin in a transcription-dependent manner. Our findings have uncovered a previously unknown role of U1 snRNP beyond the processing of precursor mRNA, and provide molecular insight into how lncRNAs are recruited to regulatory sites to carry out chromatin-associated functions.

Genomic Repeats Categorize Genes with Distinct Functions for Orchestrated Regulation.

Repetitive elements are abundantly distributed in mammalian genomes. Here, we reveal a striking association between repeat subtypes and gene function. SINE, L1, and low-complexity repeats demarcate distinct functional categories of genes and may dictate the time and level of gene expression by providing binding sites for different regulatory proteins. Importantly, imaging and sequencing analysis show that L1 repeats sequester a large set of genes with specialized functions in nucleolus- and lamina-associated inactive domains that are depleted of SINE repeats. In addition, L1 transcripts bind extensively to its DNA in embryonic stem cells (ESCs). Depletion of L1 RNA in ESCs leads to relocation of L1-enriched chromosomal segments from inactive domains to the nuclear interior and de-repression of L1-associated genes. These results demonstrate a role of L1 DNA and RNA in gene silencing and suggest a general theme of genomic repeats in orchestrating the function, regulation, and expression of their host genes.

A widely adaptable analytical method for thermal analysis of flexible electronics with complex heat source structures.

Flexible electronics, as a relatively new category of device, exhibit prodigious potential in many applications, especially in bio-integrated fields. It is critical to understand that thermal management of certain kinds of exothermic flexible electronics is a crucial issue, whether to avoid or to take advantage of the excessive temperature. A widely adaptable analytical method, validated by finite-element analysis and experiments, is conducted to investigate the thermal properties of exothermic flexible electronics with a heat source in complex shape or complex array layout. The main theoretical strategy to obtain the thermal field is through an integral along the complex curve source region. The results predicted by the analytical model enable accurate control of temperature and heat flow in the flexible electronics, which may help in the design and fabrication of flexible electronic devices in the future.

RNF8 is responsible for ATRA resistance in variant acute promyelocytic leukemia with GTF2I/RARA fusion, and inhibition of the ubiquitin-proteasome pathway contributes to the reversion of ATRA resistance.

BACKGROUND: GTF2I-RARA is a newly identified RARA fusion gene in variant acute promyelocytic leukemia (APL) patients with t(7;17)(q11;q21). Clinical manifestation in the patient showed that it is a sort of ATRA-insensitive oncogene and is different from the classic PML-RARA in terms of therapeutic reaction. METHODS: To reveal the functional characteristics and regulating mechanism of the GTF2I-RARA fusion gene, we established a GTF2I-RARA-transfected HL60 cell model and examined its sensitivity to ATRA by western blot, MTT assay, flow cytometry, and Wright-Giemsa staining. Coimmunoprecipitation and confocal microscopy were used to examine the binding of GTF2I-RARA and transcriptional corepressors. We also performed ChIP-seq to search for potential target genes. Immunoprecipitation, ubiquitination assay, western blot, luciferase assay, and real-time PCR were used to analyze the effects of RNF8 on RARA. Flow cytometry and Wright-Giemsa staining were used to study the effect of MG132 and ATRA on the GTF2I-RARA-transfected HL60 cell model. RESULT: We confirmed resistance of GTF2I-RARA to ATRA. Compared with PML-RARA, GTF2I-RARA has a higher affinity to HDAC3 under ATRA treatment. Using the ChIP-sequencing approach, we identified 221 GTF2I-RARA binding sites in model cells and found that the RING finger protein 8 (RNF8) is a target gene of GTF2I-RARA. RNF8 participates in disease progression and therapy resistance in APL with the GTF2I-RARA transcript. Elevated RNF8 expression promotes the interaction between RARA and RNF8 and induces RARA Lys-48 linkage ubiquitylation and degradation, resulting in attenuated transcriptional activation of RARA. CONCLUSION: Our results suggest that RNF8 is a key GTF2I-RARA downstream event. Using the combination of MG132 and ATRA to treat GTF2I-RARA-HL60 cells, a synergistic effect leading to GTF2I-RARA-HL60 cell differentiation was confirmed. Taken together, the targeting of RNF8 may be an alternative choice for treatment in variant APL with GTF2I-RARA fusion.