Dual-luciferase Reporter Assay Research Articles

Expression of Long Non-Coding RNA H19 in the Endometrium of Mice During Peri-Implantation and Its Regulation on Embryo Implantation

In this study, we utilized magnetic nanobeads for the extraction of nucleic acids from tissues to investigate the expression levels and correlation between lncRNA H19, miR-612, and their target gene HOXA10 in peri-implantation endometrium of mice. Furthermore, we conducted overexpression or gene knockout experiments on lncRNA H19 to observe its impact on the expression of miR-612 and HOXA10. The targeted binding relationship between lncRNA H19, miR-612, and HOXA10 was detected by dual luciferase reporter assay. The regulatory relationship between lncRNA H19, miR-612, and HOXA10 was verified through silencing or overexpression of these genes. Intrauterine transfection was used to modulate the expression of lncRNA H19 in endometria during pregnancy, followed by the detection of the expression levels of miR-612 and HOXA10 as well as ITGB3 and IGFBP-1 proteins. Compared with non-pregnant mice, we observed a significant upregulation of both lncRNA H19 and HOXA10 in the endometria of pregnant mice, while miR-612 was found to be downregulated (P < 0.05). Further analysis revealed that the expression levels of lncRNA H19 and HOXA10 increased progressively with gestational days, peaking on Day 4 (P < 0.05). Moreover, Through database analysis, we identified binding sites for lncRNA H19-miR-612 as well as HOXA10-miR-612 interactions. The dual-luciferase reporter assay further supported our conjecture that lncRNA H19 could specifically bind the miR-612, which in turn targets HOXA10 to regulate its expression (P < 0.05). In conclusion, regulations of lncRNA H19 and HOXA10 expression contribute to enhancing endometrial receptivity and facilitating decidualization of endometrial stromal cells, ultimately promoting successful embryo implantation.

Alternative splicing-related long noncoding RNA ANRIL facilitates hepatocellular carcinoma by targeting the miR-199a-5p/SRSF1 axis and impacting Anillin.

Hepatocellular carcinoma (HCC) is characterized by aberrant alternative splicing (AS), which plays an important part in the pathological process of this disease. However, available reports about genes and mechanisms involved in AS process are limited. Our previous research has identified ANRIL as a long noncoding RNA related to the AS process of HCC. Here, we investigated the exact effect and the mechanism of ANRIL on HCC progress.The ANRIL expression profile was validated using the real-time quantitative polymerase chain reactionassay. The western blot analysis and IHC assay were conducted on candidate targets, including SRSF1 and Anillin. The clinicopathological features of 97 patients were collected and analyzed. Loss-of and gain-of-function experiments were conducted. The dual-luciferase reporter assay was applied to verify the interaction between ANRIL, miR-199a-5p, and SRSF1.Anomalous upregulation of ANRIL in HCC was observed, correlating with worse clinicopathological features of HCC. HCC cell proliferation, mobility, tumorigenesis, and metastasis were impaired by depleting ANRIL. We found that ANRIL acts as a sponger of miRNA-199a-5p, resulting in an elevated level of its target protein SRSF1. The phenotypes induced by ANRIL/miR-199a-5p/SRSF1 alteration are associated with Anillin, a validated HCC promoter.ANRIL is an AS-related lncRNA promoting HCC progress by modulating the miR-199a-5p/SRSF1 axis. The downstream effector of this axis in the development of HCC is Anillin.

Knockdown of miR-361-5p promotes the induced activation of SHF-stem cells through FOXM1 mediated Wnt/β-catenin pathway in cashmere goats

The inducing activation event of secondary hair follicle (SHF)-stem cells is considered a key biological process in the SHF regeneration, and the morphogenesis of cashmere fiber in cashmere goats. The miR-361-5p was essentially implicated in the induced activation of SHF-stem cells of cashmere goats, but its functional mechanisms are unclear. Here, we confirmed miR-361-5p was significantly downregulated in anagen SHF bugle of cashmere goats compared with that at telogen, and miR-361-5p expression was significantly lower in SHF-stem cells after activation than its counterpart before activation. Further, we found that miR-361-5p could negatively regulate the induced activation event of SHF-stem cells in cashmere goats. Mechanistically, through dual-luciferase reporter assays, miR-361-5p specifically bound with FOXM1 mRNA in SHF-stem cells of cashmere goats and negatively regulated the expression of FOXM1 gene. Also, through overexpression/knockdown analysis of FOXM1 gene, our results indicated that FOXM1 upregulated the expression of Wnt/β-catenin pathway related genes in SHF-stem cells. Moreover, based on TOP/FOP-flash Wnt report assays, the knockdown of miR-361-5p promotes the Wnt/β-catenin pathway activation through upregulating the FOXM1 expression in SHF-stem cells. Finally, we demonstrated that miR-361-5p negatively regulated the induced activation of SHF-stem cells through FOXM1 mediated Wnt/β-catenin pathway in cashmere goats.

MiR-652-3p Suppressed the Protective Effects of Isoflurane Against Myocardial Injury in Hypoxia/Reoxygenation by Targeting ISL1.

This research is concentrated on investigating the role and mechanism of miR-652-3p in the protective effects of isoflurane (ISO) against myocardial ischemia-reperfusion (I/R) injury. H9c2 cells underwent pretreatment with varying concentrations of ISO, and subsequently, a hypoxia/reoxygenation (H/R) model was constructed. The levels of miR-652-3p, ISL LIM homeobox 1 (ISL1), and inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) were evaluated through reverse transcription polymerase chain reaction (RT-qPCR). Enzyme-linked immunosorbent assay was employed to investigate concentrations of myocardial injury markers, such as creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI). Cell counting kit-8 was used to evaluate cell viability, while flow cytometry was utilized to measure apoptosis. Additionally, a dual luciferase reporter assay was conducted to validate the targeting relationship between ISL1 and miR-652-3p. Herein, we confirmed thatthe level of miR-652-3p wasgradually increased with prolonged hypoxia; nevertheless, this increase was suppressed by ISO pretreatment (P < 0.05). Additionally, ISO pretreatment prevented the decrease in cell viability, increase in apoptosis, and overproduction of IL-6, TNF-α, CK-MB, and cTnI induced by H/R (P < 0.05). However, the inhibitory effects of ISO were counteracted by the increased levels of miR-652-3p (P < 0.05). ISL1 is a potential target of miR-652-3p. H/R induction suppressed ISL1 levels compared to the control, but ISO treatment increased its expression (P < 0.05). Overexpression of ISL1 inhibited the elimination of the protective effect of ISO on myocardial damage induced by the elevation of miR-652-3p (P < 0.05). The findings of this research confirm that miR-652-3p attenuated the protective effect of ISO on cardiomyocytes in myocardial ischemia by targeting ISL1.

MiR-625-5p Modulates the Metastasis and Proliferation of Gastric Cancer Cells by Governing the M6A Demethylase ALKBH5

Background: Gastric cancer is a prevalent malignant tumor globally, with metastasis and recurrence serving as primary contributors to the diminished survival rates observed in patients with advanced stages of the disease. Despite numerous investigations into the pathogenesis of gastric cancer, the significance of m6A demethylase ALKBH5 in the initiation and progression of the disease has been established, yet its precise mechanism remains incompletely understood. Methods: Immunohistochemistry was utilized for the evaluation of ALKBH5 expression in gastric cancer tissues, while RT-qPCR and WB were employed for the analysis of ALKBH5 expression in cells. Additionally, the levels of m6A methylation in RNA were assessed using the EpiQuikTM m6A methylation quantitative assay kit. The proliferative and metastatic potentials of gastric cancer cells were assessed using the CCK-8 and Transwell cell assays, respectively. The binding site between miR-625-5p and ALKBH5 was predicted through the TargetScan website, and the interaction was confirmed using a dual luciferase reporter assay. Results: The research findings indicated a significant increase in m6A methylation levels in gastric cancer cells, concomitant with a substantial decrease in ALKBH5 expression. ALKBH5 can inhibit the proliferation and metastasis of gastric cancer cells. Additionally, the interaction between miR- 625-5p and the 3'-UTR of ALKBH5 mRNA, resulting in the suppression of ALKBH5 expression, was validated. Conclusions: Through its specific interaction with ALKBH5, miR-625-5p modulates the proliferation and metastatic potential of gastric cancer cells, bridging a previously unexplored link in the pathogenesis of gastric cancer between miR-625-5p and ALKBH5. This finding suggests that miR- 625-5p could be a potential target for treating gastric cancer, with the goal of enhancing patient outcomes and quality of life.

A novel protein FNDC3B-267aa encoded by circ0003692 inhibits gastric cancer metastasis via promoting proteasomal degradation of c-Myc.

Gastric cancer (GC) ranks fifth in global cancer incidence and third in mortality rate among all cancer types. Circular RNAs (circRNAs) have been extensively demonstrated to regulate multiple malignant biological behaviors in GC. Emerging evidence suggests that several circRNAs derived from FNDC3B play pivotal roles in cancer. However, the role of circFNDC3B in GC remains elusive. We initially screened circFNDC3B with translation potential via bioinformatics algorithm prediction. Subsequently, Sanger sequencing, qRT-PCR, RNase R, RNA-FISH and nuclear-cytoplasmic fractionation assays were explored to assess the identification and localization of circ0003692, a circRNA derived from FNDC3B. qRT-PCR and ISH were performed to quantify expression of circ0003692 in human GC tissues and adjacent normal tissues. The protein-encoding ability of circ0003692 was investigated through dual-luciferase reporter assay and LC/MS. The biological behavior of circ0003692 in GC was confirmed via in vivo and in vitro experiments. Additionally, Co-IP and rescue experiments were performed to elucidate the interaction between the encoded protein and c-Myc. We found that circ0003692 was significantly downregulated in GC tissues. Circ0003692 had the potential to encode a novel protein FNDC3B-267aa, which was downregulated in GC cells. We verified that FNDC3B-267aa, rather than circ0003692, inhibited GC migration in vitro and in vivo. Mechanistically, FNDC3B-267aa directly interacted with c-Myc and promoted proteasomal degradation of c-Myc, resulting in the downregulation of c-Myc-Snail/Slug axis. Our study revealed that the novel protein FNDC3B-267aa encoded by circ0003692 suppressed GC metastasis through binding to c-Myc and enhancing proteasome-mediated degradation of c-Myc. The study offers the potential applications of circ0003692 or FNDC3B-267aa as therapeutic targets for GC.

CircSLC25A16 facilitates the development of non-small-cell lung cancer through the miR-335-5p/CISD2 axis.

Non-small-cell lung cancer (NSCLC) is a common malignancy with high morbidity and mortality. Circular RNAs are widely involved in NSCLC progression. However, the mechanism of circSLC25A16 in NSCLC has not been reported. The expressions of circSLC25A16, microRNA-335-5p (miR-335-5p), and CDGSH iron-sulfur domain-containing protein 2 (CISD2) were monitored by quantitative real-time fluorescence polymerase chain reaction. Western blot was also carried out to measure the protein levels of CISD2, hexokinase 2 (HK2), and lactate dehydrogenase A (LDHA). For functional analysis, cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine, flow cytometry, transwell, and wound healing assays were utilized to examine cell proliferation, apoptosis, and migration. Glucose uptake and lactate production were detected using commercial kits. The relationship between miR-335-5p and circSLC25A16 or CISD2 was verified by dual-luciferase reporter and RNA immunoprecipitation assays. Furthermore, tumor xenograft was established to explore the function of circSLC25A16 in vivo. CircSLC25A16 and CISD2 were overexpressed in NSCLC, but miR-335-5p was downregulated. CircSLC25A16 acted as a miR-335-5p sponge, and silencing of circSLC25A16 arrested cell proliferation, migration, and glycolysis, and promoted apoptosis, but these impacts were resumed by miR-335-5p inhibition. CISD2 was a miR-335-5p target, and overexpression of CISD2 abolished the suppressive function of miR-335-5p mimic on the malignant behavior of NSCLC cells. CircSLC25A16 could adsorb miR-335-5p to mediate CISD2 expression. Additionally, silencing circSLC25A16 restrained the growth of NSCLC tumor xenograft in vivo. CircSLC25A16 facilitated NSCLC progression via the miR-335-5p/CISD2 axis, implying that circSLC25A16 may serve as a novel biomarker for NSCLC treatment.

FOXP3 targets KIF5A to increase lactate production and promote docetaxel resistance in lung adenocarcinoma.

A prominent cause of cancer-related fatalities with a poor prognosis is lung adenocarcinoma (LUAD). KIF5A, a crucial member of the kinesin superfamily, is linked to drug resistance in malignancies. This work aims to investigate the mechanism of KIF5A in docetaxel (DTX) resistance in LUAD cells. The results of bioinformatics analysis, qRT-PCR and western blot analysis show that KIF5A, which is involved in the glycolysis pathway, is highly expressed in LUAD and is positively correlated with glycolysis-related genes. We further verify that silencing of KIF5A inhibits DTX resistance, glycolysis, and lactate production in LUAD cells via cell counting kit-8 (CCK-8), flow cytometry, Seahorse XFe 96, lactate, and glucose assays. Mechanistically, KIF5A promotes DTX resistance in LUAD, and this effect is attenuated upon the addition of an LDHA inhibitor. Chromatin immunoprecipitation and dual-luciferase reporter assays reveal that FOXP3 transcriptionally activates KIF5A. Knockdown of FOXP3 reduces lactate production and enhances DTX sensitivity in LUAD, which is restored upon simultaneous overexpression of KIF5A. Our findings reveal that FOXP3 increases DTX resistance in LUAD cells by enhancing lactate production through the upregulation of KIF5A level. In conclusion, our study provides a novel treatment target for improving chemosensitivity in LUAD.

WTAP-mediated m6A modification of lncRNA Snhg1 improves myocardial ischemia-reperfusion injury via miR-361-5p/OPA1-dependent mitochondrial fusion

BackgroundMyocardial ischemia-reperfusion injury (MIRI) is caused by reperfusion after ischemic heart disease. LncRNA Snhg1 regulates the progression of various diseases. N6-methyladenosine (m6A) is the frequent RNA modification and plays a critical role in MIRI. However, it is unclear whether lncRNA Snhg1 regulates MIRI progression and whether the lncRNA Snhg1 was modified by m6A methylation.MethodsMouse cardiomyocytes HL-1 cells were utilized to construct the hypoxia/reoxygenation (H/R) injury model. HL-1 cell viability was evaluated utilizing CCK-8 method. Cell apoptosis, mitochondrial reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were quantitated utilizing flow cytometry. RNA immunoprecipitation and dual-luciferase reporter assays were applied to measure the m6A methylation and the interactions between lncRNA Snhg1 and targeted miRNA or target miRNAs and its target gene. The I/R mouse model was constructed with adenovirus expressing lncRNA Snhg1. HE and TUNEL staining were used to evaluate myocardial tissue damage and apoptosis.ResultsLncRNA Snhg1 was down-regulated after H/R injury, and overexpressed lncRNA Snhg1 suppressed H/R-stimulated cell apoptosis, mitochondrial ROS level and polarization. Besides, lncRNA Snhg1 could target miR-361-5p, and miR-361-5p targeted OPA1. Overexpressed lncRNA Snhg1 suppressed H/R-stimulated cell apoptosis, mitochondrial ROS level and polarization though the miR-361-5p/OPA1 axis. Furthermore, WTAP induced lncRNA Snhg1 m6A modification in H/R-stimulated HL-1 cells. Moreover, enforced lncRNA Snhg1 repressed I/R-stimulated myocardial tissue damage and apoptosis and regulated the miR-361-5p and OPA1 levels.ConclusionWTAP-mediated m6A modification of lncRNA Snhg1 regulated MIRI progression through modulating myocardial apoptosis, mitochondrial ROS production, and mitochondrial polarization via miR-361-5p/OPA1 axis, providing the evidence for lncRNA as the prospective target for alleviating MIRI progression.

Nur77-IRF1 axis inhibits esophageal squamous cell carcinoma growth and improves anti-PD-1 treatment efficacy

The nuclear receptor Nur77 plays paradoxical roles in numerous cancers. However, whether Nur77 inhibits esophageal squamous cell carcinoma (ESCC) growth and affects immunological responses against ESCC has not been determined. The functional role of Nur77 in ESCC was investigated in this study using human ESCC cell lines, quantitative real-time polymerase chain reaction (PCR), cell proliferation and colony formation assays, flow cytometry analysis, western blotting and animal models. The target gene controlled by Nur77 was verified using dual-luciferase reporter assays, chromatin immunoprecipitation analysis and functional rescue experiments. To examine the clinical importance of Nur77, 72 human primary ESCC tissues were subjected to immunohistochemistry. Taken together, these findings showed that, both in vitro and in vivo, Nur77 dramatically reduced ESCC cell growth and triggered apoptosis. Nur77 directly interacts with the interferon regulatory factor 1 (IRF1) promoter to inhibit its activity in ESCC. Pharmacological induction of Nur77 using cytosporone B (CsnB) inhibited ESCC cell proliferation and promoted apoptosis both in vitro and in vivo. Furthermore, CsnB increased CD8+ T-cell infiltration and cytotoxicity to inhibit the formation of ESCC tumors in an immunocompetent mouse model. In ESCC tissues, Nur77 expression was downregulated, and IRF1 expression was increased; moreover, their expression levels were negatively related. IRF1 and Nur77 were strongly correlated with overall survival. These findings suggested that Nur77 targets and regulates the IRF1/PD-L1 axis to serve as a tumor suppressor in ESCC.Graphical abstract of the regulatory mechanism of Nur77 overexpression downregulates IRF1 in the inhibition of ESCC progression and enhance anti-PD-1 therapy efficacy.

WTAP mediated m6A-modified circ_0056856 contributes to the proliferation, migration, and invasion of IL-22-stimulated human keratinocyte by miR-197-3p/CDK1 axis.

Psoriasis is a chronic inflammation-associated skin disorder, and interleukin-22 (IL-22) is involved in psoriasis pathogenesis by boosting the proliferation and migration of keratinocytes. Mounting evidence has shown that circRNAs might play an important role in several aspects of psoriasis. This study is designed to explore the role and mechanism of circ_0056856 in regulating the phenotypes of IL-22-induced keratinocytes (HaCaT cells). Circ_0056856, microRNA-197-3p (miR-197-3p), Cyclin-dependent kinase 1 (CDK1), and Wilms tumor 1-associated protein (WTAP) levels were detected using real-time quantitative polymerase chain reaction (RT-qPCR). Cell viability, proliferation, migration, and invasion were analyzed using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), Wound scratch, and Transwell assays. After being predicted by Circinteractome or TargetScan, binding between miR-197-3p and circ_0056856 or CDK1 was verified by a dual-luciferase reporter assay. CDK1 and WTAP protein levels were determined using Western blot. Interaction between WTAP and circ_0056856 was assessed using methylated RNA immunoprecipitation (MeRIP) assay. Increased circ_0056856, CDK1, and WTAP were observed in psoriasis patients and IL-22-treated HaCaT cells. Moreover, circ_0056856 knockdown might repress IL-22-induced HaCaT cell proliferation, migration, and invasion in vitro. In mechanism, circ_0056856 might function as a sponge of miR-197-3p to modulate CDK1 expression, and WTAP improved circ_0056856 expression via m6A methylation. WTAP-guided m6A modified circ_0056856 facilitates IL-22-stimulated HaCaT cell damage through the miR-197-3p/CDK1 axis, which could provide novel insights into psoriasis treatment.

Long non-coding RNA LINC00930 targeting miR-6792-3p/ZBTB16 regulates the proliferation and EMT of pancreatic cancer

Emerging evidence suggests the dysregulation of long non-coding RNAs (lncRNAs) involved in pancreatic cancer (PC). However, the function of LINC00930 in PC has not been elaborated. In this study, we found that LINC00930 was significantly down-regulated in PC cell lines and tissues, and associated with tumor size, lymphatic metastasis, TNM stage and poor prognosis. According to the bioinformatics database, the downregulation of LINC00930 was a common event in PC associated with prognosis and EMT. Overexpression of LINC00930 inhibited the aggressive cancer phenotypes including proliferation, metastasis and epithelial-mesenchymal transition (EMT) of PC in vitro and in vivo. Bioinformatics and dual-luciferase reporter assay indicated that miR-6792-3p could directly bind to LINC00930. Additionally, the Zinc finger and BTB domain containing 16 (ZBTB16) was significantly declined in PC, which was predicted to be the downstream gene of miR-6792-3p. MiR-6792-3p mimic rescued the decreased proliferation, metastasis and EMT caused by ZBTB16 in PC cells. The LINC00930/miR-6792-3p/ZBTB16 axis was associated with the malignant progression and process of PC. The relative expression of LINC00930 was negatively correlated with the expression of miR-6792-3p and was closely linked with ZBTB16 levels in PC. LINC00930 might serve as a potential prognostic biomarker and therapeutic target for PC.

#794 LncRNA NEAT1 protects uremic toxin-induced intestinal epithelial barrier injury by regulating miR-122-5p/Occludin axis

Abstract Background and Aims Long non-coding RNA(LncRNA) has been reported to be associated with intestinal barrier damage. The aim of this study was to explore the mechanism of lncRNA Nuclear enriched abundant transcript 1 (NEAT1) in uremic toxin-induced intestinal epithelial barrier injury. Method Human colon cancer cells (Caco-2) were used to establish intestinal epithelial injury models with the urea treatment in different conditions. Cell Counting Kit-8 (CCK-8) and Western blot screening the best concentration and time. The expressions of lncRNA NEAT1 and miR-122-5p were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot and immunofluorescence were used to detect the expression of tight junction proteins Occludin, ZO-1 and Claudin-1. Sodium fluorescein was used to detect the paracellular permeability of intestinal epithelial injury models. The binding of miR-122-5p to lncRNA NEAT1 and Occludin was determined by bioinformatics analysis and dual luciferase reporter assay. Results The best condition for the injury model was urea treatment in 144 mg/dl for 48 hours. With the increase of urea intervention time and concentration, the damage degree of intestinal epithelial cells is aggravated. Based on the qRT-PCR results, lncRNA NEAT1 was significantly down-regulated in the model group. Meanwhile, the tight junction proteins Occludin, ZO-1 and Claudin-1 were significantly reduced. The permeability of sodium fluorescein was significantly increased in the model group. Overexpression of lncRNA NEAT1 can alleviate the above performances. As the target gene of lncRNA NEAT1, miR-122-5p is significantly up-regulated in the model group. The dual luciferase reporter assay proved that miR-122-5p was targets to Occludin. The protective effect of overexpression lncRNA NEAT1 on intestinal epithelial barrier function is reversed by miR-122-5p mimics. Conclusion LncRNA NEAT1 protects uremic toxin-induced intestinal epithelial barrier injury by regulating miR-122-5p/Occludin axis.

GAS5 regulated by FTO-mediated m6A modification suppresses cell proliferation via the IGF2BP2/QKI axis in breast cancer

BackgroundThe lncRNA growth arrest-specific 5 (GAS5) is involved in regulating breast cancer progression. In this study, we aimed to elucidate the function and mechanism of GAS5 in breast cancer.MethodsThe expressions of GAS5, fat mass and obesity-associated protein (FTO), insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), and Quaking (QKI) were assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot. The m6A modification level of GAS5 was detected using m6A immunoprecipitation assay (MeRIP). The interaction between IGF2BP2 and GAS5 or QKI was detected using RNA immunoprecipitation assay (RIP) and dual luciferase reporter assay. Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) assay. The biological functions of the FTO/GAS5/IGF2BP2/QKI axis was assessed using the tumor xenograft assay.ResultsLncRNA GAS5 expression decreased in breast cancer and was regulated by FTO-mediated m6A modification in an IGF2BP2-dependent manner, resulting in decreased GAS5 stability and expression. GAS5 recruited IGF2BP2 to target QKI and upregulated QKI expression in breast cancer cells. GAS5 suppressed breast cancer growth via IGF2BP2/QKI, and this inhibitory effect was modulated by FTO both in vitro and in vivo.ConclusionsGAS5 regulated by FTO-mediated m6A modification represses the growth of breast cancer via the IGF2BP2/QKI pathway, suggesting that the FTO/GAS5/IGF2BP2/QKI pathway can be a potential target for breast cancer treatment.

FOXO3 suppresses lymphoma progression through promoting miR-34b/HSPG2 axis.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma, which caused many patients to lose their precious lives. FOXO3 was a suppressor in various cancers, however, the role and mechanism of FOXO3 in DLBCL remain unclear. Bioinformatics analysis was used to offer information FOXO3 expression and its expression for prognosis of DLBCL patients. The abundance of genes and proteins was evaluated using RT-qPCR and western blot. Cell proliferation and apoptosis was detected by CCK-8 and flow cytometry. The interactions among FOXO3, miR-34b, and HSPG2 were predicted by TransmiR and Starbase and validated using dual luciferase reporter assay, ChIP assay, and RIP assay. Our findings revealed that FOXO3 expression was abnormally declined in DLBCL cells. FOXO3 upregulation restrained cell proliferation and promoted cell apoptosis of DLBCL cells, while miR-34b inhibitor eliminated these influences. Similarly, miR-34b mimic suppressed malignant behaviors of DLBCL cells, which were abolished by HSPG2 overexpression. Mechanically, FOXO3 induced miR-34b expression through interacting with miR-34b promoter and HSPG2 was a targeted gene of miR-34b. FOXO3 attenuated the capability of cell proliferation and promoted cell apoptosis rate of DLBCL cells through affecting miR-34b/HSPG2 axis, therefore inhibiting DLBCL progression.

A Novel Frameshift Variant of the ELF4 Gene in a Patient with Autoinflammatory Disease: Clinical Features, Transcriptomic Profiling and Functional Studies.

We described the diagnosis and treatment of a patient with autoinflammatory disease, named "Deficiency in ELF4, X-linked (DEX)". A novel ELF4 variant was discovered and its pathogenic mechanism was elucidated. The data about clinical, laboratory and endoscopic features, treatment, and follow-up of a patient with DEX were analyzed. Whole exome sequencing and Sanger sequencing were performed to identify potential pathogenic variants. The mRNA and protein levels of ELF4 were analyzed by qPCR and Western blotting, respectively. The association of ELF4 frameshift variant with nonsense-mediated mRNA decay (NMD) in the pathogenesis DEX was examined. Moreover, RNA-seq was performed to identify the key molecular events triggered by ELF4 variant. The relationship between ELF4 and IFN-β activity was validated using a dual-luciferase reporter assay and a ChIP-qPCR assay. An 11-year-old boy presented with a Behçet's-like phenotype. The laboratory abnormality was the most obvious in elevated inflammatory indicators. Endoscopy revealed multiple ileocecal ulcers. Intestinal histopathology showed inflammatory cell infiltrations. The patient was treated with long-term immunosuppressant and TNF-α blocker (adalimumab), which reaped an excellent response over 16 months of follow-up. Genetic analysis identified a maternal hemizygote frameshift variant (c.1022del, p.Q341Rfs*30) in ELF4 gene in the proband. The novel variant decreased the mRNA level of ELF4 via the NMD pathway. Mechanistically, insufficient expression of ELF4 disturbed the immune system, leading to immunological disorders and pathogen susceptibility, and disrupted ELF4-activating IFN-βresponses. This analysis detailed the clinical characteristics of a Chinese patient with DEX who harbored a novel ELF4 frameshift variant. For the first time, we used patient-derived cells and carried out transcriptomic analysis to delve into the mechanism of ELF4 variant in DEX.

Downregulation of circ-RAPGEF5 inhibits colorectal cancer progression by reducing the expression of polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3).

Circular RNA (circRNA) plays a crucial role in the pathogenesis and progression of colorectal cancer (CRC). However, the current understanding of the emerging function and mechanism of circ-RAPGEF5 in CRC remains poorly understood. We first evaluated the expression level of circ-RAPGEF5 in CRC tissues and cells by quantitative real-time polymerase chain reaction (qRT-PCR). Then, we analyzed cell proliferation (EdU and colony formation assay), migration (cell wound healing assay), invasion (transwell assay), and apoptosis (flow cytometry assay). To further elucidate the mechanism of circ-RAPGEF5 in CRC, bioinformatics tools, Dual-luciferase reporter assay, Ago2 RNA immunoprecipitation assay, and RNA pull-down assay were employed. Moreover, we established a CRC transplantation tumor model to evaluate the effect of circ-RAPGEF5 on tumor growth in vivo. circ-RAPGEF5 was significantly upregulated in CRC tissues and CRC cells. Furthermore, the downregulation of circ-RAPGEF5 restrained CRC cell proliferation, migration, and invasion, and promoted cell apoptosis in vitro. Mechanistically, circ-RAPGEF5 accelerated the malignant behaviors of CRC cells by sponging miR-545-5p, which targeted polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). In addition, we revealed that circ-RAPGEF5 silence curbed tumor growth in vivo. These findings revealed that circ-RAPGEF5 played an oncogenic role through the miR-545-5p/GALNT3 axis in CRC progression, providing potential therapeutic targets for the treatment of CRC.

CEBPA Restrains the Malignant Progression of Breast Cancer by Prompting the Transcription of SOCS2.

The suppressor of cytokine signaling 2 (SOCS2) has been identified to act as a tumor suppressor in breast cancer (BC) progression. However, the action of SOCS2 in macrophage polarization in BC cells has not been reported yet. The qRT-PCR and western blotting were adopted for detecting the levels of mRNAs and proteins. The macrophage M2 polarization was analyzed by flow cytometry. Analyses of cell oncogenic phenotypes and tumor growth were conducted using 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, scratch, Transwell, tube formation assays in vitro, and tumor xenograft assay in vivo, respectively. The interaction between CEBPA (CCAAT Enhancer Binding Protein Alpha) and SOCS2 was confirmed using bioinformatics analysis and dual-luciferase reporter assay. SOCS2 was lowly expressed in BC tissues and cells. Functionally, overexpression of SOCS2 inhibited macrophage M2 polarization, and impaired BC cell proliferation, angiogenesis, and metastasis. Mechanistically, CEBPA bound to the promoter region of SOCS2, and promoted its transcription. A low CEBPA expression was observed in BC tissues and cells. Forced expression of CEBPA also suppressed macrophage M2 polarization, BC cell proliferation, angiogenesis, and metastasis. Moreover, the anticancer effects mediated by CEBPA were abolished by SOCS2 knockdown. In addition, CEBPA overexpression impeded BC growth in nude mice by regulating SOCS2. CEBPA suppressed macrophage M2 polarization, BC cell proliferation, angiogenesis, and metastasis by promoting SOCS2 transcription in a targeted manner.

PRR34-AS1 promotes mitochondrial division and glycolytic reprogramming in hepatocellular carcinoma cells through upregulation of MIEF2.

LncRNA PRR34-AS1 overexpression promotes the proliferation and invasion of hepatocellular carcinoma (HCC) cells, but whether it affects HCC energy metabolism remains unclear. Mitochondrial division and glycolytic reprogramming play important roles in tumor development. In this study, the differential expression of PRR34-AS1 is explored via TCGA analysis, and higher levels of PRR34-AS1 are detected in patients with liver cancer than in healthy individuals. A series of experiments, such as CCK-8, PCR, and immunofluorescence staining, reveal that the proliferation, invasion, glycolysis, and mitochondrial division of PRR34-AS1-overexpressing hepatoma cells are significantly promoted. TCGA analysis and immunohistochemistry reveal high expression of the mitochondrial dynamin MIEF2 in liver cancer tissues. Dual-luciferase reporter assays confirm that miR-498 targets and binds to mitochondrial elongation factor 2 (MIEF2). In addition, we show that PRR34-AS1 can sponge miR-498. Therefore, we further investigate the effects of the lncRNA PRR34-AS1/miR-498/MIEF2 axis on the growth, glucose metabolism, and mitochondrial division in hepatocellular carcinoma cells. A series of experiments are performed on hepatocellular carcinoma cells after different treatments. The results show that the proliferative activity, invasive ability, and glycolytic level of hepatocellular carcinoma cells are decreased in HCC cells with low PRR34-AS1 expression, and the miR-498 expression level is increased in these cells. Inhibition of miR-498 or overexpression of MIEF2 restored the proliferative activity, invasive ability, glycolysis, and mitochondrial division in hepatocellular carcinoma cells. Thus, PRR34-AS1 regulates MIEF2 by sponging miR-498, thereby promoting mitochondrial division, mediating glycolytic reprogramming and ultimately driving the growth and invasion of HCC cells. Furthermore, in vivo mouse experiments yield results similar to those of the in vitro experiments, verifying the above results.

Resveratrol Inhibits Colorectal Cancer Cell Tumor Property by Activating the miR-769-5p/MSI1 Pathway.

Resveratrol exhibits inhibitory effects on the progression of various cancers including colorectal cancer (CRC), however, the underlying mechanism in regulating CRC development remains elusive. The present study aims to uncover the role and molecular mechanism of resveratrol in modulating CRC cell tumor properties. NCM460 cells, LoVo cells, SW480 cells, and BALB/c nude mice were utilized in this study. RNA levels of miR-769-5p and musashi RNA-binding protein 1 (MSI1) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Protein expression was assessed by western blotting or immunohistochemistry assay. Cell viability was analyzed by CCK-8 assay, while cell proliferation and apoptosis were evaluated by 5-Ethynyl-2'-deoxyuridine assay and flow cytometry analysis. Cell migration was investigated by transwell and wound-healing assays. The association between miR-769-5p and MSI1 was identified by a dual-luciferase reporter assay. Tumor formation was analyzed using a xenograft mouse model assay. Compared to control groups, miR-769-5p expression was downregulated, while MSI1 expression was upregulated in CRC tissues and cells. Resveratrol treatment led to increased miR-769-5p expression and decreased MSI1 expression in CRC cells. Resveratrol treatment or miR-769-5p upregulation inhibited CRC cell proliferation and migration, and induced apoptosis. These effects were enhanced after combined treatment with resveratrol and miR-769-5p mimics. MSI1 was identified as a target of miR-769-5p, and its overexpression attenuated the effects of miR-769-5p mimics on cell proliferation, migration, and apoptosis. Moreover, miR-769-5p overexpression enhanced the inhibitory effects of resveratrol on tumor growth in vivo. Resveratrol inhibited colorectal cancer cell tumor properties by activating the miR-769-5p/MSI1 pathway.