RNA Immunoprecipitation Assay Research Articles

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.

CircMTO1/miR-30c-5p/SOCS3 axis alleviates oral submucous fibrosis through inhibiting fibroblast-myofibroblast transition.

circRNAs have been shown to participate in diverse diseases; however, their role in oral submucous fibrosis (OSF), a potentially malignant disorder, remains obscure. Our preliminary experiments detected the expression of circRNA mitochondrial translation optimization 1 homologue (circMTO1) in OSF tissues (n = 20) and normal mucosa tissues (n = 20) collected from Hunan Xiangya Stomatological Hospital, and a significant decrease of circMTO1 expression was showed in OSF tissues. Therefore, we further explored circMTO1 expression in OSF. Target molecule expression was detected using RT-qPCR and western blotting. The migration and invasion of buccal mucosal fibroblasts (BMFs) were assessed using wound healing and Transwell assays. The interaction between miR-30c-5p, circMTO1, and SOCS3 was evaluated using dual luciferase, RNA immunoprecipitation (RIP), and RNA pull-down assays. The colocalisation of circMTO1 and miR-30c-5p was observed using fluorescence in situ hybridisation (FISH). circMTO1 and SOCS3 expression decreased, whereas miR-30c-5p expression increased in patients with OSF and arecoline-stimulated BMFs. Overexpression of circMTO1 effectively restrained the fibroblast-myofibroblast transition (FMT), as evidenced by the increase in expression of Coll I, α-SMA, Vimentin, and the weakened migration and invasion functions in BMFs. Mechanistic studies have shown that circMTO1 suppresses FMT by enhancing SOCS3 expression by sponging miR-30c-5p and subsequently inactivating the FAK/PI3K/AKT pathway. FMT induced by SOCS3 silencing was reversed by the FAK inhibitor TAE226 or the PI3K inhibitor LY294002. circMTO1/miR-30c-5p/SOCS3 axis regulates FMT in arecoline-treated BMFs via the FAK/PI3K/AKT pathway. Expanding the sample size and in vivo validation could further elucidate their potential as therapeutic targets for OSF.

SUGP2 p.(Arg639Gln) variant is involved in the pathogenesis of hemochromatosis via the CIRBP/BMPER signaling pathway.

Pathogenic variants in HFE and non-HFE genes have been identified in hemochromatosis in different patient populations, but there are still a certain number of patients with unexplained primary iron overload. We recently identified in Chinese patients a recurrent p.(Arg639Gln) variant in SURP and G-patch domain containing 2 (SUGP2), a potential mRNA splicing-related factor. However, the target gene of SUGP2 and affected iron-regulating pathway remains unknown. We aimed to investigate the pathogenicity and underlying mechanism of this variant in hemochromatosis. RNA-seq analysis revealed that SUGP2 knockdown caused abnormal alternative splicing of CIRBP pre-mRNA, resulting in an increased normal splicing form of CIRBP V1, which in turn increasedthe expression of BMPER by enhancing its mRNA stability and translation. Furthermore, RNA-protein pull-down and RNA immunoprecipitation assays revealed that SUGP2 inhibited splicing of CIRBP pre-mRNA by a splice site variant at CIRBP c.492 and was more susceptible to CIRBP c.492 C/C genotype. Cells transfected with SUGP2 p.(Arg639Gln) vector showed up-regulation of CIRBP V1 and BMPER expression and down-regulation of pSMAD1/5 and HAMP expression. CRISPR-Cas9 mediated SUGP2 p.(Arg622Gln) knock-in mice showed increased iron accumulation in the liver, higher total serum iron, and decreased serum hepcidin level. A total of 10 of 54 patients with hemochromatosis (18.5%) harbored the SUGP2 p.(Arg639Gln) variant and carried CIRBP c.492 C/C genotype, and had increased BMPER expression in the liver. Altogether, the SUGP2 p.(Arg639Gln) variant down-regulates hepcidin expression through the SUGP2/CIRBP/BMPER axis, which may represent a novel pathogenic factor for hemochromatosis.

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.

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.

TRPM4 mRNA stabilization by METTL3-mediated m6A modification promotes calcific aortic valve inflammation

BackgroundTransient receptor potential melastatin 4 (TRPM4) affects immune responses by regulating calcium homeostasis, but its role in calcific aortic valve inflammation remains unclear. This study aimed to assess the expression and function of TRPM4 in patients with or without calcific aortic valve disease (CAVD). MethodsThe mRNA and protein expression levels of TRPM4 and related factors in calcified and noncalcified tissues were measured using qRT-PCR and Western blot. The proteins interacting with TRPM4 were confirmed by RNA pull-down and RNA immunoprecipitation assays. Dual-Luciferase Reporter Assay was performed to confirm the m6A site of TRPM4. ResultsThe mRNA expression levels of TRPM4, TLR4, IL-6, MCP-1, TNF-α, and NF-κB p65 were significantly higher in calcified aortic valve tissues than in noncalcified tissues, and TRPM4 was significantly positively correlated with inflammation-related factors. The protein expression level of TRPM4, TLR4 and NF-κB p65 were significantly higher in calcified aortic valve tissues than in noncalcified tissues. N6-methyladenosine (m6A) modification of TRPM4 mRNA by METTL3-YTHDF1 up-regulated its expression in CAVD. And TRPM4 promoted the level of inflammation via activation of the JNK-MAPK signaling pathway, after knockdown TRPM4, the production of proinflammatory cytokines was significantly suppressed. ConclusionThe results indicate the pivotal role of TRPM4 in CAVD and highlight METTL3-mediated m6A modification of TRPM4 in promoting inflammation through JNK-MAPK signaling pathway. This work provides potential therapeutic strategy to impede inflammation in CAVD.

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.

#2006 CircMTND5 participated in tubulointerstitial fibrosis progression of lupus nephritis by binding with A1CF

Abstract Background and Aims lupus nephritis (LN) is the most common and severe complication of systemic lupus erythematosus (SLE). CircRNA MTND5 participated in the pathogenesis of LN by sponging miR6812. However, it remains unclear what RNA binding role of circMTND5 play in LN. We aim to investigate whether circMTND5 can interact with RBP in the development and progression in LN, and to clarify the corresponding mechanisms. Method A1CF, a RNA binding protein, E-cadherin, α-SMA, TGF-β, and Fibronectin were analyzed by immunohistochemistry on paraffin-embedded section of LN patients and NC kidney tissues. The above parameters and circMTND5 were also examined in Fcgr2b-/- spontaneous LN mice kidneys by Western blotting and qPCR. In human kidney (HK) -2 cells, the binding of circMTND5 to A1CF protein was examined by RNA immunoprecipitation (RIP) assay. After knockdown in HK-2 cells and overexpression in hTGF-β stimulated HK-2 cells, we examined the all above indices. Results the expression of A1CF was down-regulated in the kidneys of LN patients and was localized in tubular epithelial cells. E-cadherin was down-regulated while α-SMA was up-regulated. The expression of circMTND5 was down-regulated in kidneys of Fcgr2b-/- spontaneous LN mice. In HK-2 cells, the binding of circMTND5 to A1CF was confirmed by RIP. A1CF and E-cadherin were down-regulated, while α-SMA, TGF-β and FN were up-regulated in HK-2 cells with knockdown of circMTND5. The overexpression of circMTND5 reverted the changes of the those above parametered induced by hTGF-β in both mRNA and proteins levels. Conclusion The expression level of circMTND5 in renal tissue of LN mice was downregulated. CircMTND5 might play an important role in tubulointerstitial fibrosis of lupus nephritis by binding with A1CF protein.

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.

CircSCNN1A inhibits the proliferation, migration and invasion of renal cell carcinoma cells by decreasing CLDN8 expression through miR-590-5p.

Increasing evidence suggests that circular RNA (circRNA) plays a regulatory role in the progression of renal cell carcinoma (RCC). However, the precise function and underlying mechanism of circSCNN1A in RCC progression still remain unclear. The expression levels of circSCNN1A, microRNA-590-5p (miR-590-5p), claudin 8 (CLDN8), cyclin D1, matrix metalloprotein 2 (MMP2), MMP9, E-cadherin, N-cadherin and vimentin were detected by a quantitative real-time polymerase chain reaction and Western blotting analysis. Immunohistochemistry assay was performed to analyze the positive expression rate of CLDN8. Cell proliferation was investigated by cell colony formation, 5-Ethynyl-2'-deoxyuridine and DNA content quantitation assays. Cell migration and invasion were assessed by wound-healing and transwell invasion assays. Interactions among circSCNN1A, miR-590-5p and CLDN8 were identified by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay. Xenograft mouse model assay was conducted to verify the effect of circSCNN1A on tumor formation in vivo. CircSCNN1A and CLDN8 expression were significantly downregulated, while miR-590-5p was upregulated in both RCC tissues and cells. CircSCNN1A overexpression inhibited RCC cell proliferation, migration and invasion, accompanied by decreases of cyclin D1, MMP2, MMP9, N-cadherin and vimentin expression and an increase of E-cadherin expression. CircSCNN1A acted as a miR-590-5p sponge and regulated RCC cell processes by binding to miR-590-5p. CLDN8, a target gene of miR-590-5p, was involved in the regulation of the biological behaviors of RCC cells by miR-590-5p. In addition, circSCNN1A induced CLDN8 production by interacting with miR-590-5p. Further, circSCNN1A suppressed tumor formation in vivo. CircSCNN1A inhibited RCC cell proliferation, migration and invasion by regulating the miR-590-5p/CLDN8 pathway.

Histone lactylation regulates autophagy of hyperplastic scar fibroblasts by inhibiting the transcriptional activity of phosphatase and tensin homologue.

Hyperplastic scar (HS) is an overreaction of tissue to skin injury caused by local fibroblast proliferation and excessive collagen production. Histone posttranslational modification patterns are important epigenetic processes that control various biological activities. This study was designed to investigate the effects of histone lactylation on HS and the underlying mechanism. Western blot was used to analyse the lactylation level in HS patients and fibroblasts (HSFs). In vitro experiments, western blot, cell counting kit-8, and immunofluorescence staining were performed to detect the collagen level, cell viability, and autophagy, respectively. The relationship between snai2 (SLUG) and phosphatase and tensin homologue (PTEN) was assessed by RNA immunoprecipitation and dual-luciferase reporter assays. The results showed that the histone lactylation level was upregulated in HS tissues and HSFs. HSFs showed increased collagen production and cell viability, and decreased autophagy. Silencing of lactate dehydrogenase A (LDHA) promoted the transcription of PTEN by inhibiting SLUG, thus promoting autophagy. Knockdown of LDHA inhibited collagen deposition and cell viability, and increased autophagy in HSFs, and the results were reversed after PTEN inhibition. In summary, histone lactylation inhibited the transcription activity of PTEN by promoting SLUG, thereby suppressing autophagy and promoting collagen deposition and cell viability of HSFs, which might provide effective therapeutic strategies in HS.

ALKBH5 modulates bone cancer pain in a rat model by suppressing NR2B expression.

Currently, the clinical treatment of bone cancer pain (BCP) is mainly related to its pathogenesis. The aim of the present study was to elucidate the potential role of N6-methyladenosine (m6A) in BCP in the spinal cord dorsal root ganglia (DRG) of BCP rats and its specific regulatory mechanism in N-methyl-d-aspartate receptor subunit 2B (NR2B). A rat model of BCP was constructed by tibial injection of Walker256 cells, and ALKBH5 and NR2B expression in the spinal cord DRG was detected. ALKBH5 was silenced or overexpressed in PC12 cells to verify the regulatory effect of ALKBH5 on NR2B. The specific mechanism underlying the interaction between ALKBH5 and NR2B was investigated using methylated RNA immunoprecipitation and dual-luciferase reporter gene assays. The results showed increased expression of m6A, decreased expression of ALKBH5, and increased expression of NR2B in the DRG of the BCP rat model. Overexpression of ALKBH5 inhibited NR2B expression, whereas interference with ALKBH5 caused an increase in NR2B expression. In NR2B, interference with ALKBH5 caused an increase in m6A modification, which caused an increase in NR2B. Taken together, ALKBH5 affected the expression of NR2B by influencing the stability of the m6A modification site of central NR2B, revealing that ALKBH5 is a therapeutic target for BCP.

The truncated AXIN1 isoform promotes hepatocellular carcinoma metastasis through SRSF9-mediated exon 9 skipping.

Axis inhibitor protein 1 (AXIN1) is a protein recognized for inhibiting tumor growth and is commonly involved in cancer development. In this study, we explored the potential molecular mechanisms that connect alternative splicing of AXIN1 to the metastasis of hepatocellular carcinoma (HCC). Transcriptome sequencing, RT‒PCR, qPCR and Western blotting were utilized to determine the expression levels of AXIN1 in human HCC tissues and HCC cells. The effects of the AXIN1 exon 9 alternative splice isoform and SRSF9 on the migration and invasion of HCC cells were assessed through wound healing and Transwell assays, respectively. The interaction between SRSF9 and AXIN1 was investigated using UV crosslink RNA immunoprecipitation, RNA pulldown, and RNA immunoprecipitation assays. Furthermore, the involvement of the AXIN1 isoform and SRSF9 in HCC metastasis was validated in a nude mouse model. AXIN1-L (exon 9 including) expression was downregulated, while AXIN1-S (exon 9 skipping) was upregulated in HCC. SRSF9 promotes the production of AXIN1-S by interacting with the sequence of exons 8 and 10 of AXIN1. AXIN1-S significantly promoted HCC cells migration and invasion by activating the Wnt pathway, while the opposite effects were observed for AXIN1-L. In vivo experiments demonstrated that AXIN1-L inhibited HCC metastasis, whereas SRSF9 promoted HCC metastasis in part by regulating the level of AXIN1-S. AXIN1, a tumor suppressor protein that targets the AXIN1/Wnt/β-catenin signaling axis, may be a promising prognostic factor and a valuable therapeutic target for HCC.

PLAG1 interacts with GPX4 to conquer vulnerability to sorafenib induced ferroptosis through a PVT1/miR-195-5p axis-dependent manner in hepatocellular carcinoma

BackgroundSorafenib is a standard first-line treatment for advanced hepatocellular carcinoma (HCC), yet its effectiveness is often constrained. Emerging studies reveal that sorafenib triggers ferroptosis, an iron-dependent regulated cell death (RCD) mechanism characterized by lipid peroxidation. Our findings isolate the principal target responsible for ferroptosis in HCC cells and outline an approach to potentially augment sorafenib's therapeutic impact on HCC.MethodsWe investigated the gene expression alterations following sgRNA-mediated knockdown induced by erastin and sorafenib in HCC cells using CRISPR screening-based bioinformatics analysis. Gene set enrichment analysis (GSEA) and the "GDCRNATools" package facilitated the correlation studies. We employed tissue microarrays and cDNA microarrays for validation. Ubiquitination assay, Chromatin immunoprecipitation (ChIP) assay, RNA immunoprecipitation (RIP) assay, and dual-luciferase reporter assay were utilized to delineate the specific mechanisms underlying ferroptosis in HCC cells.ResultsOur study has revealed that pleiomorphic adenoma gene 1 (PLAG1), a gene implicated in pleomorphic adenoma, confers resistance to ferroptosis in HCC cells treated with sorafenib. Sorafenib leads to the opposite trend of protein and mRNA levels of PLAG1, which is not caused by affecting the stability or ubiquitination of PLAG1 protein, but by the regulation of PLAG1 at the transcriptional level by its upstream competitive endogenous long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1). Data from 139 HCC patients showed a significant positive correlation between PLAG1 and GPX4 levels in tumor samples, and PLAG1 is instrumental in redox homeostasis by driving the expression of glutathione peroxidase 4 (GPX4), the enzyme that reduces lipid peroxides (LPOs), which further leads to ferroptosis inhibition.ConclusionsFerroptosis is a promising target for cancer therapy, especially for patients resistant to standard chemotherapy or immunotherapy. Our findings indicate that PLAG1 holds therapeutic promise and may enhance the efficacy of sorafenib in treating HCC.

PiR-hsa-23533 promotes malignancy in head and neck squamous cell carcinoma via USP7

BackgroundWith regard to head and neck squamous cell carcinoma (HNSCC), its occurrence and advancement are controlled by genetic and epigenetic anomalies. PIWI-interacting RNAs (piRNAs) are recognized with significance in tumor, but the precise molecular mechanisms of piRNAs in HNSCC largely remain undisclosed. MethodsDifferentially expressed piRNAs were identified by RNA sequencing. The expression of piR-hsa-23533 was evaluated using quantitative real-time PCR and RNA in situ hybridization. The impacts of piR-hsa-23533 on the proliferation and apoptosis of HNSCC cells were investigated by a series of in vitro and in vivo assays. ResultspiR-hsa-23533 exhibits upregulation within HNSCC cells and tissues. Besides, piR-hsa-23533 overexpression promotes proliferation while inhibiting apoptosis in vitro and in vivo, while piR-hsa-23533 silencing has an opposite function. From the mechanistic perspective, piR-hsa-23533 can bind to Ubiquitin-specific protease 7 (USP7), as shown through RNA pull-down and RNA immunoprecipitation assays, promoting USP7 mRNA and protein expression. ConclusionsThese findings highlight the functional importance of piR-hsa-23533 in HNSCC and may assist in the development of anti-HNSCC therapeutic target.

CircCOX6A1 suppresses osteogenic differentiation and aggravates osteoporosis via miR-512-3p/DYRK2 axis.

Osteoporosis (OP), characterized by compromised bone integrity and increased fracture risk, poses a significant health challenge. Circular RNAs (circRNAs) have emerged as crucial regulators in various pathophysiological processes, prompting investigation into their role in osteoporosis. This study aimed to elucidate the involvement of circCOX6A1 in OP progression and understand its underlying molecular mechanisms. The primary objective was to explore the impact of circCOX6A1 on bone marrow-derived mesenchymal stem cells (BMSCs) and its potential interactions with miR-512-3p and DYRK2. GSE161361 microarray analysis was employed to assess circCOX6A1 expression in OP patients. We utilized in vitro and in vivo models, including BMSC cultures, osteogenic differentiation assays, and an OVX-induced mouse model of OP. Molecular techniques such as quantitative RT-PCR, western blotting, and functional assays like alizarin red staining (ARS) were employed to evaluate circCOX6A1 effects on BMSC proliferation, apoptosis, and osteogenic differentiation. The interaction between circCOX6A1, miR-512-3p, and DYRK2 was investigated through dual luciferase reporter assays, RNA immunoprecipitation, and RNA pull-down assays. CircCOX6A1 was found to be upregulated in osteoporosis patients, and its expression inversely correlated with osteogenic differentiation of BMSCs. CircCOX6A1 knockdown enhanced osteogenic differentiation, as evidenced by increased mineralized nodule formation and upregulation of osteogenic markers. In vivo, circCOX6A1 knockdown ameliorated osteoporosis progression in OVX mice. Mechanistically, circCOX6A1 acted as a sponge for miR-512-3p, subsequently regulating DYRK2 expression. This study provides compelling evidence for the role of circCOX6A1 in osteoporosis pathogenesis. CircCOX6A1 negatively regulates BMSC osteogenic differentiation through the miR-512-3p/DYRK2 axis, suggesting its potential as a therapeutic target for mitigating OP progression.

Pan-cancer analysis of super-enhancer-induced LINC00862 and validation as a SIRT1-promoting factor in cervical cancer and gastric cancer

Immune checkpoints inhibitors are effective but it needs more precise biomarkers for patient selection. We explored the biological significance of LINC00862 in pan-cancer by bioinformatics. And we studied its regulatory mechanisms using chromatin immunoprecipitation and RNA immunoprecipitation assays etc. TCGA and single-cell sequencing data analysis indicated that LINC00862 was overexpressed in the majority of tumor and stromal cells, which was related with poor prognosis. LINC00862 expression was related with immune cell infiltration and immune checkpoints expression, and had a high predictive value for immunotherapy efficacy. Mechanistically, LINC00862 competitively bound to miR-29c-3p to unleash SIRT1’s tumor-promoting function. SIRT1 inhibitor-EX527 were screened by virtual screening and verified by in vitro and vivo assays. Notably, acetyltransferase P300-mediated super-enhancer activity stimulated LINC00862 transcription. Collectively, LINC00862 could be a diagnostic and prognostic biomarker. LINC00862 could also be a predictive biomarker for immunotherapy efficacy. Super-enhancer activity is the driver for LINC00862 overexpression in cervical cancer and gastric cancer.

Small nucleolar RNA host gene 5 plays a role in orthodontic tooth movement by inhibiting osteoclast differentiation.

The alveolar bone remodelling promoted by reasonable mechanical force triggers orthodontic tooth movement (OTM). The generation of osteoclasts is essential in this process. However, the mechanism of mechanical force mediating osteoclast differentiation remains elusive. Small nucleolar RNA host gene 5 (SNHG5), which was reported to mediate the osteogenic differentiation of bone marrow mesenchymal stem cells in our previous study, was downregulated in human periodontal ligament cells (hPDLCs) under mechanical force. At the same time, the RANKL/OPG ratio increased. Based on this, we probed into the role of SNHG5 in osteoclast formation during OTM and the relevant mechanism. SNHG5 and the RANKL/OPG ratio under different compressive forces were detected by western blotting (WB) and qRT-PCR. Impact of overexpression or knockdown of SNHG5 on osteoclast differentiation was detected by qRT-PCR, WB and transwell experiments. The combination of SNHG5 and C/EBPβ was verified by RNA immunoprecipitation and RNA pull-down assays. The expression of SNHG5 and osteoclast markers in gingiva were analysed by qRT-PCR and the paraffin sections of periodontal tissues were used for histological analysis. Compressive force downregulated SNHG5 and upregulated the RANKL/OPG ratio in hPDLCs. Overexpression of SNHG5 inhibited RANKL's expression and osteoclast differentiation. SNHG5 combined with C/EBPβ, a regulator of osteoclast. The expression of SNHG5 in periodontal tissue decreased during OTM. SNHG5 inhibited osteoclast differentiation during OTM, achieved by affecting RANKL secretion, which may provide a new idea to interfere with bone resorption during orthodontic treatment.

Circular RNA circESYT2 serves as a microRNA-665 sponge to promote the progression of hepatocellular carcinoma through ENO2.

Circular RNAs (circRNAs) have emerged as crucial regulators in tumor progression, yet their specific role in hepatocellular carcinoma (HCC) remains largely uncharacterized. In this study, we utilized high-transcriptome sequencing to identify the upregulation of circESYT2 (hsa_circ_002142) in HCC tissues. Functional experiments carried out invivo and invitro revealed that circESYT2 played a significant role in maintaining the growth and metastatic behaviors of HCC. Through integrative analysis, we identified enolase 2 (ENO2) as a potential target regulated by circESYT2 through the competitive endogenous RNA sponge mechanism. Additional gain- or loss-of-function experiments indicated that overexpression of circESYT2 led to a tumor-promoting effect, which could be reversed by transfection of microRNA-665 (miR-665) mimic or ENO2 knockdown in HCC cells. Furthermore, the direct interaction between miR-665 and circESYT2 and between miR-665 and ENO2 was confirmed using RNA immunoprecipitation, FISH, RNA pull-down, and dual-luciferase reporter assays, highlighting the involvement of the circESYT2/miR-665/ENO2 axis in promoting HCC progression. These findings shed light on the molecular characteristics of circESYT2 in HCC tissues and suggest its potential as a biomarker or therapeutic target for HCC treatment.

PITPNA-AS1 Inhibits Cell Proliferation and Migration in Ovarian Cancer by Regulating the MIR-223-3p/RHOB Axis.

Ovarian cancer is a fatal gynecologic malignancy. Long non-coding RNA (lncRNA) has been verified to serve as key regulator in ovarian cancer tumorigenesis. The aim of the study was to study the functions and mechanism of lncRNA PITPNA-AS1 in ovarian cancer cellular process. Clinical ovarian cancer samples were collected and stored at an academic medical center. Cellular fractionation assays and fluorescence in situ hybridization were conducted to locate PITPNA-AS1 in OC cells. TUNEL staining, colony-forming assays, and Transwell assays were performed for evaluating cell apoptosis as well as proliferative and migratory abilities. Western blot was conducted for quantifying protein levels of epithelialmesenchymal transition markers. The binding relation between genes was verified by RNA pulldown, RNA immunoprecipitation, and luciferase reporter assays. Gene expression levels in ovarian cancer tissues and cells were subjected to RT-qPCR. PITPNA-AS1 level was downregulated in ovarian cancer samples and cells. PITPNA-AS1 overexpression contributed to the accelerated ovarian cancer cell apoptosis and inhibited cell migration, proliferation, and epithelial-mesenchymal transition process. In addition, PITPNA-AS1 interacted with miR-223-3p to regulate RHOB. RHOB knockdown partially counteracted the repressive impact of PITPNA-AS1 on ovarian cancer cell activities. PITPNA-AS1 inhibited ovarian cancer cellular behaviors by targeting miR-223-3p and regulating RHOB.