Dual-luciferase Reporter Assay Research Articles

RON receptor tyrosine kinase as a critical determinant in promoting tumorigenic behaviors of bladder cancer cells through regulating MMP12 and HIF-2α pathways.

The RON receptor tyrosine kinase is critical in the pathogenesis of various cancer types, however, its role in bladder cancer invasive growth is still largely unknown. Here, we found that over 90% of bladder cancer samples exhibit elevated levels of RON expression, with significantly higher expression levels observed in invasive bladder cancer compared to non-invasive bladder cancer. In vitro, RON activation resulted in increased bladder cancer cell migration and invasiveness. Results from mRNA sequencing and transcriptome analysis further demonstrated that MMP12, a downstream molecule of RON, is functionally involved in regulating RON-mediated bladder cancer cell migration and invasiveness. The underlying mechanism appeared to be the RON-mediated inhibition of HIF-2α ubiquitination, which is channeled through the activation of the JNK signaling pathway. Consequently, the activated JNK pathway increased MMP12 expression, ultimately driving bladder cancer cell migration and invasion. As evident in bioinformatics and dual-luciferase reporter assays, the RON mRNA at its 3'-untranslated regions specifically interacted with hsa-miR-659-3p. The binding of hsa-miR-659-3p downregulated the RON gene expression, attenuating the receptor-mediated tumorigenic activities of bladder cancer cells in vitro and in vivo. In conclusion, aberrant RON expression in bladder cancer cells and MMP12 and HIF-2α activities form a functional axis that causes increased bladder cancer cell migration and invasion. The fact that hsa-miR-659-3p downregulates RON expression indicates its critical role in attenuating RON-mediated tumorigenic effect on bladder cancer cells. These findings highlight the importance of RON targeting as a therapeutic means for potential bladder cancer therapy.

Dapagliflozin Suppresses High Glucose-Induced Proliferation, Oxidative Stress, and Fibrosis by Reducing Mettl3-Induced m6A Modification in Marcks mRNA.

Diabetic cardiomyopathy (DCM) is a common and severe complication of Diabetes mellitus (DM). Dapagliflozin (DAPA) is an oral anti-diabetic drug worldwide for the treatment of type 2 DM. However, the action and mechanism of DAPA in cardiac fibrosis during DCM remain vague. Primary cardiac fibroblasts (CFs) were incubated with high glucose (HG) in vitro. Cell proliferation was detected by MTT and EdU assays. Oxidative stress was evaluated by determining the production of reactive oxygen species and malondialdehyde. Cell fibrosis was assessed by detecting fibrosis-related proteins by western blotting. Levels of Mettl3 (Methyltransferase 3) and Marcks (myristoylated alanine-rich C kinase substrate) were measured using qRT-PCR and western blotting. The m6A modification profile was determined by methylated RNA immunoprecipitation assay and the interaction between Mettl3 and Marcks was verified using dual-luciferase reporter and RIP assays. DAPA treatment alleviated HG-induced proliferation, oxidative stress, and fibrosis in CFs. HG promoted the expression of Mettl3 in CFs. Knockdown of Mettl3 reversed HG-induced proliferation, oxidative stress, and fibrosis in CFs; moreover, forced expression of Mettl3 abolished the protective effects of DAPA on CFs under HG condition. Mechanistically, Mettl3 interacted with Marcks in CFs and induced Marcks mRNA m6A modification. HG induced high expression of Marcks in CFs. The overexpression of Marcks could counteract DAPA or Mettl3 knockdown-evoked inhibitory effects on CF proliferation, oxidative stress, and fibrosis under HG condition. Dapagliflozin suppressed HG-induced proliferation, oxidative stress, and fibrosis by reducing Mettl3-induced m6A modification in Marcks mRNA.

Zinc Finger Protein 263 Augments Autophagy and Promotes Intrahepatic Cholangiocarcinoma Proliferation

ABSTRACTIntrahepatic cholangiocarcinoma (ICC) is an aggressive cancer characterized by a poor prognosis. Despite Zinc finger proteins (ZNFs) importance in tumor development and progression, it is unknown how dysregulated ZNF263 contributes to intrahepatic cholangiocarcinoma. This study aimed to determine whether ZNF263 plays an oncogenic role in ICC progression. The microarray of tumor tissues from clinical intrahepatic cholangiocarcinoma was immunohistochemically analyzed for ZNF263. Based on plate colony formation, CCK8, and tumor xenograft models, ZNF263 was assessed for its biological function. Mechanistically, CUT&Tag, RNA‐seq, CHIP‐PCR, Dual luciferase reporter assay, Western blotting, transmission electron microscopy (TEM), and immunohistochemical staining were employed. ZNF263 expression was elevated in intrahepatic cholangiocarcinoma tissues compared to nontumor tissues, which negatively impacted patient outcomes. Notably, ZNF263 overexpression promoted ICC cells proliferation via enhancing autophagy, whereas ZNF263 knockdown inhibited ICC cells proliferation. Furthermore, ZNF263 binds to the enhancer region of ULK1 and mediates its expression. ULK1 over‐expressing ameliorated ZNF263 knockdown‐induced inhibition of CRC proliferation. By activating the ULK1‐autophagy axis, ZNF263 promotes proliferation of ICC and is potentially a prognostic or therapeutic target of ICC.

Identification of the Oncogenic Role of the Circ_0001326/miR-577/VDAC1 Cascade in Prostate Cancer.

Prostate cancer (PCa) is one of the leading causes of cancer death among men worldwide. Circular RNAs (circRNAs) have been implicated in the pathogenesis of PCa. However, the precise action of circ_0001326 in PCa malignant progression is still unknown. The levels of circ_0001326, miR-577 and voltage dependent anion channel 1 (VDAC1) were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell proliferation, colony formation, apoptosis, migration and invasion were evaluated by the Cell Counting Kit-8 (CCK-8), EdU staining, colony formation, flow cytometry, wound-healing and transwell assays, respectively. Targeted relationships among circ_0001326, miR-577 and VDAC1 were confirmed by dual-luciferase reporter assays. Xenograft experiments were performed to detect the role of circ_0001326 in tumor growth. Our data revealed that circ_0001326 was overexpressed in PCa tissues and cells. Circ_0001326 depletion repressed PCa cell proliferation, migration, and invasion and enhanced apoptosis in vitro, as well as hampered tumor growth in vivo. Mechanistically, circ_0001326 directly targeted miR-577, and VDAC1 was directly targeted and suppressed by miR-577. Moreover, the effects of circ_0001326 knockdown on PCa cell functional behaviors were mediated by miR-577. VDAC1 silencing phenocopied miR-577 overexpression in regulating PCa cell functional behaviors in vitro. Furthermore, circ_0001326 regulated VDAC1 expression through sponging miR-577. Our findings showed that circ_0001326 regulated PCa cell functional behaviors at least partly through targeting the miR-577/VDAC1 axis.

Human umbilical cord mesenchymal stem cells small extracellular vesicles-derived miR-370-3p inhibits cervical precancerous lesions by targeting DHCR24

Abstract Background Cervical cancer is often caused by persistent high-risk human papillomavirus (HPV) infection, causing precancerous lesions. Human umbilical cord mesenchymal stem cells-derived small extracellular vesicles (hucMSC-sEV) exhibit diverse effects on tumors. This study investigates hucMSC-sEV, the impact and mechanisms on HPV-positive cervical precancerous lesion cells to provide new treatment insights. Materials and Methods We previously obtained hucMSC and hucMSC-sEV. In vitro experiments evaluated hucMSC-sEV effects on the proliferation and migration of S12 cells (derived from cervical precancerous lesions). Bioinformatics identified key microRNA components, and their impact on S12 cell proliferation and migration was investigated. The target gene of the microRNA component was predicted and confirmed via bioinformatics and dual-luciferase reporter assays. Lentiviral systems overexpressed target gene in S12 cells to examine the effects on microRNA impacts. SH-42 inhibitor was used to investigate target gene treatment potential. Immunohistochemistry assessed target gene expression in cervical precancerous lesions tissue. Results hucMSC-sEV significantly inhibited S12 cell proliferation and migration. Bioinformatics identified miR-370-3p as an effective cargo, which also suppressed S12 cell proliferation and migration. miR-370-3p was confirmed targeting DHCR24 (24-Dehydrocholesterol Reductase). DHCR24 overexpression reversed miR-370-3p’s inhibitory effects, while SH-42 counteracted DHCR24 overexpression’s promoting effects. Clinical specimen analysis supported these findings, demonstrating a positive correlation between DHCR24 protein expression and cervical precancerous lesions’ progression. Conclusions hucMSC-sEV inhibits S12 cell proliferation and migration, mediated by miR-370-3p targeting DHCR24 to regulate cellular cholesterol content. DHCR24 inhibition reduces the cholesterol level and cell functions, suggesting its potential as a therapeutic target in cervical precancerous lesions.

The KLF16/MYC feedback loop is a therapeutic target in bladder cancer

BackgroundBladder cancer (BLCA) is a common malignancy characterized by dysregulated transcription and a lack of effective therapeutic targets. In this study, we aimed to identify and evaluate novel targets with clinical potential essential for tumor growth in BLCA.MethodsCRISPR-Cas9 screening was used to identify transcription factors essential for bladder cancer cell viability. The biological functions of KLF16 in bladder cancer were investigated both in vitro and in vivo. The regulatory mechanism between KLF16 and MYC was elucidated through a series of analyses, including RNA sequencing, quantitative polymerase chain reaction (qPCR), RNA immunoprecipitation, Western blotting, Mass spectrometry, Dual-luciferase reporter assays, Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing, OptoDroplets assays, and RNA stability assay. The clinical relevance of KLF16 and MYC in bladder cancer was evaluated through analyses of public databases and immunohistochemistry.ResultsKrüppel-like factor 16 (KLF16) was essential for BLCA cell viability. Elevated expression of KLF16 was observed in bladder cancer tissues, and higher expression levels of KLF16 were correlated with poor progression-free survival (PFS) and cancer-specific survival (CSS) probabilities in BLCA patients. Mechanistically, KLF16 mRNA competed with the mRNA of dual-specificity phosphatase 16 (DUSP16) for binding to the RNA-binding protein, WW domain binding protein 11 (WBP11), resulting in destabilization of the DUSP16 mRNA. This, in turn, led to activation of ERK1/2, which stabilized the MYC protein. Furthermore, KLF16 interacted with MYC to form nuclear condensates, thereby enhancing MYC’s transcriptional activity. Additionally, MYC transcriptionally upregulated KLF16, creating a positive feedback loop between KLF16 and MYC that amplified their oncogenic functions. Targeting this loop with bromodomain inhibitors, such as OTX015 and ABBV-744, suppressed the transcription of both KLF16 and MYC, resulting in reduced BLCA cell viability and tumor growth, as well as increased sensitivity to chemotherapy.ConclusionsOur study revealed the crucial role of the KLF16/MYC regulatory axis in modulating tumor growth and chemotherapy sensitivity in BLCA, suggesting that combining bromodomain inhibitors, such as OTX015 or ABBV-744, with DDP or gemcitabine could be a promising therapeutic intervention for BLCA patients.

CircARID1A Inhibits Tail Fat Cell Differentiation in Guangling Large-Tailed Sheep by Regulating the miR-493-3p/YTHDF2 Axis.

The Guangling Large-Tailed sheep is renowned for its unique tail fat deposition, with a significant proportion of its total body fat being localized in the tail region. Fat deposition is a complex biological process regulated by various molecular mechanisms. Our previous studies have identified a large number of differentially expressed circular RNAs (circRNAs) in the tail adipose tissue of the Guangling Large-Tailed sheep. These circRNAs may play a pivotal role in the process of fat deposition. Given the potential regulatory functions of circRNAs in adipose metabolism, investigating their roles in tail fat deposition is of significant scientific importance. In this study, we identified novel circARID1A. Using various experimental methods, including lentivirus infection, RNase R treatment, actinomycin D assay, qPCR, western blotting, and dual-luciferase reporter assays, we determined that circARID1A inhibits the expression of miR-493-3p through competitive binding, thereby regulating adipocyte differentiation. Further research revealed that miR-493-3p promotes adipocyte differentiation by targeting YTH domain family 2 (YTHDF2), and this regulatory effect is also influenced by circARID1A. In conclusion, our findings suggest that circARID1A inhibits tail fat cell differentiation in the Guangling Large-Tailed sheep through the circARID1A/miR-493-3p/YTHDF2 axis, providing theoretical support for improving meat quality and fat deposition in sheep.

A Set of Proximal Regulatory Elements Contribute to the Transcriptional Activity of the Human Lipoprotein Lipase Promoter.

Lipoprotein lipase (LPL) is a multifunctional protein that catalyzes the hydrolysis of plasma triglycerides, releasing free fatty acids, which play critical roles in the metabolism and transport of lipids. The transcription of LPL in response to cell types and regulatory factors is a complex process that starts with its promoter. In previous studies, several proximal regulatory elements within the human LPL promoter were individually characterized. This study was designed to characterize the effect of 12 proximal regulatory elements as a combined unit on the transcriptional activity of the LPL promoter. The hypothesis was that these proximal regulatory elements collectively result in the optimal transcriptional activity of the human LPL promoter. Full and partial LPL promoter sequences, which contained and excluded the 12 regulatory elements, respectively, were cloned and inserted into a promoterless luciferase reporter vector. The functional activities of these constructs were tested in vitro using a dual-luciferase reporter assay. Our results showed that HEK-293 cells transfected with the full LPL promoter exhibited significantly greater luciferase activity than cells transfected with partial LPL promoters. Our results indicate that the proximal regulatory elements within the LPL promoter, including four TATA boxes, two Oct-1 sites, one CT element, two C/EBPα sites, one SP1 site, and two cis-acting regions (LP-α and LP-β), are essential for its transcriptional activity.

NOTCH3 promotes malignant progression of bladder cancer by directly regulating SPP1 and activating PI3K/AKT pathway

The biological role and precise molecular mechanisms of Notch receptor 3 (NOTCH3) in the malignant progression of bladder cancer (BLCA) remain unclear. In this study, we found that NOTCH3 was significantly upregulated and associated with poor prognosis in BLCA patients. Functional experiments demonstrated that NOTCH3 knockdown inhibited BLCA cell proliferation, migration, invasion and significantly suppressed tumor growth and metastasis in vivo as well. Mechanically, chromatin immunoprecipitation and dual-luciferase reporter assays confirmed that NOTCH3 could promote the transcription of secreted phosphoprotein 1 (SPP1), a potential downstream target gene of NOTCH3, by binding to the CSL elements in the SPP1 promoter. Moreover, we also found that targeting NOTCH3 inhibited BLCA growth and metastasis by suppressing the SPP1-PI3K/AKT axis. Our study highlights the critical role of NOTCH3-SPP1-PI3K/AKT axis in the malignant progression of BLCA, suggesting that NOTCH3 may be a potential therapeutic target for BLCA.

ADAMTS16 drives epithelial-mesenchymal transition and metastasis through a feedback loop upon TGF-β1 activation in lung adenocarcinoma

Lung adenocarcinoma (LUAD) is the major subtype of lung cancer. The poor prognosis of LUAD patients is attributed primarily to metastasis. ADAMTS16 is a crucial member of the ADAMTS family and is involved in tumor progression. However, its role and regulatory mechanism in LUAD remain unexplored. In this study, ADAMTS16 was identified as a crucial oncogene and survival predictor in LUAD via analyses of public datasets. Clinical specimens and tissue microarrays confirmed the differential expression and prognostic value of ADAMTS16 in LUAD patients. Transcriptome data and in vitro experiments demonstrated that ADAMTS16 was positively associated with epithelial-mesenchymal transition (EMT) and the migration abilities of LUAD cells. Knockdown of ADAMTS16 attenuated lung and pleural metastasis in an animal model. Mechanistically, the results of the enzyme-linked immunosorbent assay (ELISA) and western blot (WB) suggested that ADAMTS16 activated the TGF-β signaling pathway by facilitating the conversion of LAP-TGF-β1 to active TGF-β1. Co-Immunoprecipitation (co-IP) indicated an interaction between ADAMTS16 and LAP-TGF-β1. Inhibition of ADAMTS16 impaired EMT and aggressiveness of LUAD cells, while treatment with recombinant TGF-β1 reversed this inhibition. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays indicated that SOX4 acted as a transcriptional activator of ADAMTS16 and that TGF-β1 regulated the expression of ADAMTS16 by increasing the binding of SOX4 to the promoter of ADAMTS16. Suppressing the TGF-β signaling pathway inhibited ADAMTS16 expression, EMT, and lung metastasis, whereas overexpressing SOX4 reversed this inhibition. Therefore, ADAMTS16 forms a positive feedback loop with the TGF-β1/SOX4 axis to regulate EMT and metastasis, and disruption of this feedback loop inhibits tumor progression. These findings underscore the potential of ADAMTS16 as a prognostic biomarker and therapeutic target in LUAD and offer novel insight into the mechanism of EMT and metastasis.

Role of the CTCF/p300 axis in osteochondrogenic-like differentiation of polyploid giant cancer cells with daughter cells

BackgroundPolyploid giant cancer cells (PGCCs) have properties of cancer stem cells (CSCs). PGCCs with daughter cells (PDCs) undergo epithelial–mesenchymal transition and show enhanced cellular plasticity. This study aimed to elucidate the mechanisms underlying the osteo/chondrogenic-like differentiation of PDCs, which may be exploited therapeutically by transdifferentiation into post-mitotic and functional cells.MethodsCobalt chloride was used to induce PGCC formation in MDA-MB-231 and HEY cells, and PDCs were cultured in osteo/chondrogenic differentiation media. Alcian blue staining was used to confirm osteo/chondrogenic differentiation, and the cell cycle was detected using flow cytometry. The expression of osteo/chondrogenic differentiation-related proteins was compared, and a co-immunoprecipitation assay was used to demonstrate the interactions between proteins. Bioinformatic analysis was used to explore the regulatory mechanism of osteo/chondrogenic differentiation, and a dual-luciferase reporter assay was performed to validate the interaction between transcriptional factors and target genes. Animal xenograft models were used to confirm the osteo/chondrogenic differentiation of PDCs.ResultsWhen cultured in osteo/chondrogenic medium, the stemness of PDCs decreased, and the expression of osteo/chondrogenic-related markers increased. This osteo/chondrogenic-like process was regulated by the transforming growth factor-β pathway in a time-dependent manner. A concurrent increase in the expression of histone acetyltransferase p300 and the transcription factor CCCTC-binding factor (CTCF) was observed. Co-immunoprecipitation assays revealed that p300 acetylated the osteo/chondrogenic marker RUNT-related transcription factor 2 (RUNX2). Analysis of chromatin immunoprecipitation sequencing datasets revealed that both CTCF and histone H3 lysine 27 acetylation (H3K27ac) were enriched in the promoter region of E1A-associated protein p300 (P300). The four predicted binding sites for CTCF and P300 were validated using dual-luciferase reporter assays. We examined the interaction between CTCF and H3K27ac and found that these two proteins had a combined effect on the transactivation of P300.ConclusionCTCF, in synergy with H3K27ac, amplified the expression of P300, facilitating acetyl group transfer to RUNX2. This acetylation stabilized RUNX2 and promoted osteo/chondrogenic differentiation, thereby reducing the incidence of PDC malignancies.

MicroRNA-429 overexpression overcomes imatinib resistance of glioma cells by negatively regulating lysophosphatidic acid receptor 1

ABSTRACT Background Glioma is one of the most aggressive and lethal malignancies in central nervous system. It has been reported that miR-429 is declined in glioma and functions as a tumor suppressor. Nonetheless, the potential role of miR-429 in drug resistance of glioma is still ambiguous. Methods Stable imatinib-resistant lines U251-AR and T98G-AR were established using glioma cell lines U251 and T98G. Cell apoptosis and cycle were analyzed by flow cytometry, and CCK-8 assay was utilized to measure cell viability. Protein and RNA levels were tested with western blot and RT-qPCR. The predicted binding site was confirmed by dual luciferase reporter assay. Results Imatinib-resistant U251-AR and T98G-AR cells presented lower level of miR-429 and higher level of LPAR1. MiR-429 overexpression obviously promoted imatinib sensitivity in glioma cells, indicated by the reduced IC50 value, facilitated cell apoptosis and cell cycle arrest at G0/G1 phase, and downregulated multidrug resistance-related proteins. LPAR1 was verified as a direct target of miR-429 and its expression was negatively regulated by miR-429. Additionally, overexpression of LPAR1 restrained the biological function of miR-429 on imatinib chemoresistance. Conclusion MiR-429 partly sensitized glioma cells to imatinib via downregulation LPAR1, which might provide an approach to overcome imatinib chemoresistance during glioma treatment.

N6-methyladenosine modification of circular RNA circASH2L suppresses growth and metastasis in hepatocellular carcinoma through regulating hsa-miR-525-3p/MTUS2 axis

BackgroundCircRNAs have been demonstrated to play a crucial role in regulating the growth and progression of various cancers, including hepatocellular carcinoma (HCC). Nevertheless, the circRNA's expression pattern and function in HCC need more investigation.MethodsBioinformatics techniques were used to identify differentially expressed circRNAs in HCC. CircASH2L expression in HCC tissues was assessed through qRT-PCR and ISH analysis. To assess circASH2L's impact on HCC progression, a variety of experiments were carried out both in vitro and in vivo, such as CCK8, colony formation, EdU assay, flow cytometry, transwell assay, and xenograft mouse model. Various experimental techniques including qRT-PCR, dual luciferase reporter assay, FISH, RNA pull-down, and RIP experiments were utilized to evaluate the relationship between circASH2L, miR-525-3p, and MTUS2. Additionally, experiments were conducted to explore the impact of m6A modification on circASH2L expression, including RNA stability assay, m6A RNA immunoprecipitation assay (MeRIP), and Co-IP experiments.ResultsWe found that circASH2L was downregulated in HCC tissues and the downregulation of circASH2L was significantly correlated with malignant characteristics as well as poor overall survival of patients with HCC. CircASH2L was found to inhibit cells growth, migration and invasion as well as tumorigenesis and metastasis in vivo. Mechanistically, we established that circASH2L directly interacted with miR-525-3p to enhance MTUS2 expression, subsequently leading to tumor suppression. Moreover, the influence of circASH2L on tumor suppression was attenuated by increasing miR-525-3p levels, and MTUS2 was recognized as an essential intermediary in circASH2L-induced tumor suppression. Additionally, N6−methyladenosine (m6A) modification was identified in circASH2L. Our data suggested that METTL3 was responsible for mediating m6A methylation of circASH2L, ultimately regulating circASH2L expression through the promotion of its degradation. These findings collectively highlight the role of circASH2L as a tumor suppressor through a unique circASH2L/miR-525-3p/MTUS2 axis, shedding light on the significance of m6A modification in regulating circASH2L function.ConclusionThe work emphasizes circASH2L as a promising therapeutic target for treating HCC, offering new insights into the role of circRNAs in HCC development.

Irisin Improves Preeclampsia by Promoting Embryo Implantation and Vascular Remodeling.

Preeclampsia is a pregnancy-specific disorder with unclear pathogenesis. Irisin, a recently identified exercise-induced factor, significantly influences lipid metabolism and cardiovascular function. Nonetheless, its role in trophoblast development during human placentation and the related intracellular signaling pathways remain poorly understood. We assessed peripheral blood irisin expression in early pregnancy among patients with preeclampsia and its correlation with key clinical indicators. In trophoblast cell lines and mice, we used exogenous irisin and viral knockdown to investigate functional changes. Phosphorylation-specific antibody arrays and dual-luciferase reporter assays were used to explore downstream molecular mechanisms, which were subsequently validated in trophoblast cell lines and relevant gene knockout mice. In early pregnancy, patients with preeclampsia exhibit decreased peripheral blood irisin levels, occurring earlier than traditional predictive markers, such as PLGF (placental growth factor) and sFlt-1 (soluble fms-like tyrosine kinase-1). Furthermore, irisin concentration is positively correlated with proteinuria and abnormal blood pressure during pregnancy. Exogenous irisin significantly enhanced trophoblast cell migration, invasion, and proliferation while inhibiting apoptosis. It also increased STAT (signal transducers and activators of transcription) 4 phosphorylation and its binding to the GLUT (glucose transporter)-3 promoter, resulting in elevated GLUT-3 expression and glucose uptake in trophoblast cells. In vivo, increased peripheral irisin promoted embryo implantation, vascular remodeling, and enhanced glucose uptake, whereas reduced irisin resulted in a preeclampsia-like phenotype characterized by elevated blood pressure, proteinuria, renal-placental dysfunction, adipose accumulation, and restricted fetal growth. Peripheral irisin improves preeclampsia by promoting embryo implantation and vascular remodeling through the activation of the STAT4/GLUT-3 pathway. Reduced peripheral irisin may contribute to preeclampsia-like pathologies. This study supports the advocacy for appropriate exercise during early pregnancy and provides new insights for preeclampsia prevention.

ABC Family Gene Polymorphisms and Cognitive Functions Interact to Influence Antidepressant Efficacy.

The importance of identifying relevant indicators of antidepressant efficacy is highlighted by the low response rates to antidepressant treatment for depression. The ABC gene family, encoding ATP-dependent transport proteins facilitating the transport of psychotropic drugs, has drawn attention. This study delved into the relationship between antidepressant efficacy and seven single nucleotide polymorphisms of ABCB1 and ABCB6 genes. A total of 549 depressed patients participated in the study, and all completed a 6-week course of antidepressant treatment. Cognitive function was assessed at baseline and post-treatment. Patients were categorized based on post-treatment HAMD-17 scores (with HAMD≤7 indicating remission), and comparisons were made between different groups in terms of allelic gene frequencies and genotypes. Logistic regression was used to explore the interaction between cognitive function and genotype on efficacy. Dual-luciferase reporter assays were performed to compare the regulatory effects of rs1109866 allele variants on the ABCB6 promoter. There were no notable differences in allelic gene frequencies and genotypes between the remission and non-remission groups. Nonetheless, a significant interaction was identified between the rs1109866 genotype and language fluency-related indicators concerning efficacy (p=0.029) before correction. The dual-luciferase reporter assays demonstrated markedly higher fluorescence intensity of rs1109866-C compared to that of rs1109866-T (p<0.001). Relying solely on genetic polymorphisms of ABC family genes as predictors of antidepressant treatment response may not be sufficient. However, the interaction between the rs1109866 and cognition plays a pivotal role. The potentially enhanced transcriptional activity of rs1109866-C might offer insight into its impact on antidepressant efficacy.

The LINC01094/miR-545-3p/SLC7A11 Signaling Axis Promotes the Development of Gastric Cancer by Regulating Cell Growth and Ferroptosis.

This study aimed to investigate the role and mechanism of action of LINC01094 in the development of gastric cancer (GC). The expression levels of LINC01094 in GC patients and healthy individuals were analyzed online using the Cancer Genome Atlas database. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses were performed to determine the expression of LINC01094/miR-545-3p/SLC7A11 in GC tissues and cells. Functional experiments (MTT assay, colony formation assay, and flow cytometry) were conducted to assess the effect of LINC01094 and miR-545-3p on cell proliferation, viability, apoptosis, cell cycle, and reactive oxygen species. Correlations between LINC01094 and miR-545-3p, as well as SLC7A11, were analyzed and validated using the dual-luciferase reporter assay and RNA immunoprecipitation. The levels of Fe2+, malondialdehyde, and glutathione in the cells were measured biochemically, and the protein expression levels of Bcl-2, cleaved caspase3, Cyclin D1, and p21 were detected by Western blotting. LINC01094 was significantly upregulated in the GC tissues and cells with a targeting relationship with miR-545-3p; the expression levels of LINC01094 and miR-545-3p were negatively correlated. Knockdown of LINC01094 notably inhibited the proliferation and viability of GC cells and promoted cell ferroptosis, which, however, was abrogated by the silencing of miR-545-3p. These findings indicate that miR-545-3p could target and positively correlate with SLC7A11 expression. Additionally, LINC01094 could promote GC cell progression and affect cellular ferroptosis by regulating the miR-545-3p/SLC7A11 signaling axis.

DDX3 participates in miRNA biogenesis and RNA interference through translational control of PACT and interaction with AGO2.

DDX3 is a DEAD-box RNA helicase that plays multiple roles in RNA metabolism, including translation. We previously reported that DDX3 is required for translation of PACT, a binding partner of Dicer, suggesting a role for DDX3 in microRNA (miRNA) biogenesis and RNA interference (RNAi). Emerging evidence suggests that DDX3 plays a vital role in tumorigenesis and cancer progression, however, its underlying mechanism is still not fully understood. Here, we showed that the control of PACT by DDX3 is conserved in human cells and Caenorhabditis elegans. Using a miRNA microarray, we found that DDX3 regulates the expression of a small subset of cancer-related miRNAs. These oncogenic miRNAs were down-regulated by knockdown of DDX3 or PACT and up-regulated by overexpression of DDX3 or PACT in HEK293Tcells. Similar results were obtained in human cancer HCT116 and HeLa cells. Dual luciferase reporter assay showed that DDX3 and PACT are required for short hairpin RNA (shRNA)-induced RNAi. We also performed co-immunoprecipitation to confirm the interaction between DDX3 and AGO2, a significant component of the RNA-induced silencing complex, supporting a role for DDX3 in the RNAi pathway. We further examined the effects of DDX3 and PACT on cell proliferation, and stable overexpression of DDX3 in HEK293 cells results in loss of contact inhibition of cell growth. Hence, we propose that DDX3 may participate in cancer development by regulating the RNAi pathway.

FTO suppresses cardiac fibrosis after myocardial infarction via m6A-mediated epigenetic modification of EPRS

BackgroundCardiac fibrosis is common in myocardial infarction (MI), leading to progressive cardiac dysfunction. Studies suggested that the abnormal N6-methyladenosine (m6A) modification induced by fat mass and obesity protein (FTO) is vital in MI. However, the effects of FTO on post-infarction cardiac fibrosis have not been detected.MethodsWestern blot and quantitative real-time PCR were performed to detect the expression of FTO in the fibrotic tissue of rats. The functions of FTO on collagen biosynthesis were analyzed in vitro and in vivo. The underlying targets of FTO were selected through RNA-seq with m6A-seq. The following dual luciferase reporter assay and RNA stability assay were conducted to investigate the mechanisms of FTO-mediated m6A regulation.ResultsThe expression of FTO was decreased in the fibrotic tissue of post-infarction rats. The HIF-1 signal pathway was enriched after MI. HIF-1α could bind to the promoter of FTO and inhibit its expression. Functionally, FTO inhibited collagen synthesis after MI in vitro and in vivo. Mechanistically, EPRS was selected as the underlying target of FTO-induced m6A regulation. IGF2BP3 recognized and bound to the m6A sites of EPRS mRNA, which improved its stability. EPRS was required for cardiac fibrosis induced by FTO silencing.ConclusionsFTO, identified as a cardioprotective factor, suppressed collagen synthesis in post-infarction cardiac fibrosis via m6A modification, which provided a new therapeutic strategy for cardiac fibrosis.

LINC01614 Promotes Oral Squamous Cell Carcinoma by Regulating FOXC1.

Background: Long non-coding RNAs (lncRNAs) are pivotal mediators during the development of carcinomas; however, it remains to be investigated whether lncRNAs are implicated in oral squamous cell carcinoma (OSCC). Methods: In this study, quantitative real-time PCR was conducted for detecting the expression of LINC01614 in OSCC cell lines. The biological functions of LINC01614 were assessed by loss- and gain-of-function experiments conducted both in vivo and in vitro. Cellular proliferation, migration, and invasion were investigated herein, and dual luciferase reporter assays were additionally performed to explore the relationships among LINC01614, miR-138-5p, and Forkhead box C1 (FOXC1). Results: The research presented herein revealed that OSCC cells express high levels of LINC01614. Functional experiments employing cellular and animal models demonstrated that LINC01614 knockdown repressed the malignant phenotypes of OSCC cells, including their growth, invasiveness, and migration. Further investigation revealed that LINC01614 absorbs miR-138-5p miRNA by functioning as a competing endogenous RNA to downregulate the abundance of FOXC1. Conclusions: The findings revealed that LINC01614 contributes to the progression of OSCC by targeting the FOXC1 signaling pathway. The study provides insights into a novel mechanistic process to regulate the development of OSCC, and established a possible target for the therapeutic management of OSCC.

A positive feedback loop of SRSF9/USP22/ZEB1 promotes the progression of ovarian cancer

ABSTRACT Ovarian cancer (OC) is recognized as the most lethal type of gynecological malignancy, making treatment options challenging. Discovering novel therapeutic targets will benefit OC patients. This study aimed to reveal the mechanism by which SRSF9 regulates OC progression. Cell proliferation was determined via CCK-8 assays, whereas cell migration and invasion were monitored via Transwell assays. Western blotting and qPCR assays were used to detect protein and mRNA alterations. RNA pull-down, RNA immunoprecipitation (RIP), and actinomycin D experiments were performed to investigate the relationships between SRSF9 and USP22. Co-IP was used to validate the interaction between USP22 and ZEB1. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were used to verify the regulatory effect of ZEB1 on the transcription of SRSF9. Subcutaneous xenograft models were established to evaluate the impact of SRSF9 on tumor development. Knockdown of SRSF9 significantly suppressed the proliferation, invasion, migration, tumorigenicity, and epithelial‒mesenchymal transition (EMT) of OC cells. SRSF9 can bind to USP22 mRNA, increasing its stability. Moreover, the overexpression of USP22 reversed the impact of SRSF9 silencing on malignant phenotypes. USP22 can mediate the deubiquitination of ZEB1, thereby enhancing the progression of OC. Furthermore, ZEB1 upregulated SRSF9 expression through transcriptional activation, thus establishing a positive feedback loop. SRSF9 enhanced the malignant characteristics of OC through a positive feedback loop of SRSF9/USP22/ZEB1. This functional circuit may help in the development of novel therapeutic approaches for treating OC.