MicroRNA-92b targets tumor suppressor gene FBXW7 in glioblastoma

IntroductionGlioblastoma (GBM) is a highly aggressive and lethal primary brain tumor. Despite limited treatment options, the overall survival of GBM patients has shown minimal improvement over the past two decades. Factors such as delayed cancer diagnosis, tumor heterogeneity, cancer stem cell survival, infiltrative nature of GBM cells, metabolic reprogramming, and development of therapy resistance contribute to treatment failure. To address these challenges, multitargeted therapies are urgently needed for improved GBM treatment outcomes. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. Dysregulated miRNAs have been identified in GBM, playing roles in tumor initiation, progression, and maintenance. Among these miRNAs, miR-92b (miRNA-92b-3p) has been found to be overexpressed in various cancers, including GBM. However, the specific target genes of miR-92b and its therapeutic potential in GBM remain poorly explored.MethodsSamples encompassed T98G, U87, and A172 human GBM cell lines, GBM tumors from Puerto Rican patients, and murine tumors. In-situ hybridization (ISH) assessed miR-92b expression in patient tumors. Transient and stable transfections modified miR-92b levels in GBM cell lines. Real-time PCR gauged gene expressions. Caspase 3 and Trypan Blue assays evaluated apoptosis and viability. Bioinformatics tools (TargetScanHuman 8.0, miRDB, Diana tools, miRWalk) predicted targets. Luciferase assays and Western Blots validated miRNA-target interactions. A subcutaneous GBM Xenograft mouse model received intraperitoneal NC-OMIs or miR92b-OMIs encapsulated in liposomes, three-times per week for two weeks. Analysis utilized GraphPad Prism 8; statistical significance was assessed using 2-tailed, unpaired Student’s t-test and two-way ANOVA as required.ResultsThis study investigated the expression of miR-92b in GBM tumors compared to normal brain tissue samples, revealing a significant upregulation. Inhibition of miR-92b using oligonucleotide microRNA inhibitors (OMIs) suppressed GBM cell growth, migration, and induced apoptosis, while ectopic expression of miR-92b yielded opposite effects. Systemic administration of liposomal-miR92b-OMIs in GBM xenograft mice resulted in reductions in tumor volume and weight. Subsequent experiments identified F-Box and WD Repeat Domain Containing 7 (FBXW7) as a direct target gene of miR-92b in GBM cells.DiscussionFBXW7 acts as a tumor suppressor gene in various cancer types, and analysis of patient data demonstrated that GBM patients with higher FBXW7 mRNA levels had significantly better overall survival compared to those with lower levels. Taken together, our findings suggest that the dysregulated expression of miR-92b in GBM contributes to tumor progression by targeting FBXW7. These results highlight the potential of miR-92b as a therapeutic target for GBM. Further exploration and development of miR-92b-targeted therapies may offer a novel approach to improve treatment outcomes in GBM patients

Increased Expression of the RBPMS Splice Variants Inhibits Cell Proliferation in Ovarian Cancer Cells

RNA-Binding Protein with Multiple Splicing (RBPMS) is a member of family proteins that bind to nascent RNA transcripts and regulate their splicing, localization, and stability. Evidence indicates that RBPMS controls the activity of transcription factors associated with cell growth and proliferation, including AP-1 and Smads. Three major RBPMS protein splice variants (RBPMSA, RBPMSB, and RBPMSC) have been described in the literature. We previously reported that reduced RBPMS levels decreased the sensitivity of ovarian cancer cells to cisplatin treatment. However, little is known about the biological role of the RBPMS splice variants in ovarian cancer cells. We performed RT-PCR and Western blots and observed that both RBPMSA and RBPMSC are reduced at the mRNA and protein levels in cisplatin resistant as compared with cisplatin sensitive ovarian cancer cells. The mRNA and protein levels of RBPMSB were not detectable in any of the ovarian cancer cells tested. To better understand the biological role of each RBPMSA and RBPMSC, we transfected these two splice variants in the A2780CP20 and OVCAR3CIS cisplatin resistant ovarian cancer cells and performed cell proliferation, cell migration, and invasion assays. Compared with control clones, a significant reduction in the number of colonies, colony size, cell migration, and invasion was observed with RBPMSA and RBPMSC overexpressed cells. Moreover, A2780CP20-RBPMSA and A2780CP20-RBPMSC clones showed reduced senescence-associated β-galactosidase (β-Gal)-levels when compared with control clones. A2780CP20-RBPMSA clones were more sensitive to cisplatin treatment as compared with A2780CP20-RBPMSC clones. The A2780CP20-RBPMSA and A2780CP20-RBPMSC clones subcutaneously injected into athymic nude mice formed smaller tumors as compared with A2780CP20-EV control group. Additionally, immunohistochemical analysis showed lower proliferation (Ki67) and angiogenesis (CD31) staining in tissue sections of A2780CP20-RBPMSA and A2780CP20-RBPMSC tumors compared with controls. RNAseq studies revealed many common RNA transcripts altered in A2780CP20-RBPMSA and A2780CP20-RBPMSC clones. Unique RNA transcripts deregulated by each RBPMS variant were also observed. Kaplan–Meier (KM) plotter database information identified clinically relevant RBPMSA and RBPMSC downstream effectors. These studies suggest that increased levels of RBPMSA and RBPMSC reduce cell proliferation in ovarian cancer cells. However, only RBPMSA expression levels were associated with the sensitivity of ovarian cancer cells to cisplatin treatment

Dental Sleep Medicine among dental practitioners: Preliminary findings from the National Dental Practice-Based Research Network.

OBJECTIVES/INTRODUCTION: Dental sleep medicine (DSM) focuses on oral appliance therapy (OAT) to manage sleep-disordered breathing (SDB), including obstructive sleep apnea (OSA). This brief poll aimed to assess the interest and practices of dental practitioners in DSM. To this day lack of data exists regarding how DSM practices function in the clinical setting. Therefore, identifying knowledge gaps in DSM among dental practitioners may improve patient outcomes. METHODS: A preliminary brief questionnaire ("Quick Poll") on DSM was conducted through the National Dental Practice-Based Research Network (Network) members (n=311). The poll contained five questions about DSM. RESULTS: Results showed that 66% of practitioners have involvement in DSM patient care. A total of 44% of practitioners who answered the Quick Poll do not screen for snoring or SDBs. About 40% of respondents are either interested in continuing education courses on the topic or had already taken multiple courses on DSM. The top three topics of DSM research of interest to practitioners were various DSM practice models, response to OAT, and compliance with OAT. CONCLUSION: Network dental practitioner respondents face challenges regarding the treatment of SDBs. Despite these challenges, most practitioners are interested in engaging in DSM. Based on these preliminary findings, there are informational needs regarding the current state of clinical care, side effects of OAT, choice of OAT, titration protocols, and patient outcomes

Targeting MicroRNA-143 Leads to Inhibition of Glioblastoma Tumor Progression

Glioblastoma (GBM) is the most common and aggressive of all brain tumors, with a median survival of only 14 months after initial diagnosis. Novel therapeutic approaches are an unmet need for GBM treatment. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level. Several dysregulated miRNAs have been identified in all cancer types including GBM. In this study, we aimed to uncover the role of miR-143 in GBM cell lines, patient samples, and mouse models. Quantitative real-time RT-PCR of RNA extracted from formalin-fixed paraffin-embedded (FFPE) samples showed that the relative expression of miR-143 was higher in GBM patients compared to control individuals. Transient transfection of GBM cells with a miR-143 oligonucleotide inhibitor (miR-143-inh) resulted in reduced cell proliferation, increased apoptosis, and cell cycle arrest. SLC30A8, a glucose metabolism-related protein, was identified as a direct target of miR-143 in GBM cells. Moreover, multiple injections of GBM tumor-bearing mice with a miR-143-inh-liposomal formulation significantly reduced tumor growth compared to control mice. The reduced in vitro cell growth and in vivo tumor growth following miRNA-143 inhibition suggests that miR-143 is a potential therapeutic target for GBM therapy

Brain Targeted Gold Liposomes Improve RNAi Delivery for Glioblastoma.

INTRODUCTION: Glioblastoma (GBM) is the most common and lethal of the central nervous system (CNS) malignancies. The initiation, progression, and infiltration ability of GBMs are attributed in part to the dysregulation of microRNAs (miRNAs). Thus, targeting dysregulated miRNAs with RNA oligonucleotides (RNA interference, RNAi) has been proposed for GBM treatment. Despite promising results in the laboratory, RNA oligonucleotides have clinical limitations that include poor RNA stability and off-target effects. RNAi therapies against GBM confront an additional obstacle, as they need to cross the blood-brain barrier (BBB). METHODS: Here, we developed gold-liposome nanoparticles conjugated with the brain targeting peptides apolipoprotein E (ApoE) and rabies virus glycoprotein (RVG). First, we functionalized gold nanoparticles with oligonucleotide miRNA inhibitors (OMIs), creating spherical nucleic acids (SNAs). Next, we encapsulated SNAs into ApoE, or RVG-conjugated liposomes, to obtain SNA-Liposome-ApoE and SNA-Liposome-RVG, respectively. We characterized each nanoparticle in terms of their size, charge, encapsulation efficiency, and delivery efficiency into U87 GBM cells in vitro. Then, they were administered intravenously (iv) in GBM syngeneic mice to evaluate their delivery efficiency to brain tumor tissue. RESULTS: SNA-Liposomes of about 30-50 nm in diameter internalized U87 GBM cells and inhibited the expression of miRNA-92b, an aberrantly overexpressed miRNA in GBM cell lines and GBM tumors. Conjugating SNA-Liposomes with ApoE or RVG peptides increased their systemic delivery to the brain tumors of GBM syngeneic mice. SNA-Liposome-ApoE demonstrated to accumulate at higher extension in brain tumor tissues, when compared with non-treated controls, SNA-Liposomes, or SNA-Liposome-RVG. DISCUSSION: SNA-Liposome-ApoE has the potential to advance the translation of miRNA-based therapies for GBM as well as other CNS disorders

Reduced RBPMS Levels Promote Cell Proliferation and Decrease Cisplatin Sensitivity in Ovarian Cancer Cells

Worldwide, the number of cancer-related deaths continues to increase due to the ability of cancer cells to become chemotherapy-resistant and metastasize. For women with ovarian cancer, a staggering 70% will become resistant to the front-line therapy, cisplatin. Although many mechanisms of cisplatin resistance have been proposed, the key mechanisms of such resistance remain elusive. The RNA binding protein with multiple splicing (RBPMS) binds to nascent RNA transcripts and regulates splicing, transport, localization, and stability. Evidence indicates that RBPMS also binds to protein members of the AP-1 transcription factor complex repressing its activity. Until now, little has been known about the biological function of RBPMS in ovarian cancer. Accordingly, we interrogated available Internet databases and found that ovarian cancer patients with high RBPMS levels live longer compared to patients with low RBPMS levels. Similarly, immunohistochemical (IHC) analysis in a tissue array of ovarian cancer patient samples showed that serous ovarian cancer tissues showed weaker RBPMS staining when compared with normal ovarian tissues. We generated clustered regularly interspaced short palindromic repeats (CRISPR)-mediated RBPMS knockout vectors that were stably transfected in the high-grade serous ovarian cancer cell line, OVCAR3. The knockout of RBPMS in these cells was confirmed via bioinformatics analysis, real-time PCR, and Western blot analysis. We found that the RBPMS knockout clones grew faster and had increased invasiveness than the control CRISPR clones. RBPMS knockout also reduced the sensitivity of the OVCAR3 cells to cisplatin treatment. Moreover, β-galactosidase (β-Gal) measurements showed that RBPMS knockdown induced senescence in ovarian cancer cells. We performed RNAseq in the RBPMS knockout clones and identified several downstream-RBPMS transcripts, including non-coding RNAs (ncRNAs) and protein-coding genes associated with alteration of the tumor microenvironment as well as those with oncogenic or tumor suppressor capabilities. Moreover, proteomic studies confirmed that RBPMS regulates the expression of proteins involved in cell detoxification, RNA processing, and cytoskeleton network and cell integrity. Interrogation of the Kaplan–Meier (KM) plotter database identified multiple downstream-RBPMS effectors that could be used as prognostic and response-to-therapy biomarkers in ovarian cancer. These studies suggest that RBPMS acts as a tumor suppressor gene and that lower levels of RBPMS promote the cisplatin resistance of ovarian cancer cells

Presentation_1_MicroRNA-92b targets tumor suppressor gene FBXW7 in glioblastoma.pptx

IntroductionGlioblastoma (GBM) is a highly aggressive and lethal primary brain tumor. Despite limited treatment options, the overall survival of GBM patients has shown minimal improvement over the past two decades. Factors such as delayed cancer diagnosis, tumor heterogeneity, cancer stem cell survival, infiltrative nature of GBM cells, metabolic reprogramming, and development of therapy resistance contribute to treatment failure. To address these challenges, multitargeted therapies are urgently needed for improved GBM treatment outcomes. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. Dysregulated miRNAs have been identified in GBM, playing roles in tumor initiation, progression, and maintenance. Among these miRNAs, miR-92b (miRNA-92b-3p) has been found to be overexpressed in various cancers, including GBM. However, the specific target genes of miR-92b and its therapeutic potential in GBM remain poorly explored.MethodsSamples encompassed T98G, U87, and A172 human GBM cell lines, GBM tumors from Puerto Rican patients, and murine tumors. In-situ hybridization (ISH) assessed miR-92b expression in patient tumors. Transient and stable transfections modified miR-92b levels in GBM cell lines. Real-time PCR gauged gene expressions. Caspase 3 and Trypan Blue assays evaluated apoptosis and viability. Bioinformatics tools (TargetScanHuman 8.0, miRDB, Diana tools, miRWalk) predicted targets. Luciferase assays and Western Blots validated miRNA-target interactions. A subcutaneous GBM Xenograft mouse model received intraperitoneal NC-OMIs or miR92b-OMIs encapsulated in liposomes, three-times per week for two weeks. Analysis utilized GraphPad Prism 8; statistical significance was assessed using 2-tailed, unpaired Student’s t-test and two-way ANOVA as required.ResultsThis study investigated the expression of miR-92b in GBM tumors compared to normal brain tissue samples, revealing a significant upregulation. Inhibition of miR-92b using oligonucleotide microRNA inhibitors (OMIs) suppressed GBM cell growth, migration, and induced apoptosis, while ectopic expression of miR-92b yielded opposite effects. Systemic administration of liposomal-miR92b-OMIs in GBM xenograft mice resulted in reductions in tumor volume and weight. Subsequent experiments identified F-Box and WD Repeat Domain Containing 7 (FBXW7) as a direct target gene of miR-92b in GBM cells.DiscussionFBXW7 acts as a tumor suppressor gene in various cancer types, and analysis of patient data demonstrated that GBM patients with higher FBXW7 mRNA levels had significantly better overall survival compared to those with lower levels. Taken together, our findings suggest that the dysregulated expression of miR-92b in GBM contributes to tumor progression by targeting FBXW7. These results highlight the potential of miR-92b as a therapeutic target for GBM. Further exploration and development of miR-92b-targeted therapies may offer a novel approach to improve treatment outcomes in GBM patients.</p