Plasma EV-miRNAs as Potential Biomarkers of COVID-19 Vaccine Immune Response in Cancer Patients.

Cancer patients, prone to severe COVID-19, face immune challenges due to their disease and treatments. Identifying biomarkers, particularly extracellular vesicle (EV)-derived microRNAs (miRNAs), is vital for comprehending their response to COVID-19 vaccination. Therefore, this study aimed to investigate specific EV-miRNAs in the plasma of cancer patients under active treatment who received the COVID-19 booster vaccine. The selected miRNAs (EV-hsa-miR-7-5p, EV-hsa-miR-15b-5p, EV-hsa-miR-24-3p, EV-hsa-miR-145- 5p, and EV-hsa-miR-223-3p) are involved in regulating SARS-CoV-2 spike protein and cytokine release, making them potential biomarkers for vaccination response. The study involved 54 cancer patients. Plasma and serum samples were collected at pre-boost vaccination, and at 3 and 6 months post-boost vaccination. Anti-spike antibody levels were measured. Additionally, RNA was extracted from EVs isolated from plasma and the expression levels of miRNAs were assessed. The results showed a significantly positive antibody response after COVID-19 boost vaccination. The expression levels of EV-hsa-miR-7-5p, EV-hsa-miR-15b-5p, EV-hsa-miR-24-3p, and EV-hsa-miR-223-3p increased significantly after 6 months of COVID-19 booster vaccination. Interestingly, an increased expression of certain EV-hsa-miRNAs was positively correlated. Bioinformatic analysis revealed that these correlated miRNAs play a critical role in regulating the targets present in antiviral responses and cytokine production. These findings suggest that EV-hsa-miR-15b-5p, EV-hsa-miR-24-3p, and EV-hsa-miR-223-3p may be crucial in immune response induced by mRNA vaccines.

Expression of hsa-miRNA-15b, -99b, -181a and Their Relationship to Angiogenesis in Renal Cell Carcinoma.

BACKGROUND: MicroRNAs (miRNAs) play a regulatory role in various human cancers. The roles of hsa-miR-15a-5p, hsa-miR-99b-5p, and hsa-miR-181a-5p have not been fully explored in the angiogenesis of renal cell carcinoma (RCC). AIMS: The present study aimed to evaluate the expression of these miRNAs in tumorous and adjacent healthy tissues of RCC. METHODS: Paired tumorous and adjacent normal kidney tissues from 20 patients were studied. The expression levels of hsa-miR-15b-5p, hsa-miR-99b-5p, and hsa-miR-181a-5p were quantified by TaqMan miRNA Assays. Putative targets were analyzed by qRT-PCR. RESULTS: Significant downregulation of all three miRNAs investigated was observed in tumorous samples compared to adjacent normal kidney tissues. Spearman analysis showed a negative correlation between the expression levels of miRNAs and the pathological grades of the patients. Increased expression of vascular endothelial growth factor-A (VEGF-A) and hypoxia-inducible factor-1α (HIF-1α), a tissue inhibitor of metalloproteinases-1 (TIMP-1), was observed in tumorous samples compared to adjacent normal tissues. Depletion of tissue inhibitors of metalloproteinase-2 (TIMP-2) and metalloproteinase-2 (MMP-2) was detected compared to normal adjacent tissues. The examined miRNAs might function as contributing factors to renal carcinogenesis. However, more prospective studies are warranted to evaluate the potential role of miRNAs in RCC angiogenesis.

Delivery of miR-15b-5p via magnetic nanoparticle-enhanced bone marrow mesenchymal stem cell-derived extracellular vesicles mitigates diabetic osteoporosis by targeting GFAP.

Diabetic osteoporosis (DO) presents significant clinical challenges. This study aimed to investigate the potential of magnetic nanoparticle-enhanced extracellular vesicles (GMNPE-EVs) derived from bone marrow mesenchymal stem cells (BMSCs) to deliver miR-15b-5p, thereby targeting and downregulating glial fibrillary acidic protein (GFAP) expression in rat DO models. Data was sourced from DO-related RNA-seq datasets combined with GEO and GeneCards databases. Rat primary BMSCs, bone marrow-derived macrophages (BMMs), and osteoclasts were isolated and cultured. EVs were separated, and GMNPE targeting EVs were synthesized. Bioinformatic analysis revealed a high GFAP expression in DO-related RNA-seq and GSE26168 datasets for disease models. Experimental results confirmed elevated GFAP in rat DO bone tissues, promoting osteoclast differentiation. miR-15b-5p was identified as a GFAP inhibitor, but was significantly downregulated in DO and enriched in BMSC-derived EVs. In vitro experiments showed that GMNPE-EVs could transfer miR-15b-5p to osteoclasts, downregulating GFAP and inhibiting osteoclast differentiation. In vivo tests confirmed the therapeutic potential of this approach in alleviating rat DO. Collectively, GMNPE-EVs can effectively deliver miR-15b-5p to osteoclasts, downregulating GFAP expression, and hence, offering a therapeutic strategy for rat DO.

Linc00662 sponges miR-15b-5p to promote hypopharyngeal squamous cell carcinoma progression by facilitating cancer stem cell-like phenotypes.

Background: Long non-coding RNAs (lncRNAs) are associated with multiple head and neck tumors and play important roles in cancer. This study explored the molecular mechanism of Linc00662 in hypopharyngeal squamous cell carcinoma (HSCC). Methods: Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect gene expression in HSCC tissues. The viability and proliferation of tumor cells were measured using CCK-8 assays. HSCC cell apoptosis was measured using flow cytometry and western blotting. Cell stemness was examined using the sphere formation assay. A xenograft tumor model was established to investigate the role of Linc00662 in vivo. Results: The expression level of Linc00662 in HSCC tissues was significantly higher than that in adjacent normal tissues. The expression of Linc00662 had no significant relationship with the tumor stage. Patients with high Linc00662 expression were found to have shorter overall survival than those with low Linc00662 expression. Linc00662 over-expression promoted cell viability and inhibited apoptosis. Using online databases and a dual luciferase reporter, miR-15b-5p was confirmed as a potential downstream sponge of Linc00662. Moreover, Linc00662 was negatively associated with miR-15b-5p in HSCC cells. Depletion of miR-15b-5p can reverse the function of Linc00662 in vivo and in vitro. Furthermore, Linc00662 promotes tumor growth, which was abolished by miR-15b-5p mimics. Importantly, the stemness of cancer stem cells was mediated by the Linc00662/miR-15b-5p axis. Conclusion: Patients with HSCC with high Linc00662 showed poor prognosis and high Linc00662 induced stemness of tumor cells by targeting miR-15b-5p. Linc00662 may serve as a novel diagnostic and target marker for head and neck squamous cell carcinoma.

MicroRNAs predict early complications of autologous hematopoietic stem cell transplantation.

Autologous hematopoietic stem cell transplantation (AHSCT) remains the most prevalent type of stem cell transplantation. In our study, we investigated the changes in circulating miRNAs in AHSCT recipients and their potential to predict early procedure-related complications. We collected serum samples from 77 patients, including 54 with multiple myeloma, at four key time points: before AHSCT, on the day of transplantation (day 0), and at days + 7 and + 14 post-transplantation. Through serum miRNA-seq analysis, we identified altered expression patterns and miRNAs associated with the AHSCT procedure. Validation using qPCR confirmed deviations in the levels of miRNAs at the beginning of the procedure in patients who subsequently developed bacteremia: hsa-miR-223-3p and hsa-miR-15b-5p exhibited decreased expression, while hsa-miR-126-5p had increased level. Then, a neural network model was constructed to use miRNA levels for the prediction of bacteremia. The model achieved an accuracy of 93.33% (95%CI: 68.05-99.83%), with a sensitivity of 100% (95%CI: 67.81-100.00%) and specificity of 90.91% (95%CI: 58.72-99.77%) in predicting bacteremia with mean of 6.5 ± 3.2 days before occurrence. In addition, we showed unique patterns of miRNA expression in patients experiencing platelet engraftment delay which involved the downregulation of hsa-let-7f-5p and upregulation of hsa-miR-96-5p; and neutrophil engraftment delay which was associated with decreased levels of hsa-miR-125a-5p and hsa-miR-15b-5p. Our findings highlight the significant alterations in serum miRNA levels during AHSCT and suggest the clinical utility of miRNA expression patterns as potential biomarkers that could be harnessed to improve patient outcomes, particularly by predicting the risk of bacteremia during AHSCT.

miR-15b-5p promotes HgCl2-induced chicken embryo kidney cells ferroptosis by targeting ß-TrCP-mediated ATF4 ubiquitin degradation.

Mercuric chloride (HgCl2), a widespread environmental pollutant, induces ferroptosis in chicken embryonic kidney (CEK) cells. Whereas activating transcription factor 4 (ATF4), a critical mediator of oxidative homeostasis, plays a dual role in ferroptosis, but its precise mechanisms in HgCl2-induced ferroptosis remain elusive. This study aims to investigate the function and molecular mechanism of ATF4 in HgCl2-induced ferroptosis. Our results revealed that ATF4 was downregulated during HgCl2-induced ferroptosis in CEK cells. Surprisingly, HgCl2 exposure has no significant impact on ATF4 mRNA level. Further investigation indicated that HgCl2 enhanced the expression of the E3 ligase beta-transducin repeat-containing protein (ß-TrCP) and increased ATF4 ubiquitination. Subsequent findings identified that miR-15b-5p as an upstream modulator of ß-TrCP, with miR-15b-5p downregulation observed in HgCl2-exposed CEK cells. Importantly, miR-15b-5p mimics suppressed ß-TrCP expression and reversed HgCl2-induced cellular ferroptosis. Mechanistically, HgCl2 inhibited miR-15b-5p, and promoted ß-TrCP-mediated ubiquitin degradation of ATF4, thereby inhibited the expression of antioxidant-related target genes and promoted ferroptosis. In conclusion, our study highlighted the crucial role of the miR-15b-5p/ß-TrCP/ATF4 axis in HgCl2-induced nephrotoxicity, offering a new therapeutic target for understanding the mechanism of HgCl2 nephrotoxicity.

LncRNA Malat1 regulates iPSC-derived ß-cell differentiation by targeting the miR-15b-5p/Ihh axis.

BACKGROUND: Differentiation of induced pluripotent stem cells (iPSCs)-derived ß-like cells is a novel strategy for treatment of type 1 diabetes. Elucidation of the regulatory mechanisms of long noncoding RNAs (lncRNAs) in ß-like cells derived from iPSCs is important for understanding the development of the pancreas and pancreatic ß-cells and may improve the quality of ß-like cells for stem cell therapy. METHODS: ß-like cells were derived from iPSCs in a three-step protocol. RNA sequencing and bioinformatics analysis were carried out to screen the differentially expressed lncRNAs and identify the putative target genes separately. LncRNA Malat1 was chosen for further research. Series of loss and gain of functions experiments were performed to study the biological function of LncRNA Malat1. Quantitative real-time PCR (qRT-PCR), Western blot (WB) analysis and immunofluorescence (IF) staining were carried out to separately detect the functions of pancreatic ß-cells at the mRNA and protein levels. Cytoplasmic and nuclear RNA fractionation and fluorescence in situ hybridization (FISH) were used to determine the subcellar location of lncRNA Malat1 in ß-like cells. Enzyme-linked immunosorbent assays (ELISAs) were performed to examine the differentiation and insulin secretion of ß-like cells after stimulation with different glucose concentrations. Structural interactions between lncRNA Malat1 and miR-15b-5p and between miR-15b-5p/Ihh were detected by dual luciferase reporter assays (LRAs). RESULTS: We found that the expression of lncRNA Malat1 declined during differentiation, and overexpression (OE) of lncRNA Malat1 notably impaired the differentiation and maturation of ß-like cells derived from iPSCs in vitro and in vivo. Most importantly, lncRNA Malat1 could function as a competing endogenous RNA (ceRNA) of miR-15b-5p to regulate the expression of Ihh according to bioinformatics prediction, mechanistic analysis and downstream experiments. CONCLUSION: This study established an unreported regulatory network of lncRNA Malat1 and the miR-15b-5p/Ihh axis during the differentiation of iPSCs into ß-like cells. In addition to acting as an oncogene promoting tumorigenesis, lncRNA Malat1 may be an effective and novel target for treatment of diabetes in the future.

The regulatory role and mechanism of USP14 in endothelial cell pyroptosis induced by coronary heart disease.

OBJECTIVE: The present study probes into the role and mechanism of ubiquitin specific peptidase 14 (USP14) in coronary heart disease (CHD)-triggered endothelial cell pyroptosis. METHODS: An in vitro CHD model was established by inducing human coronary artery endothelial cells (HCAECs) with oxidized low-density lipoprotein (ox-LDL). HCAECs were transfected with si-USP14, followed by evaluation of cell viability by CCK-8 assay, detection of lactate dehydrogenase (LDH) activity by assay kit, detection of USP14, miR-15b-5p, NLRP3, GSDMD-N, and Cleaved-Caspase-1 expressions by qRT-PCR or Western blot, as well as IL-1ß and IL-18 concentrations by ELISA. Co-IP confirmed the binding between USP14 and NLRP3. The ubiquitination level of NLRP3 in cells was measured after protease inhibitor MG132 treatment. Dual-luciferase reporter assay verified the targeting relationship between miR-15b-5p and USP14. RESULTS: USP14 and NLRP3 were highly expressed but miR-15b-5p was poorly expressed in ox-LDL-exposed HCAECs. USP14 silencing strengthened the viability of ox-LDL-exposed HCAECs, reduced the intracellular LDH activity, and diminished the NLRP3, GSDMD-N, Cleaved-Caspase-1, IL-1ß, and IL-18 expressions. USP14 bound to NLRP3 protein and curbed its ubiquitination. Repression of NLRP3 ubiquitination counteracted the inhibitory effect of USP14 silencing on HCAEC pyroptosis. miR-15b-5p restrained USP14 transcription and protein expression. miR-15b-5p overexpression alleviated HCAEC pyroptosis by suppressing USP14/NLRP3. CONCLUSION: USP14 stabilizes NLRP3 protein expression through deubiquitination, thereby facilitating endothelial cell pyroptosis in CHD. miR-15b-5p restrains endothelial cell pyroptosis by targeting USP14 expression.

miR-15b-5p transcription mediated by CREB1 protects against inflammation and apoptosis in Parkinson disease models by inhibiting AXIN2 and activating Wnt/ß-catenin.

Parkinson disease (PD) is a major neurodegenerative disease that greatly undermines people's health and for which effective therapeutic strategies are currently limited. This study dissected the effects of expression changes of AXIN2, a modulator of the Wnt/beta-catenin signaling pathway, the transcription factor CREB1, and of the microRNA miR-15b-5p on apoptosis and the inflammatory response in a PD mouse model in vivo and in a cellular PD model in vitro. The analyses demonstrated low CREB1 and miR-15b-5p expression and high AXIN2 expression in both models. miR-15b-5p overexpression or AXIN2 knockdown alleviated the inflammatory response indicated by decreased levels of TNF-α, IL-6, and IL-1ß and apoptosis indicated by decreased levels of cleaved caspase-3 and Bax and elevated Bcl-2. Protection by miR-15b-5p upregulation was counteracted by the simultaneous overexpression of AXIN2. miR-15b-5p targeted AXIN2. CREB1 promoted miR-15b-5p expression, which activated the Wnt/ß-catenin pathway by inhibiting AXIN2. Collectively, the data indicate that transcriptional expression of miR-15b-5p can be promoted by CREB1 to inhibit AXIN2 and activate Wnt/ß-catenin, thereby reducing the inflammatory response and apoptosis in these PD models. These data suggest the CREB1/miR-15b-5p/AXIN2 axis is a potential therapeutic target in PD patients.

Increased Levels of miR-15b-5p and miR-20b-5p in Pancreatic Ductal Adenocarcinoma with Hepatic Metastases.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal forms of cancer. The symptoms appear in advanced stages, and diagnostic and prognostic tests for the early detection of PDAC and disease evolution are not available. The dysregulation of microRNAs (miRNAs) has been associated with cancer development and progression, and some miRNAs have been reported to promote specific metastasis. In this study we aimed to identify the miRNAs dysregulated in PDAC tumoral tissues and a subset of miRNAs associated with tumoral characteristics, mainly metastasis presence and site. For this, the expression of 84 miRNAs was evaluated by qPCR in 30 tumoral tissues and 16 samples of non-tumoral pancreatic tissues. The comparison revealed 32 dysregulated miRNAs (19 upregulated and 13 downregulated) in the PDAC group. Reactome pathway over-representation analysis revealed that these miRNAs are involved in several biological pathways, including "ESR-mediated signaling", "PIP3 activates AKT signaling", and "Regulation of PTEN", among others. Moreover, our study identified an upregulation of miR-15b-5p and miR-20b-5p in the tumoral tissues of patients with hepatic metastasis, outlining these miRNAs as potential markers for hepatic metastasis. No significant difference in miRNA expression was observed in relation to anatomic location, lymphovascular invasion, lung metastasis, and the presence of diabetes.

MiR-15b-5p inhibits castration-resistant growth of prostate cancer cells by targeting the muscarinic cholinergic receptor CHRM3.

Cholinergic receptor muscarinic 3 (CHRM3)-mediated focal adhesion kinase/YES-associated protein (YAP) signalling is essential for the growth of castration-resistant prostate cancer (CRPC) cells. Here, we evaluated the molecular mechanisms through which CHRM3 overexpression facilitates castration-resistant growth. Small RNA sequencing combined with in silico analyses revealed that CHRM3 was a putative target of miR-15b-5p. Notably, androgen deprivation suppressed miR-15b-5p expression and increased CHRM3 expression. Moreover, miR-15b-5p bound directly to CHRM3 and inhibited YAP activation induced by CHRM3 stimulation. Furthermore, miR-15b-5p abolished the growth of CRPC cells induced by CHRM3 stimulation. We conclude that the miR-15b-5p/CHRM3/YAP signalling axis promotes the castration-resistant growth of prostate cancer.

LncRNA PVT1 is a novel mediator promoting the angiogenesis response associated with collateral artery formation.

AIMS: Angiogenesis plays a key role in coronary collateral circulation (CCC), the compensatory formation of new blood vessels during chronic total coronary occlusion. This study aimed to determine whether plasmacytoma variant translocation 1 (PVT1), a long non-coding (lnc) RNA involved in tumor angiogenesis, plays a role in regulating angiogenesis during chronic coronary ischemia. MAIN METHODS: Patients with coronary artery disease, and ≥ 90% stenosis, were examined and divided into "Good" and "Poor" CCC groups based on Rentrop Cohen classification. RNA samples were obtained from all patients, as well as from oxygen and glucose-deprived (OGD) HUVECs. PVT1, miR-15b-5p and AKT3 levels were measured with RT-qPCR or Western blot, while HUVEC migration and angiogenesis were detected by, respectively, wound-healing and tube formation assays. Luciferase reporter assay confirmed direct PVT1-miR-15b-5p binding. KEY FINDINGS: Increased PVT1 was found in "Good CCC" patient plasma, along with being highly expressed among OGD HUVECs; PVT1 knockdown reduced HUVEC migration, tube formation, and pro-angiogenic factor expression. Conversely, OGD HUVECs had downregulated miR-15b-5p, and miR-15b-5p overexpression significantly depressed their angiogenic capabilities. These PVT1 knockdown- or miR-15b-5p overexpression-associated reductions in angiogenic effects were reversed by AKT3 overexpression. In vivo, neovascularization and functioning in both ischemic mice hind-limbs and infarcted myocardium injected with ADV-sh-PVT1 were reduced, which were ameliorated by concurrent antagomiR-15b-5p injections. SIGNIFICANCE: Circulating PVT1 may serve as a useful biomarker to distinguish between good versus poor CCC, as it is involved in orchestrating angiogenesis via the miR-15b-5p-AKT3 axis; it thus has potential as a target for treating ischemic disease.

A Comprehensive Review on Function of miR-15b-5p in Malignant and Non-Malignant Disorders.

miR-15b-5p is encoded by MIR15B gene. This gene is located on cytogenetic band 3q25.33. This miRNA participates in the pathogenesis of several cancers as well as non-malignant conditions, such as abdominal aortic aneurysm, Alzheimer's and Parkinson's diseases, cerebral ischemia reperfusion injury, coronary artery disease, dexamethasone induced steatosis, diabetic complications and doxorubicin-induced cardiotoxicity. In malignant conditions, both oncogenic and tumor suppressor impacts have been described for miR-15b-5p. Dysregulation of miR-15b-5p in clinical samples has been associated with poor outcome in different kinds of cancers. In this review, we discuss the role of miR-15b-5p in malignant and non-malignant conditions.

Downregulation of Kcnq1ot1 attenuates ß-cell proliferation and insulin secretion via the miR-15b-5p/Ccnd1 and Ccnd2 axis.

AIM: To examine the effect of lncRNA Kcnq1ot1 on pancreatic ß cells in the development of diabetes. METHODS: The expression levels of Kcnq1ot1 were detected in the islets of diabetes mouse models and the serum of patients with type 2 diabetes by qRT-PCR. CCK8, Ki67 staining, immunohistochemical analyses, glucose-stimulated insulin secretion and intraperitoneal glucose tolerance test were performed to detect the effect of Kcnq1ot1 on ß-cell proliferation and insulin secretion in vitro and in vivo. The relationship between Kcnq1ot1 and miR-15b-5p was predicted by bioinformatics prediction, which was confirmed by luciferase reporter assay. RESULTS: Kcnq1ot1 was more abundant in the pancreas. The expression of Kcnq1ot1 was decreased in the islets of db/db mice and diet-induced obese mice and in the serum of patients with type 2 diabetes. Silencing Kcnq1ot1 inhibited the ß-cell proliferation concomitant with a reduction in the levels of Ccnd1 and Ccnd2. Insulin synthesis and secretion were impaired, along with the decreased expression of Ins1, Ins2, and insulin-related transcription factors. Moreover, Kcnq1ot1 knockdown in vivo reduced glucose tolerance and decreased insulin secretion, consistent with the reduction in the relative islet area and Ki67-positive ß-cells detected by immunochemistry and immunofluorescence staining, respectively. Mechanistically, Kcnq1ot1 directly targeted miR-15b-5p which regulated ß-cell proliferation and insulin secretion through Ccnd1 and Ccnd2. Notably, the suppression of miR-15b-5p attenuated the inhibition of Min6 proliferation and insulin production induced by Kcnq1ot1 knockdown. CONCLUSION: Kcnq1ot1 regulated ß-cell proliferation and insulin secretion via the miR-15b-5p/Ccnd1 and Ccnd2 axis, which is worthy of further investigation considering its potential in diabetes treatment.

MicroRNA-15b-5p inhibits tumor necrosis factor alpha-induced proliferation, migration, and extracellular matrix production of airway smooth muscle cells via targeting yes-associated protein 1.

The excessive proliferation and the deposition of extracellular matrix (ECM) of airway smooth muscle (ASM) cells facilitates airway remodeling in asthma. This study explores how microRNA-15b-5p (miR-15b-5p) functions in modulating the proliferation, migration, inflammatory response, and ECM deposition of ASM cells. MiR-15b-5p and yes-associated protein 1 (YAP1) mRNA expression levels in tumor necrosis factor alpha (TNF-α)-induced ASM cells were, respectively, examined by real-time quantitative polymerase-chain reaction. Besides, the proliferative ability and migrative potential of ASM cells were examined by cell counting kit-8 assay, 5-bromo-2 '-deoxyuridine assay, and transwell assays, respectively. Interleukin-6 and interleukin-8 levels in ASM cells were detected by enzyme-linked immunosorbent assay. YAP1, collagen I, and collagen III expressions in ASM cells were detected by Western blot. With dual-luciferase reporter gene assay, the relations between miR-15b-5p and YAP1 3'UTR in ASM cells was examined. MiR-15b-5p expression level was reduced in ASM cells treated with TNF-α. MiR-15b-5p repressed TNF-α-initiated growth and migration of ASM cells and also suppressed IL-6 and IL-8 secretion, and inhibited collagen I and collagen III expressions in ASM cells. Furthermore, it was validated that YAP1 was a downstream target of miR-15b-5p in ASM cells. Notably, YAP1 overexpression attenuated the inhibitory effects of miR-15b-5p up-regulation on the proliferation, migration, and inflammatory response, as well as ECM deposition of TNF-α-induced ASM cells. In conclusion, miR-15b-5p/YAP1 axis modulates the growth, migration, inflammatory response, and ECM deposition of ASM cells, thus participating in the pathogenesis of asthma.

Treatment of Human Glioblastoma U251 Cells with Sulforaphane and a Peptide Nucleic Acid (PNA) Targeting miR-15b-5p: Synergistic Effects on Induction of Apoptosis.

Glioblastoma multiforme (GBM) is a lethal malignant tumor accounting for 42% of the tumors of the central nervous system, the median survival being 15 months. At present, no curative treatment is available for GBM and new drugs and therapeutic protocols are urgently needed. In this context, combined therapy appears to be a very interesting approach. The isothiocyanate sulforaphane (SFN) has been previously shown to induce apoptosis and inhibit the growth and invasion of GBM cells. On the other hand, the microRNA miR-15b is involved in invasiveness and proliferation in GBM and its inhibition is associated with the induction of apoptosis. On the basis of these observations, the objective of the present study was to determine whether a combined treatment using SFN and a peptide nucleic acid interfering with miR-15b-5p (PNA-a15b) might be proposed for increasing the pro-apoptotic effects of the single agents. To verify this hypothesis, we have treated GMB U251 cells with SFN alone, PNA-a15b alone or their combination. The cell viability, apoptosis and combination index were, respectively, analyzed by calcein staining, annexin-V and caspase-3/7 assays, and RT-qPCR for genes involved in apoptosis. The efficacy of the PNA-a15b determined the miR-15b-5p content analyzed by RT-qPCR. The results obtained indicate that SFN and PNA-a15b synergistically act in inducing the apoptosis of U251 cells. Therefore, the PNA-a15b might be proposed in a "combo-therapy" associated with SFN. Overall, this study suggests the feasibility of using combined treatments based on PNAs targeting miRNA involved in GBM and nutraceuticals able to stimulate apoptosis.

CERS6-AS1 contributes to the malignant phenotypes of colorectal cancer cells by interacting with miR-15b-5p to regulate SPTBN2.

Accumulating evidence indicates that long noncoding RNAs (lncRNAs) act as tumor promoters or suppressors in various types of cancer. Previous investigations suggest that ceramide synthase 6 (CERS6) antisense RNA 1 (CERS6-AS1) acts as an oncogene in breast cancer; however, its role in colorectal cancer is unknown. This study aimed to explore the molecular mechanism of CERS6-AS1 in colorectal cancer. Gene expression in colorectal cancer was examined using reverse transcription-quantitative polymerase chain reaction and western blot analyses. The viability and proliferation of colorectal cancer cells were measured by Cell Counting Kit-8 assays and colony formation assays. The migratory and invasive capacities of the colorectal cancer cells were assessed by Transwell assay. Cell stemness was examined by sphere-formation assay. Mechanistically, RNA pull-down assays, RNA immunoprecipitation assays, and luciferase reporter assays were performed to explore the relationship among CERS6-AS1, miR-15b-5p and spectrin beta, non-erythrocytic 2 (SPTBN2). Moreover, a xenograft tumor model was established to investigate the role of CERS6-AS1 in vivo. We found that CERS6-AS1 and SPTBN2 were highly expressed in colorectal cancer tissues and cells. CERS6-AS1 depletion inhibited cell viability, proliferation, migration, and invasion; the epithelial-mesenchymal transition process and stemness. It suppressed xenograft tumor growth in colorectal cancer. Moreover, SPTBN2 levels were positively regulated by CERS6-AS1 and negatively regulated by miR-15b-5p in colorectal cancer cells. Rescue assays revealed that SPTBN2 reversed the inhibitory effect of CERS6-AS1 deficiency on the malignant behaviors of colorectal cancer cells. Overall, the lncRNA CERS6-AS1 facilitates malignant phenotypes of colorectal cancer cells by targeting miR-15b-5p to upregulate SPTBN2.

Circular RNA circCHFR downregulation protects against oxidized low-density lipoprotein-induced endothelial injury via regulation of microRNA-15b-5p/growth arrest and DNA damage inducible gamma.

Atherosclerosis is the leading cause of coronary heart disease. In recent years, circ_0029589 (circCHFR) has been found to be associated with atherosclerosis development. However, the molecular mechanism of circCHFR action in atherosclerosis development is unknown. This study was aimed to investigate the function and action mechanism of circCHFR in atherosclerosis development. An atherosclerosis cell model was created by exposing human vascular endothelial cells (HUVECs) to oxidized low-density lipoprotein. The expression of circCHFR, microRNA(miR)-15b-5p, growth arrest and DNA damage inducible gamma (GADD45G), and their associated proteins was evaluated using quantitative reverse transcription-polymerase chain reaction and Western blotting. Additionally, cell viability, apoptosis, and cytokine levels were determined using Cell Counting Kit-8 (CCK8) assay, flow cytometry, and enzyme-linked immunosorbent assay, respectively. circCHFR expression was upregulated in patients with atherosclerosis and oxidized low-density lipoprotein (ox-LDL)-exposed HUVECs, whereas miR-15b-5p expression was downregulated. circCHFR silencing significantly improved viability and reduced apoptosis of HUVECs. In addition, the pro-apoptotic protein Bax and atherosclerosis-associated cytokines (interleukin-1ß, interleukin-6, and tumor necrosis factor-α) were significantly downregulated, whereas the anti-apoptotic protein Bcl-2 was upregulated. Further, we discovered that circCHFR serves as a molecular sponge of miR-15b-5p. GADD45G was found to be an important target of miR-15b-5p; miR-15b-5p mimic inhibited GADD45G expression, reduced apoptosis and proinflammatory cytokine secretion, and improved cell survival. However, these effects of miR-15b-5p on (ox-LDL) induced HUVECs were reversed with GADD45G plasmid co-transfection. In conclusion, circCHFR promotes atherosclerosis progression via the miR-15b-5p/GADD45G axis and may be an important target for atherosclerosis treatment.

MiR-15p-5p Mediates the Coordination of ICAM-1 and FAK to Promote Endothelial Cell Proliferation and Migration.

Intercellular adhesion molecule-1 (ICAM-1) in endothelial cells is critical for neutrophil adhesion and transmigration across the endothelium. Focal adhesion kinase (FAK), which controls the turnover of focal adhesion to regulate cell adhesion and migration, plays a role in the resolution of inflammation. However, the coordinated involvement of ICAM-1 and FAK during endothelial inflammation has yet to be elucidated. This study reports that, as part of an inflammatory response, ICAM-1 controls FAK expression in endothelial cells via the microRNA miR-15b-5p. Induction of lung injury by lipopolysaccharide (LPS) resulted in higher levels of FAK expression in inflammatory tissues, while in ICAM-1 knockout mice, FAK expression was reduced in the lungs. FAK expression was also reduced in endothelial cells following ICAM-1 siRNA downregulation. Furthermore, ICAM-1 inhibited miR-15b-5p expression while increasing FAK mRNA and protein expression via binding of miR-15b-5p to the 3' untranslated region (UTR) of FAK. ICAM-1 inhibited miR-15b-5p promoter activity and hence reduced miR-15b-5p expression. FAK increased endothelial cell proliferation and migration, whereas miR-15b-5p inhibited cell proliferation and migration. These findings indicate that the inflammatory molecule ICAM-1 regulates FAK expression via miR-15b-5p levels, which in turn controls endothelial cell proliferation and migration.

TRPM2-AS promotes the malignancy of osteosarcoma cells by targeting miR-15b-5p/PPM1D axis.

Osteosarcoma (OS) is a malignant tumor with a low survival rate and a high incidence rate worldwide. Although research has reported the involvement of long non-coding RNAs (lncRNAs) in the pathogenesis of OS cells, the role of TRPM2-AS, miR-15b-5p, and PPM1D in OS progression remains unclear. This study aimed to examine the interaction of the TRPM2-AS/miR-15b-5p/PPM1D axis in OS cells to gain new insights into the molecular mechanism and pathogenesis of OS. After performing in vitro functional assays, we discovered that TRPM2-AS was overexpressed in OS cells. TRPM2-AS silencing impaired OS cell viability, proliferation, and migration, while it induced apoptosis in OS cells in vitro. Our experimental analysis also revealed that PPM1D is a direct target of miR-15b-5p. TRPM2-AS silencing was found to reverse the tumorigenic effect of the miR-15b-5p inhibitor, while the miR-15b-5p inhibitor restored the inhibition of OS caused by silencing PPM1D. Moreover, our findings revealed that miR-15b-5p exerted its tumor-suppressive role by directly targeting PPM1D. In conclusion, this study suggests that TRPM2-AS could promote OS cell malignancy by sponging miR-15b-5p/PPM1D axis.