Combination of miR-99b-5p and Enzalutamide or Abiraterone Synergizes the Suppression of EMT-Mediated Metastasis in Prostate Cancer.

Prostate cancer (PCa) is the most frequently diagnosed cancer and second leading cause of cancer deaths among American men. Androgen deprivation therapy (ADT) has been systemically applied as a first-line therapy for PCa patients. Despite the initial responses, the majority of patients under ADT eventually experienced tumor progression to castration-resistant prostate cancer (CRPC), further leading to tumor metastasis to distant organs. Therefore, identifying the key molecular mechanisms underlying PCa progression remains crucial for the development of novel therapies for metastatic PCa. Previously, we identified that tumor-suppressive miR-99b-5p is frequently downregulated in aggressive African American (AA) PCa and European American (EA) CRPC, leading to upregulation of mTOR, androgen receptor (AR), and HIF-1α signaling. Given the fact that mTOR and HIF-1α signaling are critical upstream pathways that trigger the activation of epithelial-mesenchymal transition (EMT), we hypothesized that miR-99b-5p may play a critical functional role in regulating EMT-mediated PCa metastasis. To test this hypothesis, a series of cell biology, biochemical, and in vitro functional assays (wound healing, transwell migration, cell/ECM adhesion, and capillary-like tube formation assays) were performed to examine the effects of miR-99b-5p mimic on regulating EMT-mediated PCa metastasis processes. Our results have demonstrated that miR-99b-5p simultaneously targets MTOR and AR signaling, leading to upregulation of E-cadherin, downregulation of Snail/N-cadherin/Vimentin, and suppression of EMT-mediated PCa metastasis. MiR-99b-5p alone and in combination with enzalutamide or abiraterone significantly inhibits the EMT-mediated metastasis of AA PCa and EA CRPC.

The effect of STAT1, miR-99b, and MAP2K1 in alcoholic liver disease (ALD) mouse model and hepatocyte.

Alcoholic liver disease (ALD) serves as the leading cause of chronic liver diseases-related morbidity and mortality, which threatens the life of millions of patients in the world. However, the molecular mechanisms underlying ALD progression remain unclear. Here, we applied microarray analysis and experimental approaches to identify miRNAs and related regulatory signaling that associated with ALD. Microarray analysis identified that the expression of miR-99b was elevated in the ALD mouse model. The AML-12 cells were treated with EtOH and the expression of miR-99b was enhanced in the cells. The expression of miR-99b was positively correlated with ALT levels in the ALD mice. The microarray analysis identified the abnormally expressed mRNAs in ALD mice and the overlap analysis was performed with based on the differently expressed mRNAs and the transcriptional factors of miR-99b, in which STAT1 was identified. The elevated expression of STAT1 was validated in ALD mice. Meanwhile, the treatment of EtOH induced the expression of STAT1 in the AML-12 cells. The expression of STAT1 was positively correlated with ALT levels in the ALD mice. The positive correlation of STAT1 and miR-99b expression was identified in bioinformatics analysis and ALD mice. The expression of miR-99b and pri-miR-99b was promoted by the overexpression of STAT1 in AML-12 cells. ChIP analysis confirmed the enrichment of STAT1 on miR-99b promoter in AML-12 cells. Next, we found that the expression of mitogen-activated protein kinase kinase 1 (MAP2K1) was negatively associated with miR-99b. The expression of MAP2K1 was downregulated in ALD mice. Consistently, the expression of MAP2K1 was reduced by the treatment of EtOH in AML-12 cells. The expression of MAP2K1 was negative correlated with ALT levels in the ALD mice. We identified the binding site of MAP2K1 and miR-99b. Meanwhile, the treatment of miR-99b mimic repressed the luciferase activity of MAP2K1 in AML-12 cells. The expression of MAP2K1 was suppressed by miR-99b in the cells. We observed that the expression of MAP2K1 was inhibited by the overexpression of STAT1 in AML-12 cells. Meanwhile, the apoptosis of AML-12 cells was induced by the treatment of EtOH, while miR-99b mimic promoted but the overexpression of MAP2K1 attenuated the effect of EtOH in the cells. In conclusion, we identified the correlation and effect of STAT1, miR-99b, and MAP2K1 in ALD mouse model and hepatocyte. STAT1, miR-99b, and MAP2K1 may serve as potential therapeutic target of ALD.

Regulation of Zbp1 by miR-99b-5p in microglia controls the development of schizophrenia-like symptoms in mice.

Current approaches to the treatment of schizophrenia have mainly focused on the protein-coding part of the genome; in this context, the roles of microRNAs have received less attention. In the present study, we analyze the microRNAome in the blood and postmortem brains of schizophrenia patients, showing that the expression of miR-99b-5p is downregulated in both the prefrontal cortex and blood of patients. Lowering the amount of miR-99b-5p in mice leads to both schizophrenia-like phenotypes and inflammatory processes that are linked to synaptic pruning in microglia. The microglial miR-99b-5p-supressed inflammatory response requires Z-DNA binding protein 1 (Zbp1), which we identify as a novel miR-99b-5p target. Antisense oligonucleotides against Zbp1 ameliorate the pathological effects of miR-99b-5p inhibition. Our findings indicate that a novel miR-99b-5p-Zbp1 pathway in microglia might contribute to the pathogenesis of schizophrenia.

miR-99b/let-7e/miR-125a cluster suppresses pancreatic cancer through regulation of NR6A1.

This experiment investigates how the miR-99b/let-7e/miR-125a cluster regulates the mechanism of NR6A1 involved in the invasive and metastatic effects of pancreatic cancer (PCa). Bioinformatics prediction and dual luciferase reporter gene assay were applied to verify the targeted relationship between miR-99b/let-7e/miR-125a and NR6A1. ASPC1 cells underwent transfection with lentiviruses to overexpress miR-99b/let-7e/miR-125a (individual or together) to explore functions of miR-99b/let-7e/miR-125a cluster governing NR6A1 in PCa. The detection of tumorigenesis was verified by tumor formation assay in nude mice in vivo, and mouse models of liver metastasis of PCa observed cell metastasis of PCa. MiR-99b/let-7e/miR-125a cluster was screened for differential expression in PCa. NR6A1 was confirmed as a target gene of the miR-99b/let-7e/miR-125a cluster. Findings demonstrated that overexpression of the miR-99b/let-7e/miR-125a cluster inhibited cell invasion, metastasis, proliferation, and tumorigenesis in PCa. Conversely, overexpressed NR6A1, a crucial gene in the miR-99b/let-7e/miR-125a cluster, promoted cell invasion, migration, and proliferation in PCa. Moreover, the overexpression of the miR-99b/let-7e/miR-125a cluster inhibited liver metastases and tumor formation. Thus, the study concludes that the miR-99b/let-7e/miR-125a cluster impedes the invasion and metastasis of PCa cells via targeting the NR6A1 gene.

Bone Marrow Mesenchymal Stem Cell-derived Exosomal microRNA- 99b-5p Promotes Cell Growth of High Glucose-treated Human Umbilical Vein Endothelial Cells by Modulating THAP Domain Containing 2 Expression.

INTRODUCTION: Bone marrow mesenchymal stem cell-derived exosomes (BMSC-exos) may function as novel candidates for treating diabetic wounds due to their ability to promote angiogenesis. MATERIALS AND METHODS: This study investigated the effects of BMSC-exos on the growth and metastasis of human umbilical vein endothelial cells (HUVECs) treated with high glucose (HG). The exosomes were separated from BMSCs and identified. The cell phenotype was detected by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and 5-ethynyl-2'-deoxyuridine, wound healing, and transwell assays, while the number of tubes was measured via tube formation assay. RESULT: The RNA and protein expression levels were studied using reverse transcription-quantitative polymerase chain reaction and western blotting, whereas integration of microRNA-99b-5p (miR-99b-5p) with THAP domain containing 2 (THAP2) was confirmed via dual-luciferase reporter and RNA pull-down assays. Results of transmission electron microscopy, nanoparticle tracking analysis, and laser scanning confocal microscopy revealed that exosomes were successfully separated from BMSCs and endocytosed into the cytoplasm by HUVECs. Similarly, BMSC-exos were found to promote the growth of HG-treated HUVECs, while their growth was inhibited by suppressing miR-99b-5p. THAP2 was found to bind to miR-99b-5p, where THAP2 inhibition reversed the miR-99b-5p-induced effects on cell growth, migration, and tube numbers. CONCLUSION: In conclusion, miR-99b-5p in BMSC-exo protects HUVECs by negatively regulating THAP2 expression.

miR-99b-3p/Mmp13 axis regulates NLRP3 inflammasome-dependent microglial pyroptosis and alleviates neuropathic pain via the promotion of autophagy.

BACKGROUND: Neuropathic pain significantly impairs quality of life, and effective interventions are limited. NOD-like receptor thermal protein domain associated protein 3 (NLRP3)-mediated microglial pyroptosis and the subsequent proinflammatory cytokine production are critical in exacerbating pain. Considering microglial pyroptosis as a potential target for developing specific analgesic interventions for neuropathic pain, our study investigated the pathogenesis and therapeutic targets in this condition. METHODS: In vitro experiments involved the co-culture of the immortalized BV-2 microglia cell line with lipopolysaccharide (LPS) to induce microglial pyroptosis. Differentially expressed microRNAs (miRNAs) were identified using high-throughput sequencing analysis. The downstream target genes of these miRNAs were determined through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and the downstream target genes, combined with miRNAs, were predicted and verified through dual luciferase reporter gene assays. In vivo experiments were conducted to construct a chronic constriction injury (CCI) neuropathic pain model in rats and evaluate the analgesic effects of intrathecal injection of an adeno-associated virus vector (AAV) carrying miR-99b-3p. Gene expression was modulated through mimic or siRNA transfection. Western blot analysis assessed the expression of microglial pyroptosis and autophagy-related proteins, whereas RT-qPCR measured changes in proinflammatory cytokines expression. RESULTS: LPS-stimulated up-regulation of proinflammatory cytokines in microglia, accompanied by NLRP3-dependent pyroptosis, including increased NLRP3, GSDMD-N, Caspase1-p20, and mature-IL-1ß expression. High-throughput sequencing analysis revealed 16 upregulated and 10 downregulated miRNAs in LPS-stimulated microglia, with miR-99b-3p being the most downregulated. KEGG analysis revealed that the target genes of these miRNAs are primarily enriched in calcium, FoxO, and mitogen-activated protein kinase (MAPK) signal pathways. Furthermore, overexpression of miR-99b-3p through mimic transfection significantly inhibited the inflammatory response and NLRP3-mediated pyroptosis by promoting autophagy levels in activated microglia. In addition, we predicted that the 3' untranslated region (UTR) of matrix metalloproteinase-13 (Mmp13) could bind to miR-99b-3p, and knockdown of Mmp13 expression through siRNA transfection similarly ameliorated enhanced proinflammatory cytokines expression and microglial pyroptosis by enhancing autophagy. In vivo, Mmp13 was co-localized with spinal dorsal horn microglia and was suppressed by intrathecal injection of the AAV-miR-99b-3p vector. Moreover, overpressed miR-99b-3p alleviated CCI-induced mechanical allodynia and neuroinflammation while suppressing pyroptosis by enhancing autophagy in the spinal cord of CCI rats. CONCLUSION: miR-99b-3p exerts analgesic effects on neuropathic pain by targeting Mmp13. These antinociceptive effects are, at least in part, attributed to the promotion of autophagy, thereby inhibiting neuroinflammation and NLRP3-mediated pyroptosis in activated microglia.

Tumor suppressive miR-99b-5p as an epigenomic regulator mediating mTOR/AR/SMARCD1 signaling axis in aggressive prostate cancer.

Introduction: African American (AA) men exhibited 2.3-fold higher PCa incidence and 1.7-fold higher PCa mortality rates when compared to the European American (EA) men. Besides the socioeconomic factors, emerging evidence has highlighted that biological risk factors may play critical roles in the AA PCa disparities. Previously, we have shown that downregulated miR-99b-5p and upregulated mTOR cooperatively promotes the AA PCa aggressiveness and drug resistance. Methods: In this study, we aimed to explore the miR-99b-5p/mTOR/AR/SMARCD1 signaling axis in AA PCa aggressiveness. The analyses used in the study included immunofluorescence, western blot, in-vitro functional assays (TUNEL, colony forming, and MTT), and chromatin immunoprecipitation (ChIP)-qPCR assays in 2D and/or 3D culture model of EA PCa and AA PCa cell lines. Results: Specifically, the immunofluorescence staining, and western blot analysis has revealed that nuclear mTOR, AR, and SMARCD1 were highly expressed in AA PCa (MDA PCa 2b) compared to EA PCa (LNCaP) cell line. Western blot analysis further revealed that miR-99b-5p inhibited protein levels of mTOR, AR/AR-V7 and SMARCD1 in cytoplasm and nuclei of EA and AA PCa. The in-vitro functional (MTT, TUNEL, and clonogenic) assays have demonstrated that miR-99b-5p effectively inhibited cell proliferation/survival and induced cell apoptosis in EA and AA PCa cells. Moreover, combination of miR-99b-5p and enzalutamide (Enz) synergistically enhances the cytotoxicity against aggressive AA PCa and castration-resistant prostate cancer (CRPC). mTOR ChIP-qPCR assays further demonstrated that miR-99b-5p or miR-99b-5p/Enz significantly reduces the recruitment of mTOR to the genes involved in the metabolic reprogramming in CRPC. Discussion: Taken together, miR-99b-5p may function as an epigenomic driver to modulate the mTOR/AR/SMARCD1 signaling axis in AA PCa and resistant CRPC.

Effect of the MiR-99b and MiR-135b on peritoneal carcinomatosis and liver metastasis in colorectal cancer.

AIM: This study aimed to evaluate the expression levels of miR-99b and miR-135b in peritoneal carcinoma and liver metastases associated with Colorectal Cancer (CRC), assess their association with the intracellular signaling pathway proteins Kirsten Rat Sarcoma Virus (KRAS) and Akt, and investigate their effects on survival. MATERIALS AND METHODS: Changes in the KRAS gene and Akt proteins, expression levels of miR-99b and miR-135b, and factors affecting survival were compared between colorectal cancer-associated peritoneal carcinomatosis and liver metastasis. RESULTS: The expression levels of miR-99b and miR-135b and the immunohistochemical grade classification score of Akt were higher in colorectal cancer, peritoneal carcinomatosis, and liver metastasis than in normal tissues (p < 0.05). MiR-99b expression was highest in CRC, whereas miR-135b expression was highest in peritoneal carcinomatosis (p < 0.05). The expression level of miR-99b decreased and that of miR-135b increased in peritoneal and liver metastases compared with that in the tumor tissue. MiR-99b, Akt, and recurrence were risk factors that affected the overall survival rate in the model of clinical predictions (p = 0.045, p = 0.006, and p = 0.012, respectively). CONCLUSION: While the expression of miR-99b was highest in the primary tumor, its decrease in liver metastasis and peritoneal carcinomatosis suggests that miR-99b has a protective effect against liver metastasis and peritoneal carcinomatosis. However, the detection of miR-135b expression was highest in peritoneal carcinomatosis and liver metastasis compared with that in the colorectal cancer tissues suggesting that it facilitates peritoneal carcinomatosis and liver metastasis. Furthermore, miR-99b, KRAS mutations, and Akt are risk factors for the overall survival of colorectal cancer.

Exosomal miR-99b-5p Secreted from Mesenchymal Stem Cells Can Retard the Progression of Colorectal Cancer by Targeting FGFR3.

Human bone marrow mesenchymal stem cells (BMSCs) are efficient mass producers of exosomes that can potentially be utilized for delivery of miRNAs in cancer therapy. The current study aimed to assess the role of MSC-exosomal miR-99b-5p during the development of colorectal cancer (CRC). The potential value of using plasma levels of exosomal miR-99b-5p for predicting the liver metastasis of colorectal cancer was also assessed. In this study, we found that overexpression of fibroblast growth factor receptor 3 (FGFR3) was associated with tumor progression in CRC and FGFR3 was the target gene of miR-99b-5p, which was down-regulated in CRC tissues. Furthermore, we observed that elevated miR-99b-5p inhibited CRC cell proliferation, invasion and migration, while reduced levels had the opposite effect on CRC cells. Moreover, exosomal miR-99b-5p delivered by BMSCs was able to limit the proliferation, invasion and migration of CRC cells in vitro, as well as suppressing tumor growth in vivo. Collectively, these findings revealed that MSC-derived exosomal miR-99b-5p can be transferred into CRC cells and which can suppress tumor progression by targeting FGFR3. This highlights the potential of using exosomal miR-99b-5p as a novel diagnostic marker for CRC, while providing a therapeutic target to combat CRC.

Effect of the MiR-99b and MiR-135b on peritoneal carcinomatosis and liver metastasis in colorectal cancer

Abstract Aim This study aimed to evaluate the expression levels of miR-99b and miR-135b in peritoneal carcinoma and liver metastases associated with Colorectal Cancer (CRC), assess their association with the intracellular signaling pathway proteins Kirsten Rat Sarcoma Virus (KRAS) and Akt, and investigate their effects on survival. Materials and methods Changes in the KRAS gene and Akt proteins, expression levels of miR-99b and miR-135b, and factors affecting survival were compared between colorectal cancer-associated peritoneal carcinomatosis and liver metastasis. Results The expression levels of miR-99b and miR-135b and the immunohistochemical grade classification score of Akt were higher in colorectal cancer, peritoneal carcinomatosis, and liver metastasis than in normal tissues (p< 0.05). MiR-99b expression was highest in CRC, whereas miR-135b expression was highest in peritoneal carcinomatosis (p< 0.05). The expression level of miR-99b decreased and that of miR-135b increased in peritoneal and liver metastases compared with that in the tumor tissue. MiR-99b, Akt, and recurrence were risk factors that affected the overall survival rate in the model of clinical predictions (p= 0.045, p= 0.006, and p= 0.012, respectively). Conclusion While the expression of miR-99b was highest in the primary tumor, its decrease in liver metastasis and peritoneal carcinomatosis suggests that miR-99b has a protective effect against liver metastasis and peritoneal carcinomatosis. However, the detection of miR-135b expression was highest in peritoneal carcinomatosis and liver metastasis compared with that in the colorectal cancer tissues suggesting that it facilitates peritoneal carcinomatosis and liver metastasis. Furthermore, miR-99b, KRAS mutations, and Akt are risk factors for the overall survival of colorectal cancer.

Downregulation of miR-99b-5p and Upregulation of Nuclear mTOR Cooperatively Promotes the Tumor Aggressiveness and Drug Resistance in African American Prostate Cancer.

Mammalian target of rapamycin (mTOR) regulates various fundamental cellular events including cell proliferation, protein synthesis, metabolism, apoptosis, and autophagy. Tumor suppressive miR-99b-5p has been implicated in regulating PI3K/AKT/mTOR signaling in a variety of types of cancer. Our previous study suggested the reciprocal miR-99b-5p/MTOR (downregulated/upregulated) pairing as a key microRNA-mRNA regulatory component involved in the prostate cancer (PCa) disparities. In this study, we further validated the expression profiles of mTOR and miR-99b-5p in the PCa, colon, breast, and lung cancer specimens and cell lines. The immunohistochemistry (IHC), immunofluorescence, Western blot, and RT-qPCR assays have confirmed that mTOR is upregulated while miR-99b-5p is downregulated in different patient cohorts and a panel of cancer cell lines. Intriguingly, elevated nuclear mTOR expression was observed in African American PCa and other advanced cancers. Transfection of the miR-99b-5p mimic resulted in a significant reduction in nuclear mTOR and androgen receptor (AR), while a slight/moderate to no decrease in cytoplasmic mTOR and AR in PCa and other cancer cells, suggesting that miR-99b-5p inhibits mTOR and AR expression and their nuclear translocation. Moreover, overexpression of miR-99b-5p targets/inhibits AR-mTOR axis, subsequently initiating cell apoptosis and sensitizing docetaxel-induced cytotoxicity in various cancers. In conclusion, our data suggest that reciprocal miR-99b-5p/nuclear mTOR pairing may be a more precise diagnostic/prognostic biomarker for aggressive PCa, than miR-99b-5p/MTOR pairing or mTOR alone. Targeting the AR-mTOR axis using miR-99b-5p has also been suggested as a novel therapeutic strategy to induce apoptosis and overcome chemoresistance in aggressive PCa.

chi-miR-99b-3p Regulates the Proliferation of Goat Skeletal Muscle Satellite Cells In Vitro by Targeting Caspase-3 and NCOR1.

We previously found that chi-miR-99b-3p was highly expressed in the skeletal muscle of 7-month-old (rapid growth period) goats and speculated that it may be associated with muscle development. To further investigate the role of chi-miR-99b-3p in goats, we found that chi-miR-99b-3p acted as a myogenic miRNA in the regulation of skeletal muscle development. Dual-luciferase reporter assays, qRT-PCR, and Western blot results confirmed that Caspase-3 and nuclear receptor corepressor 1 were direct targets for chi-miR-99b-3p as their expression was inhibited by this miR. Cell proliferation and qRT-PCR assays showed that chi-miR-99b-3p promoted proliferation through relevant targets and intrinsic apoptosis-related genes in goat skeletal muscle satellite cells (SMSCs), whereas inhibition of chi-miR-99b-3p had the opposite effect. Furthermore, integrative transcriptomic analysis revealed that overexpression of chi-miR-99b-3p induced various differentially expressed (DE) genes mainly associated with the cell cycle, relaxin signaling pathway, DNA replication, and protein digestion and absorption. Notably, most of the cell-cycle-related genes were downregulated in SMSCs after miR-99b-3p upregulation, including the pro-apoptosis-related gene BCL2. In addition, 47 DE miRNAs (16 upregulated and 31 downregulated) were determined by Small RNA-sequencing in SMSCs after chi-miR-99b-3p overexpression. Based on the KEGG enrichment analysis, we found that these DE miRNAs were involved in the biological pathways associated with the DE genes. Our study demonstrated that chi-miR-99b-3p was an effective facilitator of goat SMSCs and provided new insights into the mechanisms by which miRNAs regulate skeletal muscle growth in goats.

MicroRNA-99b-5p targets mTOR/AR axis, induces autophagy and inhibits prostate cancer cell proliferation.

OBJECTIVES: MicroRNAs (miRNAs) are the small non-coding regulatory RNA molecules involved in gene regulation via base-pairing with complementary sequences in mRNAs. The dysregulation of specific miRNAs, such as miR-99b-5p (miR-99b), is associated with prostate cancer (PCa) progression. However, the mechanistic role of miR-99b in PCa remains to be determined. In this study, we aimed to investigate the functional and clinical significance of miR-99b in PCa. STUDY DESIGN: The expression of miR-99b and its downstream targets mTOR/AR in the PCa samples were analyzed by RT/qPCR. The effects of miR-99b overexpression/inhibition on PCa cell survival/proliferation, spheroid formation, and cell migration were examined by specific assays. Luciferase reporter assays were performed to determine the binding of miR-99b to 3' untranslated region (UTR) of the mTOR gene. The effects of miR-99b on the expression of mTOR, AR, and PSA proteins, as well as on AKT/mTOR signaling, autophagy, and neuroendocrine differentiation markers were analyzed by western blotting. The expression of miR-99b, mTOR, AR, PSA in AR-negative PC3 and AR-positive LNCaP cells was analyzed by RT/qPCR. The effect of miR-99b on global gene expression in PC3 cells was analyzed by RNA-seq. RESULTS: The expression of miR-99b was downregulated in tumor samples from PCa patients, whereas the expression of mTOR and AR was upregulated. In PCa cell lines, overexpression of miR-99b inhibited cell proliferation and cell colony/spheroid formation; induced apoptosis, and increased sensitivity towards docetaxel (DTX). In contrast, inhibition of miR-99b by miR-99b inhibitor resulted in increased cell growth in PCa cells. Mechanistically, miR-99b inhibited the expression of the mammalian target of the rapamycin (mTOR) gene by binding to its 3' UTR and induced autophagy. Furthermore, miR-99b inhibited androgen receptor (AR) activity in LNCaP cells and induced apoptosis. Activation of AR signaling by dihydrotestosterone (DHT) downregulated miR-99b expression and promoted cell PCa cell growth/survival, whereas inactivation of mTOR by rapamycin or AR by enzalutamide decreased miR-99b mediated PCa cell growth. CONCLUSION: Our data suggest that miR-99b functions as a tumor suppressor by targeting the mTOR/AR axis in PCa cells, implicating miR-99b as a novel biomarker and therapeutic target for PCa management.

Long Non-coding RNA UCA1 Regulates SRPK1 Expression Through miR- 99b-3p in Ovarian Cancer.

BACKGROUND: Ovarian carcinoma (OC) is one of the most common malignancies of the female reproductive organs, with a low survival rate primarily due to the lack of effective methods for early diagnosis and prognosis. OBJECTIVE: In this article, our motivation is to explore the lncRNA-related network mechanisms involved in the pathogenesis of OC. METHODS: Public lncRNAs and mRNA expression datasets for OC were collected from the Gene Expression Omnibus (GEO) database. By integrated bioinformatics analysis, we constructed a UCA1-miRNA-mRNA network. We studied lncRNA-related molecular modulation mechanism in ovarian cancer cells based on MTT assay, dual luciferase reporter gene assays, quantitative realtime PCR, and western blotting. RESULTS: UCA1 was higher in ovarian tumor tissues and cells than normal tissues and cells. It was demonstrated in this study that knockdown of UCA1 inhibited ovarian cancer cell viability, which a miR-99b-3p inhibitor could reverse in vitro. Further, UCA1 was shown to regulate the expression of SRPK1 by directly binding to miR-99b-3p. CONCLUSION: These results suggest that UCA1 functions as an oncogene in ovarian cancer. Inhibition of UCA1/miR-99b-3p/SRPK1 axis may become a novel target for treating ovarian cancer.

miR-99b-5p, miR-380-3p, and miR-485-3p are novel chemosensitizing miRNAs in high-risk neuroblastoma.

Neuroblastoma is a deadly childhood cancer arising in the developing sympathetic nervous system. High-risk patients are currently treated with intensive chemotherapy, which is curative in only 50% of children and leaves some surviving patients with life-long side effects. microRNAs (miRNAs) are critical regulators of neural crest development and are deregulated during neuroblastoma tumorigenesis, making miRNA-based drugs an attractive therapeutic avenue. A functional screen of >1,200 miRNA mimics was conducted in neuroblastoma cell lines to discover miRNAs that sensitized cells to low doses (30% inhibitory concentration [IC30]) of doxorubicin and vincristine chemotherapy used in the treatment of the disease. Three miRNAs, miR-99b-5p, miR-380-3p, and miR-485-3p, had potent chemosensitizing activity with doxorubicin in multiple models of high-risk neuroblastoma. These miRNAs underwent genomic loss in a subset of neuroblastoma patients, and low expression predicted poor survival outcome. In vitro functional assays revealed each of these miRNAs enhanced the anti-proliferative and pro-apoptotic effects of doxorubicin. We used RNA sequencing (RNA-seq) to show that miR-99b-5p represses neuroblastoma dependency genes LIN28B and PHOX2B both in vitro and in patient-derived xenograft (PDX) tumors. Luciferase reporter assays demonstrate that PHOX2B is a direct target of miR-99b-5p. We anticipate that restoring the function of the tumor-suppressive miRNAs discovered here may be a valuable therapeutic strategy for the treatment of neuroblastoma patients.

Human bone marrow mesenchymal stem cells-derived exosomes attenuated prostate cancer progression via the miR-99b-5p/IGF1R axis.

MicroRNA-99b-5p (miR-99b-5p) has been shown to be enriched in serum exosomes of prostate cancer (PCa) patients treated with radiotherapy, while its function in PCa progression remains unclear. The expression levels of miR-99b-5p in PCa tissues, cancer cell lines and human bone marrow mesenchymal stem cells (HBMSCs), as well as HBMSCs-derived exosomes were assessed by quantitative real-time PCR (qRT-PCR). MiR-99b-5p mimics or inhibitor was transfected into HBMSCs, and HBMSCs-derived exosomes with abnormal expression of miR-99b-5p were used to stimulate PCa cell-line LNCaP cells. Cell proliferative rate was evaluated using Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) staining assays. Cell migration and invasion were analyzed by Transwell assay. The epithelial-mesenchymal transition (EMT) was evaluated by detecting EMT-related markers using Western blot analysis. The animal model was constructed to confirm the function of miR-99b-5p in vivo. The expression levels of MiR-99b-5p were decreased in PCa tissues and cell lines, while elevated in HBMSCs-derived exosomes. HBMSCs-derived exosomes significantly inhibited cell malignant phenotypes of PCa cells, and miR-99b-5p mimics transfected HBMSCs further enhanced the inhibitory effects of HBMSCs on PCa progression. In addition, miR-99b-5p inhibitor transfected HBMSCs-derived exosomes promoted the progression of PCa in vitro. Insulin-like growth factor 1 receptor (IGF1R) was identified as a downstream target of miR-99b-5p. Moreover, miR-99b-5p mimics transfected HBMSCs obviously inhibited tumor progression by downregulating IGF1R in animal model in vivo. Our results demonstrated that HBMSCs could attenuate PCa progression, and exosomal miR-99b-5p and IGF1R participated in the regulatory process, contributing to our understanding of the pathogenic mechanism of PCa.

[miR-99b-5p inhibits the activation of NLRP3 inflammasome to alleviate the neurotoxicity induced by paclitaxel chemotherapy].

OBJECTIVE: To study the effects of miR-99b-5p (non-coding RNA) in alleviating pathological neuropathic pain after paclitaxel chemotherapy by inhibiting NLRP3 inflammatory vesicle activation and the effects on neuronal cells pyrosis and apoptosis. METHODS: SD rats were randomly divided into blank group, model group, agomiR-99b-5P treatment group, and agomiR-NC group, 6 rats in each group. The blank group received saline treatment as a control, the model group established a pain model induced by paclitaxel, and the rats in agomiR-99b-5p treatment group and agomiR-NC group were treated with agomiR-99b-5p and agomiR-NC injections, respectively. The expressions of miR-99b-5p in the blank group, model group, and treatment group were detected by RT-qPCR. The mechanical foot retraction threshold (MWT) of the blank group, model group, and treatment group were detected. TUNEL was used to detect the apoptosis of spinal dorsal horn cells. The levels of ROS, MDA, and SOD were detected by ELISA kits. The protein expressions of NLRP3, caspase-1, and IL-1ß were detected by immunofluorescence staining. RESULTS: Compared with the model group, the expression level of miR-99b-5p and the MWT were increased significantly in agomiR-99b-5p treatment group (P＜0.05), the apoptosis of dorsal horn cells was inhibited (P＜0.05), the level of antioxidant stress was increased in rats, the levels of ROS and MDA were decreased (P＜0.05), while the level of SOD was increased (P＜0.05). Immunofluorescence showed that the expressions of NLRP3, caspase-1, and IL-1ß were inhibited by miR-99b-5p. CONCLUSION: miR-99b-5p can alleviate the apoptosis and pyroptosis of neurons after paclitaxel chemotherapy by inhibiting the activation of NLRP3 and improving oxidative stress in vivo.

Megakaryocyte-specific knockout of the Mir-99b/let7e/125a cluster lowers platelet count without altering platelet function.

The purpose of this research was to assess the effects of a microRNA (miRNA) cluster on platelet production. Human chromosome 19q13.41 harbors an evolutionarily conserved cluster of three miRNA genes (MIR99B, MIRLET7E, MIR125A) within 727 base-pairs. We now report that levels of miR-99b-5p, miR-let7e-5p and miR-125a-5p are strongly correlated in human platelets, and all are positively associated with platelet count, but not white blood count or hemoglobin level. Although the cluster regulates hematopoietic stem cell proliferation, the function of this genomic locus in megakaryocyte (MK) differentiation and platelet production is unknown. Furthermore, studies of individual miRNAs do not represent broader effects in the context of a cluster. To address this possibility, MK/platelet lineage-specific Mir-99b/let7e/125a knockout mice were generated. Compared to wild type littermates, cluster knockout mice had significantly lower platelet counts and reduced MK proplatelet formation, but no differences in MK numbers, ploidy, maturation or ultra-structural morphology, and no differences in platelet function. Compared to wild type littermates, knockout mice showed similar survival after pulmonary embolism. The major conclusions are that the effect of the Mir-99b/let7e/125a cluster is confined to a late stage of thrombopoiesis, and this effect on platelet number is uncoupled from platelet function.

MiR-99b-5p suppressed proliferation of human osteoblasts by targeting FGFR3 in osteoporosis.

Osteoporosis is a common skeletal disease characterized by reduced bone mass partially caused by an imbalance between bone resorption and formation. Considering the potential role of microRNAs (miRNAs) in osteoporosis, we attempted to identify deregulated miRNA that participates in the pathogenesis of osteoporosis. We analyzed online datasets for differentially expressed miRNAs and predicted deregulated miRNA target genes, applied these genes for signaling pathway enrichment annotation, and selected the possible miR-99b-5p/FGFR3 axis. Within osteoporosis bone tissues, miR-99b-5p was upregulated and FGFR3 was downregulated. miR-99b-5p overexpression inhibited osteoblast proliferation and osteogenesis-related genes expression, whereas FGFR3 overexpression exerted opposite effects upon the proliferation of osteoblasts and osteogenesis-related genes expression. By direct targeting, miR-99b-5p inhibited FGFR3 expression. Moreover, FGFR3 silencing significantly reversed the roles of miR-99b-5p inhibition in the proliferation of osteoblasts and osteogenesis-related genes expression. In conclusion, we identify a deregulated miRNA/mRNA axis in osteoporosis and osteogenic differentiation, namely the miR-99b-5p/FGFR3 axis; through targeting FGFR3, miR-99b-5p inhibits osteoblast proliferation and activity, which might subsequently affect the bone formation in osteoporosis progression.

MiR-99b-5p Attenuates Adipogenesis by Targeting SCD1 and Lpin1 in 3T3-L1 Cells.

The number and distribution of adipocytes directly affect the quality of livestock meat products. The analysis of the adipogenesis mechanism is the basis for improving meat quality. The formation of adipocytes is regulated by many factors, including a class of endogenous small RNAs, named microRNA (miRNA). Previous studies have shown that miRNAs could affect adipogenesis by post-transcriptional regulation of target genes. In our study, a decreased miR-99b-5p expression level was found in the adipose tissue of obese mice. Overexpression of miR-99b-5p could increase cell proliferation by promoting the cell cycle while inhibiting cell differentiation. In addition, interference with miR-99b-5p obtained the opposite result. Furthermore, the proteomics sequencing analysis screened 1154 differentially expressed proteins, which are closely related to adipocyte differentiation and fatty acid metabolism. In addition, the results of the dual-luciferase test showed that miR-99b-5p can directly target the proteins SCD1 and Lpin1 with significantly different expression levels in proteomic sequencing. Then, this result was verified at the level of mRNA and protein in a further study. Collectively, these results suggested that miR-99b-5p may be a target for improving meat quality.