Genetic Heterogeneity in Cellular Angiofibromas.

BACKGROUND: Cellular angiofibroma, a rare benign mesenchymal neoplasm, is classified within the 13q/RB1 family of tumors due to morphological, immunohistochemical, and genetic similarities with spindle cell lipoma. Here, genetic data reveal pathogenetic heterogeneity in cellular angiofibroma. METHODS: Three cellular angiofibromas were studied using G-banding/Karyotyping, array comparative genomic hybridization, RNA sequencing, and direct cycling sequencing. RESULTS: The first tumor carried a del(13)(q12) together with heterozygous loss and minimal expression of the RB1 gene. Tumors two and three displayed chromosome 8 abnormalities associated with chimeras of the pleomorphic adenoma gene 1 (PLAG1). In tumor 2, the cathepsin B (CTSB) fused to PLAG1 (CTSB::PLAG1) while in tumor 3, the mir-99a-let-7c cluster host gene (MIR99AHG) fused to PLAG1 (MIR99AHG::PLAG1), both leading to elevated expression of PLAG1 and insulin growth factor 2. CONCLUSION: This study uncovers two genetic pathways contributing to the pathogenetic heterogeneity within cellular angiofibromas. The first aligns with the 13q/RB1 family of tumors and the second involves PLAG1-chimeras. These findings highlight the diverse genetic landscape of cellular angiofibromas, providing insights into potential diagnostic strategies.

MiR-99a-3p downregulates TRIM21 to promote gastric cancer development.

Gastric cancer (GC) stands as one of the most formidable malignancies worldwide. It is well-established that miRNAs play a crucial role in the initiation and progression of various human cancers. Among these, miR-99a-3p has been implicated in the pathogenesis of GC. In the context of our study, we embarked on the comprehensive examination of miR-99a-3p expression in GC cells. Additionally, we sought to establish a correlation between miR-99a-3p expression levels and the overall survival (OS) of GC patients, and our findings hinted at its potential role in predicting an unfavorable prognosis. To further investigate the functional implications of miR-99a-3p in GC, we conducted a series of cell-based experiments after successfully knocking down miR-99a-3p. These investigations uncovered a substantial inhibition of cellular events associated with tumor progression. Moreover, employing TargetScan, we identified Tripartite motif-containing protein 21 (TRIM21) as a putative target with a binding site for miR-99a-3p. Subsequent dual-luciferase reporter gene assay confirmed the direct interaction between miR-99a-3p and TRIM21. Western blot analysis validated the alteration in TRIM21 expression levels, revealing an upregulation upon miR-99a-3p knockdown. Building on these molecular findings, we extended our investigations to human GC tissues, where we observed a downregulation of TRIM21, which, notably, correlated with shorter overall survival. Lastly, to further solidify our conclusions, we conducted a series of in vitro and in vivo rescue experiments, collectively suggesting that miR-99a-3p promoted the progression of GC cells through the downregulation of TRIM21. In summary, our study comprehensively explored the role of miR-99a-3p in GC, revealing its association with unfavorable patient outcomes, functional implications in tumor progression, and a direct regulatory relationship with TRIM21. These findings collectively underscore the significance of miR-99a-3p in the pathogenesis of GC and present a potential therapeutic avenue for further investigation.

Reduced miR-99a-3p levels in systemic lupus erythematosus may promote B cell proliferation via NCAPG and the PI3K/AKT signaling pathway.

BACKGROUND: Systemic lupus erythematosus is an immunologically dysregulated disease characterized by the presence of multiple autoantibodies. In SLE, B lymphocytes contribute to the dysregulated production of autoantibodies and cytokines. Recently, we discovered that miR-99a-3p binds to both EIF4EBP1 and NCAPG mRNA and that lowering miR-99a-3p can promote B cell autophagy in SLE by increasing EIF4EBP1 expression. However, the functions of miR-99a-3p and NCAPG in SLE have not been extensively investigated. OBJECTIVE: This work aims to evaluate the levels of miR-99a-3p and NCAPG expression in SLE B cells and to determine whether the aberrant expression of miR-99a-3p and NCAPG contributes to the pathological mechanisms in SLE. METHODS: B lymphocytes were obtained through immunomagnetic negative selection. Using RT-qPCR, miR-99a-3p and NCAPG mRNA expressions in B lymphocytes and in the BALL-1 cell line were measured. To determine the relative abundance of NCAPG, PI3K, p-PI3K, AKT, and p-AKT, we normalize them to the level of ß-actin using Western blotting. Evaluation of miR-99a-3p and NCAPG's impact on cell proliferation was done utilizing CCK-8 assay. Using flow cytometry, the cell cycle and apoptosis were both measured. RESULTS: Comparing SLE B cells to healthy controls, miR-99a-3p expression was significantly downregulated. Additionally, it was observed that SLE B cells had significantly higher NCAPG mRNA expression. Blocking miR-99a-3p expression in BALL-1 cells with an antagomir elevated NCAPG expression, facilitated PI3K/AKT pathway activation, improved cell proliferation, raised the fraction of S-phase cells, and prevented cell apoptosis. The opposite effects of upregulated miR-99a-3p levels on BALL-1 cells were observed by using an agomir. Furthermore, the effect of decreased miR-99a-3p expression on cell proliferation was partially mediated by elevating NCAPG levels and activating the PI3K/AKT pathway. CONCLUSION: Our research indicates that lower miR-99a-3p expression in SLE B cells appears to boost B cell number via the NCAPG and PI3K/AKT pathways.

Synergistic effects of miR-143 with miR-99a inhibited cell proliferation and induced apoptosis in breast cancer.

Breast cancer (BC) is the most common cancer type and the fifth leading cause of cancer-related deaths. The primary goals of BC treatment are to remove the tumor and prevent metastasis. Despite advances in BC treatment, more effective therapies are required. miRNAs can regulate many targets involved in biological processes and tumor progression; these molecules have emerged as a promising cancer treatment strategy. In the present study, we investigated the effects of miR-99a and miR-143 in single expression plasmids for BC inhibition. In this study, the precursor structure of miRNAs in the expression vector pEGFP-N1 entered single and double states, and MCF7 and T47D cells were transfected. The miRNAs expression level after transfection was then measured using qPCR. The MultiMiR package was used to obtain predicted and validated miRNA targets. MTT assay, qRT-PCR, migration test, and flow cytometry were used to assess the effect of miRNA and gene modulation. The qPCR results revealed that miRNA constructs were significantly expressed after the transfection of both cell lines. The biological function of miRNAs showed that upregulation of miR-99a and miR-143 in any of the two selected BC cells inhibited their proliferation and migration rate, significantly inducing apoptosis (p < 0.01). Also, miR-99a/miR-143 co-treatment has a synergistic anticancer effect in cancer cells via Akt1 and CDK6 targeting. These findings suggest that miR-99a/miR-143 plays synergistic regulatory roles in BC, possibly via a shared signaling pathway, providing a therapeutic strategy for BC treatment.

Differential effects of microRNAs miR-21, miR-99 and miR-145 on lung regeneration and inflammation during recovery from influenza pneumonia.

In a mouse model of influenza pneumonia, we previously documented that proliferating alveolar type II (AT2) cells are the major stem cells involved in early lung recovery. Profiling of microRNAs revealed significant dysregulation of specific ones, including miR-21 and miR-99a. Moreover, miR-145 is known to exhibit antagonism to miR-21. This follow-up study investigated the roles of microRNAs miR-21, miR-99a, and miR-145 in the murine pulmonary regenerative process and inflammation during influenza pneumonia. Inhibition of miR-21 resulted in severe morbidity, and in significantly decreased proliferating AT2 cells due to impaired transition from innate to adaptive immune responses. Knockdown of miR-99a culminated in moderate morbidity, with a significant increase in proliferating AT2 cells that may be linked to PTEN downregulation. In contrast, miR-145 antagonism did not impact morbidity nor the proliferating AT2 cell population, and was associated with downregulation of TNF-alpha, IL1-beta, YM1, and LY6G. Hence, a complex interplay exists between expression of specific miRNAs, lung regeneration, and inflammation during recovery from influenza pneumonia. Inhibition of miR-21 and miR-99a (but not miR-145) can lead to deleterious cellular and molecular effects on pulmonary repair and inflammatory processes during influenza pneumonia.

EZH2-mediated epigenetic silencing of tumor-suppressive let-7c/miR-99a cluster by hepatitis B virus X antigen enhances hepatocellular carcinoma progression and metastasis.

BACKGROUND: Hepatitis B virus (HBV)-encoded X antigen, HBx, assists in the development of hepatocellular carcinoma (HCC) through complex mechanisms. Our results provide new insights into the EZH2 epigenetic repression of let-7c that promotes HCC migration induced by HBx. Thus, let-7c and HMGA2 represent key diagnostic markers and potential therapeutic targets for the treatment of HBV-related HCC. RESULTS: We investigated the epigenetic regulation of let-7c, an important representative miRNA in liver tumor metastasis, in human HCC cells to verify the effect of HBx. Based on quantitative PCR (qPCR) of mRNA isolated from tumor and adjacent non-tumor liver tissues of 24 patients with HBV-related HCC, EZH2 expression was significantly overexpressed in most HCC tissues (87.5%). We executed a miRNA microarray analysis in paired HBV-related HCC tumor and adjacent non-tumorous liver tissue from six of these patients and identified let-7c, miR-199a-3p, and miR-99a as being downregulated in the tumor tissue. Real-time PCR analysis verified significant downregulation of let-7c and miR-99a in both HepG2X and Hep3BX cells, which stably overexpress HBx, relative to parental cells. HBX enhanced EZH2 expression and attenuated let-7c expression to induce HMGA2 expression in the HCC cells. Knockdown of HMGA2 significantly downregulated the metastatic potential of HCC cells induced by HBx. CONCLUSIONS: The deregulation of let-7c expression by HBx may indicate a potential novel pathway through deregulating cell metastasis and imply that HMGA2 might be used as a new prognostic marker and/or as an effective therapeutic target for HCC.

Dynamic and static circulating cancer microRNA biomarkers - a validation study.

For cancers and other pathologies, early diagnosis remains the most promising path to survival. Profiling of longitudinal cohorts facilitates insights into trajectories of biomarkers. We measured microRNA expression in 240 serum samples from patients with colon, lung, and breast cancer and from cancer-free controls. Each patient provided at least two serum samples, one prior to diagnosis and one following diagnosis. The median time interval between the samples was 11.6 years. Using computational models, we evaluated the circulating profiles of 21 microRNAs. The analysis yielded two sets of biomarkers, static ones that show an absolute difference between certain cancer types and controls and dynamic ones where the level over time provided higher diagnostic information content. In the first group, miR-99a-5p stands out for all three cancer types. In the second group, miR-155-5p allows to predict lung cancers and colon cancers. Classification in samples from cancer and non-cancer patients using gradient boosted trees reached an average accuracy of 79.9%. The results suggest that individual change over time or an absolute value at one time point may predict a disease with high specificity and sensitivity.

Plasma microRNA Profiling in Type 2 Diabetes Mellitus: A Pilot Study.

Type 2 diabetes mellitus (T2D) is a chronic metabolic disease characterized by insulin resistance and ß-cell dysfunction and leading to many micro- and macrovascular complications. In this study we analyzed the circulating miRNA expression profiles in plasma samples from 44 patients with T2D and 22 healthy individuals using next generation sequencing and detected 229 differentially expressed miRNAs. An increased level of miR-5588-5p, miR-125b-2-3p, miR-1284, and a reduced level of miR-496 in T2D patients was verified. We also compared the expression landscapes in the same group of patients depending on body mass index and identified differential expression of miR-144-3p and miR-99a-5p in obese individuals. Identification and functional analysis of putative target genes was performed for miR-5588-5p, miR-125b-2-3p, miR-1284, and miR-496, showing chromatin modifying enzymes and apoptotic genes being among the significantly enriched pathways.

MiR-99a alleviates apoptosis and extracellular matrix degradation in experimentally induced spine osteoarthritis by targeting FZD8.

BACKGROUND: Our previous study identified miR-99a as a negative regulator of early chondrogenic differentiation. However, the functional role of miR-99a in the pathogenesis of osteoarthritis (OA) remains unclear. METHODS: We examined the levels of miR-99a and Frizzled 8 (FZD8) expression in tissue specimens. Human SW1353 chondrosarcoma cells were stimulated with IL-6 and TNF-α to construct an in vitro OA environment. A luciferase reporter assay was performed to analyze the relationship between miR-99a and FZD8. CCK-8 assays, flow cytometry, and ELISA assays were used to assess cell viability, apoptosis, and inflammatory molecule expression, respectively. Percutaneous intra-spinal injections of papain mixed solution were performed to create an OA Sprague-Dawley rat model. Alcian Blue staining, Safranin O Fast Green staining, and Toluidine Blue O staining were performed to detect the degrees of cartilage injury. RESULTS: MiR-99a expression was downregulated in the severe spine OA patients when compared with the mild spine OA patients, and was also decreased in the experimentally induced in vitro OA environment when compared with the control environment. Functionally, overexpression of miR-99a significantly suppressed cell apoptosis and extracellular matrix degradation stimulated by IL-6 and TNF-α. FZD8 was identified as a target gene of miR-99a. Furthermore, the suppressive effects of miR-99a on cell injury induced by IL-6 and TNF-α were reversed by FZD8 overexpression. Moreover, the levels of miR-99a expression were also reduced in the induced OA model rats, and miR-99a agomir injection relieved the cartilage damage. At the molecular level, miR-99a overexpression downregulated the levels of MMP13, ß-catenin, Bax, and caspase-3 protein expression and upregulated the levels of COL2A1 and Bcl-2 protein expression in the in vitro OA-like chondrocyte model and also in the experimental OA model rats. CONCLUSIONS: Our data showed that miR-99a alleviated apoptosis and extracellular matrix degradation by targeting FZD8, and thereby suppressed the development and progression of experimentally induced spine osteoarthritis.

Targeting of EIF4EBP1 by miR-99a-3p affects the functions of B lymphocytes via autophagy and aggravates SLE disease progression.

Excessive activation of immune cells plays a key role in the pathogenesis of systemic lupus erythematosus (SLE). The regulation of immune cells by miRNAs is a research hotspot. In this study, second-generation high-throughput sequencing revealed a reduction in miR-99a-3p expression in patients with SLE; however, the specific mechanism underlying this phenomenon remains unclear. After transfection with an miR-99a-3p agomir, the proliferation of Ball-1 cells decreased and the levels of their apoptosis increased. The opposite effects were observed in cells transfected with the miR-99a-3p antagomir. Luciferase reporter assay indicated that miR-99a-3p directly targeted EIF4EBP1. Rescue experiments confirmed the proposed interaction between miR-99a-3p and EIF4EBP1. In vitro, in vivo and clinical investigations further confirmed that the miR-99a-3p agomir reduced the expression of EIF4EBP1, LC3B and LAMP-2A. In the in vivo experiments, serum levels of anti-nuclear antibodies, double-stranded DNA, IgE, IgM, IL-6, IL-10 and B lymphocyte stimulator were higher in mice from the antagomir group than those in mice from the MRL/lpr group. Furthermore, the protein and mRNA levels of EIF4EBP1, LC3B and LAMP-2A, the intensity of immunohistochemical staining of EIF4EBP1, LC3B and LAMP-2A, the urinary protein levels, and the C3 immunofluorescence deposition increased in mice from the antagomir group. The upregulation of miR-99a-3p expression protected B cells from EIF4EBP1-mediated autophagy, whilst the downregulation of miR-99a-3p expression induced autophagy via the EIF4EBP1-mediated regulation of the autophagy signalling pathway in B cells isolated from individuals with SLE. Based on these results, miR-99a-3p and EIF4EBP1 may be considered potential targets for SLE treatment.

MicroRNA-99a-3p/GRP94 axis affects metastatic progression of human papillary thyroid carcinoma by regulating ITGA2 expression and localization.

Papillary thyroid cancer (PTC) usually has favorable prognosis; however, distant metastasis is a leading cause of death associated with PTC. MicroRNA-99a-3p (miR-99a-3p) is a member of the miR-99 family that is shown to be a tumor suppressor in various human cancers including the anaplastic thyroid cancer, another type of thyroid cancer. The Cancer Genome Atlas database and our previous study reported that miR-99a-3p is downregulated in human PTC tissues as well as human papillary thyroid carcinoma B-CPAP and TPC-1 cell lines. However, its pathological role in PTC remains unclear, especially its impact on PTC metastasis. In the present study, the role of miR-99a-3p in PTC metastasis was molecularly evaluated in in vitro and in vivo models. Our functional study revealed that overexpressing miR-99a-3p significantly suppresses epithelial-mesenchymal transition (EMT) and anoikis resistance as well as migration and invasion of B-CPAP and TPC-1 cells. The mechanical study indicated that glucose-regulated protein 94 (GRP94) is the direct target of miR-99a-3p. Moreover, GRP94 overexpression reverses the inhibitory effect of miR-99a-3p on PTC metastasis. In addition, the miR-99a-3p/GRP94 axis exerts its effect via inhibiting the expression and cytoplasmic relocation of integrin 2α (ITGA2). Furthermore, in vivo experiments confirmed that miR-99a-3p significantly inhibits tumor growth and lung metastasis in PTC xenograft mice. Overall, our findings suggested that the miR-99a-3p/GRP94/ITGA2 axis may be a novel therapeutic target for the prevention of PTC metastasis.

Circular RNA circFNDC3B protects renal carcinoma by miR-99a downregulation.

Various circular RNAs (circRNAs) have been reported to involve in carcinoma. This study explored the role and mechanism of circRNA circFNDC3B (circFNDC3B) in renal carcinoma (RC). The detection indicators in this paper were viability, colony, and migration, which respectively investigated by Cell Counting Kit-8, colony formation, and migration assay. Reverse transcriptase quantitative polymerase chain reaction tested and cell transfection changed circFNDC3B and miR-99a expression. Moreover, western blot tested relate-proteins of proliferation, migration, and cell pathways were examined by western blot. circFNDC3B was upregulated at RC tissues. circFNDC3B enhanced cell viability, colony and migration, and miR-99a mimic played reverse impacts. Furthermore, circFNDC3B negatively regulated miR-99aand circFNDC3B restrained the janus kinase 1/signal transducer and activator of transcription 3 (JAK1/STAT3) and extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathways by miR-99a downregulation. Overexpression of circFNDC3B enhanced cell viability, colony formation and migration by miR-99a downregulation via JAK1/STAT3 and MEK/ERK pathways.

Benzyl isothiocyanate promotes miR-99a expression through ERK/AP-1-dependent pathway in bladder cancer cells.

Benzyl isothiocyanate (BITC), a bioactive natural product present in cruciferous vegetables, has been proved to prevent cancer progression through various mechanisms. In our previous report, we proved that BITC exhibits antitumor effects in bladder cancer by suppressing IGF1R, FGFR3, and mTOR, which is mediated by miR-99a expression. In this study, we identified the signal pathway involved in regulating miR-99a expression after BITC exposure in bladder cancer. Treatment with different BITC concentrations resulted in induction of miR-99a expression in bladder cancer cell lines. Activation of extracellular signal-regulated protein kinase (ERK) and c-jun N-terminal kinase was observed in bladder cancer after BITC treatment for 24 hours. Interestingly, by using a chemical inhibitor of candidate pathways, we found that only the ERK signal pathway is required for miR-99a expression. Furthermore, we evaluated the transcription factor that may contribute to miR-99a expression in response to BITC treatment. The results indicated that c-Jun/AP-1 was activated after BITC treatment. Moreover, we confirmed c-Jun/AP-1 activation through immunofluorescence and the luciferase reporter assay. The results showed that BITC treatment markedly improved nuclear translocation of c-Jun/AP-1 and luciferase activity dose dependently. Finally, pretreatment with the ERK inhibitor U0126 diminished c-Jun phosphorylation and transcriptional activation, suggesting that BITC elicits ERK/c-Jun signal transduction, which is responsible for miR-99a expression in bladder cancer. The present work identifies the mechanism involved in upregulation miR-99a after BITC treatment, which provides an explanation for BITC biological function in our previous work.

Pathway Analysis of Selected Circulating miRNAs in Plasma of Breast Cancer Patients: A Preliminary Study.

MicroRNAs in the circulation of breast cancer (BC) patients have great potential for the early diagnosis, treatment and monitoring of breast cancer. The aim of this preliminary study was to obtain the expression profile of selected miRNAs in the plasma of BC patients that could discriminate BC patients from healthy volunteers and may be useful in early detection of BC. Significantly deregulated miRNAs were evaluated by pathway analysis with the prediction of potential miRNA targets. The study enrolled plasma samples from 65 BC patients and 34 healthy volunteers. Selected miRNAs were screened in pilot testing by the real-time PCR (qPCR) method, and the most appropriate reference genes were selected for normalisation by the geNorm algorithm. In the final testing, we detected miR-99a, miR-130a, miR-484 and miR-1260a (p < 0.05) as significantly up-regulated in the plasma of BC patients. Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis revealed that all significantly deregulated miRNAs are involved in the Hippo and Transforming Growth Factor-beta (TGF-beta) signalling pathways. Our study confirmed a different profile of selected circulating miRNAs in the plasma of BC patients with an emphasis on some critical points in the analysis process.

MicroRNA-99a-5p suppresses breast cancer progression and cell-cycle pathway through downregulating CDC25A.

In this study, we aimed to explore the association between miR-99a-5p and CDC25A in breast cancer and the regulatory mechanisms of miR-99a-5p on breast cancer. The expressions of messenger RNA and microRNAs in breast cancer tissues and adjacent tissues were analyzed by the Cancer Genome Atlas microarray analysis. Quantitative real-time polymerase chain reaction was conducted to find out the expression levels of miR-99a-5p and CDC25A. The expression levels of proteins (CDC25A, ki67, cyclin D1, p21, BAX, BCL-2, BCL-XL, MMP2, and MMP9) were determined by Western blot analysis. The relationship between miR-99a-5p and CDC25A was predicted and verified by bioinformatics analysis and dual luciferase assay. After transfection, cell proliferation, invasion, and apoptosis of breast cancer tissues were, respectively, observed by cell counting kit-8 assay, transwell assay, and flow cytometry (FCM). Furthermore, the relationship among miR-99a-5p, CDC25A, and cell-cycle progression was determined by FCM assay. The nude mouse transplantation tumor experiment was performed to verify the influence of miR-99a-5p on breast cancer cell in vivo. The expression of miR-99a-5p in breast cancer tissues and cells was significantly downregulated, whereas CDC25A expression was upregulated. MiR-99a-5p targeted CDC25A and suppressed its expression in breast cancer cells. Overexpression of miR-99a-5p and decreased expression of CDC25A could suppress breast cancer cell proliferation and invasion and facilitate apoptosis. Cell-cycle progression was significantly activated by downregulated miR-99a-5p and upregulated CDC25A. Moreover, miR-99a-5p overexpression repressed the expressions of CDC25A, marker ki67, and Cyclin D1 proteins, whereas it upregulated the expression of p21 protein. MicroRNA-99a-5p suppresses breast cancer progression and cell-cycle pathway through downregulating CDC25A.

Refinement of breast cancer molecular classification by miRNA expression profiles.

BACKGROUND: Accurate classification of breast cancer using gene expression profiles has contributed to a better understanding of the biological mechanisms behind the disease and has paved the way for better prognostication and treatment prediction. RESULTS: We found that miRNA profiles largely recapitulate intrinsic subtypes. In the case of HER2-enriched tumors a small set of miRNAs including the HER2-encoded mir-4728 identifies the group with very high specificity. We also identified differential expression of the miR-99a/let-7c/miR-125b miRNA cluster as a marker for separation of the Luminal A and B subtypes. High expression of this miRNA cluster is linked to better overall survival among patients with Luminal A tumors. Correlation between the miRNA cluster and their precursor LINC00478 is highly significant suggesting that its expression could help improve the accuracy of present day's signatures. CONCLUSIONS: We show here that miRNA expression can be translated into mRNA profiles and that the inclusion of miRNA information facilitates the molecular diagnosis of specific subtypes, in particular the clinically relevant sub-classification of luminal tumors.

MiR-99a-5p inhibits bladder cancer cell proliferation by directly targeting mammalian target of rapamycin and predicts patient survival.

Bladder cancer is a common malignancy and miR-99a-5p has been reported to be downregulated in bladder cancer, but its function and the underlying mechanism in bladder cancer development remains largely unclear. Here, we report that miR-99a-5p expression was decreased in bladder cancer compared with the adjacent normal tissues. Receiver operating characteristic curve revealed that miR-99a-5p expression signature had area under curve value of 0.7989 in differing bladder cancer from the adjacent normal tissues. Bladder cancer patients with low expression of miR-99a-5p had a poor survival rate. Gain-of-function and loss-of-function approaches demonstrated that miR-99a-5p inhibited bladder cell proliferation and cell cycle. Furthermore, we identified that mammalian target of rapamycin (mTOR) was a direct target of miR-99a-5p and mTOR restore could rescue the proliferative ability of bladder cancer cells. Moreover, miR-99a-5p/mTOR axis regulated S6K1 phosphorylation. These suggested that miR-99a-5p/mTOR axis might be a therapeutic target for bladder cancer.

MiRNA-99a can regulate proliferation and apoptosis of human granulosa cells via targeting IGF-1R in polycystic ovary syndrome.

PURPOSE: We aimed to evaluate the regulation of miR-99a to the biological functions of granulosa cells in polycystic ovary syndrome (PCOS) via targeting IGF-1R. METHODS: We collected aspirated follicular fluid in both patients with and without PCOS. Granulosa cells (GCs) were isolated through Percoll differential centrifugation to detect both miR-99a and IGF-1R expressions. We further transfected COV434 cells with miR-99a mimics to establish a miRNA-99a (miR-99a) overexpression model. We explored the regulation of miR-99a to the proliferation and apoptosis of human GCs via IGF-1R in COV434. The effect of different insulin concentrations on miR-99a expression was also evaluated. RESULTS: MiR-99a was significantly downregulated while IGF-1R was upregulated in patients with PCOS. MiR-99a can regulate IGF-1R on a post-transcriptional level. After transfection of miR-99a mimics, the proliferation rate was decreased and apoptosis rate was increased significantly in COV434. Exogenous insulin-like growth factor 1 (IGF-1) treatment could reverse the effect of miR-99a. MiR-99a was negatively and dose-dependently regulated by insulin in vitro. CONCLUSIONS: MiR-99a expression was downregulated in patients with PCOS, the degree of which may be closely related to insulin resistance and hyperinsulinemia. MiR-99a could attenuate proliferation and promote apoptosis of human GCs through targeting IGF-1R, which could partly explain the abnormal folliculogenesis in PCOS.

MicroRNA-99a is a novel regulator of KDM6B-mediated osteogenic differentiation of BMSCs.

Skeletal tissue originates from mesenchymal stem cells (MSCs) with differentiation potential into the osteoblast lineage regulated by essential transcriptional and post-transcriptional mechanisms. Recently, miRNAs and histone modifications have been identified as novel key regulators of osteogenic differentiation of MSCs. Here, we identified miR-99a and its target lysine (K)-specific demethylase 6B (KDM6B) gene as novel modulators of osteogenic differentiation of bone mesenchymal stem cells (BMSCs). Microarray profiling and further validation by quantitative real-time RT-PCR revealed that miR-99a was up-regulated during osteoblastic differentiation of BMSCs, and decreased in differentiated osteoblasts. Transfection of miR-99a mimics inhibited osteoblastic commitment and differentiation of BMSCs, whereas inhibition of miR-99a by inhibitors enhances these processes. KDM6B was determined as one of important targets of miR-99a, which was further confirmed by luciferase assay of 3'-UTR of KDM6B. Moreover, HOX gene level decreased after transfection of miR-99a mimics in BMSCs, which indicated that KDM6B is a bona fide target of miR-99a. Furthermore, in a model of in vivo bone regeneration, osteoblast-specific gain- and loss-of-function experiments performed using cranial bone defects revealed that miR-99a mimics-transfected BMSCs reduced bone formation, and conversely, miR-99a inhibitors-transfected BMSCs increased in vivo bone formation. Tissue-specific inhibition of miR-99a may be a potential novel therapeutic approach for enhancing BMSCs-based bone formation and regeneration.

Downregulation of circ_008018 protects against cerebral ischemia-reperfusion injury by targeting miR-99a.

Circular RNAs (circRNAs) are highly expressed in eukaryotic cells and regulate physiological and pathophysiological processes. However, the role of circRNAs in cerebral ischemia-reperfusion (I/R) injury remains largely unknown. In this study, we found that circ_008018 level was higher in the cortical tissue of mice with middle cerebral artery occlusion as compared to those in the sham group 24 h after reperfusion. Knockdown of circ_008018 attenuated cerebral I/R-induced brain tissue damage and neurological deficits in mice by inducing microRNA miR-99a overexpression. The decreased phosphorylation of Akt and glycogen synthase kinase 3ß caused by I/R was partly reversed by circ_008018 silencing or miR-99a overexpression. Taken together, these results provide new insight into the mechanisms of apoptosis resulting from cerebral I/R injury and suggest that targeted inhibition of circ_008018 can protect against subsequent neurological damage.