The differential effects of blocking retinal orexin receptors on the expression of retinal c-fos and hypothalamic Vip, PACAP, Bmal1, and c-fos in Male Wistar Rats.

Orexin A and B (OXA and OXB) and their receptors are expressed in the majority of retinal neurons in humans, rats, and mice. Orexins modulate signal transmission between the different layers of the retina. The suprachiasmatic nucleus (SCN) and the retina are central and peripheral components of the body's biological clocks; respectively. The SCN receives photic information from the retina through the retinohypothalamic tract (RHT) to synchronize bodily functions with environmental changes. In present study, we aimed to investigate the impact of inhibiting retinal orexin receptors on the expression of retinal Bmal1 and c-fos, as well as hypothalamic c-fos, Bmal1, Vip, and PACAP at four different time-points (Zeitgeber time; ZT 3, 6, 11, and ZT-0). The intravitreal injection (IVI) of OX1R antagonist (SB-334867) and OX2R antagonist (JNJ-10397049) significantly up-regulated c-fos expression in the retina. Additionally, compared to the control group, the combined injection of SB-334867 and JNJ-10397049 showed a greater increase in retinal expression of this gene. Moreover, the expression of hypothalamic Vip and PACAP was significantly up-regulated in both the SB-334867 and JNJ-10397049 groups. In contrast, the expression of Bmal1 was down-regulated. Furthermore, the expression of hypothalamic c-fos was down-regulated in all groups treated with SB-334867 and JNJ-10397049. Additionally, the study demonstrated that blocking these receptors in the retina resulted in alterations in circadian rhythm parameters such as mesor, amplitude, and acrophase. Finally, it affected the phase of gene expression rhythms in both the retina and hypothalamus, as identified through cosinor analysis and the zero-amplitude test. This study represents the initial exploration of how retinal orexin receptors influence expression of rhythmic genes in the retina and hypothalamus. These findings could provide new insights into how the retina regulates the circadian rhythm in both regions and illuminate the role of the orexinergic system expression within the retina.

7SK small nuclear RNA (Rn7SK) induces apoptosis through intrinsic and extrinsic pathways in human embryonic kidney cell line.

BACKGROUND: Rn7SK, a highly conserved small nuclear non-coding RNA, controls Polymerase II transcription machinery by activating of the Positive Transcriptional Elongation Factor b (P-TEFb). Apart from its role in transcriptional regulation, the potential functions of Rn7SK in cell apoptosis are poorly understood. In a previous study, we demonstrated that overexpression of 7SK induces apoptosis in HEK cells. However, it remains unclear whether 7SK-mediated apoptosis induction is exerted through the intrinsic or extrinsic pathways. METHODS AND RESULTS: Rn7SK was overexpressed in HEK 293T cell line using Lipofectamine 2000 reagent to investigate its potential apoptotic functions. The overexpression of Rn7SK resulted in reduced cell viability through the induction of apoptosis, as evidenced by MTT assay and Annexin V/PI staining. Concurrently, alterations in the expression levels of key apoptosis-related genes were observed, as determined by quantitative RT-PCR. Furthermore, Rn7SK overexpression led to a decrease in cell proliferation, as assessed by colony formation assay and growth curve analysis. This reduction was associated with downregulated expression of key proliferative-related genes. Additionally, the migration and invasion capabilities of cells were significantly inhibited upon upregulation of Rn7SK, as demonstrated by transwell assays. CONCLUSIONS: This study suggests the apoptotic role of 7SK through both intrinsic and extrinsic pathways, necessitating further investigation into its underlying mechanisms.

Rapid identification of bacteria by the pattern of redox reactions rate using 2',7'-dichlorodihydrofluorescein diacetate.

Bacterial infection is a life-threatening situation, and its rapid diagnosis is essential for treatment. Apart from medical applications, rapid identification of bacteria is vital in the food industry or the public health system. There are various bacterial identification techniques, including molecular-based methods, immunological approaches, and biosensor-based procedures. The most commonly used methods are culture-based methods, which are time-consuming. The objective of this study is to find a fingerprint of bacteria to identify them. Three strains of bacteria were selected, and seven different concentrations of each bacterium were prepared. The bacteria were then treated with two different molar concentrations of the fluorescent fluorophore, dichlorodihydrofluorescein diacetate for 30 minutes. Then, using the fluorescence mode of a multimode reader, the fluorescence emission of each bacterium is scanned twice during 60 minutes. Plotting the difference between two scans versus the bacteria concentration results in a unique fluorescence pattern for each bacterium. Observation of the redox state of bacteria, during 90 minutes, results in a fluorescence pattern that is clearly a fingerprint of different bacteria. This pattern is independent of fluorophore concentration. Mean Squares Errors (MSE) between the fluorescence patterns of similar bacteria is less than that of different bacteria, which shows the method can properly identify the bacteria. In this study, a new label-free method is developed to detect and identify different species of bacteria by measuring the redox activity and using the fluorescence fluorophore, dichlorodihydrofluorescein diacetate. This robust and low-cost method can properly identify the bacteria, uses only one excitation and emission wavelength, and can be simply implemented with current multimode plate readers.

Autophagy, a critical element in the aging male reproductive disorders and prostate cancer: a therapeutic point of view.

Autophagy is a highly conserved, lysosome-dependent biological mechanism involved in the degradation and recycling of cellular components. There is growing evidence that autophagy is related to male reproductive biology, particularly spermatogenic and endocrinologic processes closely associated with male sexual and reproductive health. In recent decades, problems such as decreasing sperm count, erectile dysfunction, and infertility have worsened. In addition, reproductive health is closely related to overall health and comorbidity in aging men. In this review, we will outline the role of autophagy as a new player in aging male reproductive dysfunction and prostate cancer. We first provide an overview of the mechanisms of autophagy and its role in regulating male reproductive cells. We then focus on the link between autophagy and aging-related diseases. This is followed by a discussion of therapeutic strategies targeting autophagy before we end with limitations of current studies and suggestions for future developments in the field.

Investigating the Effects of Maternal Separation on Hypothalamic-Pituitary-Adrenal Axis and Glucose Homeostasis under Chronic Social Defeat Stress in Young Adult Male Rat Offspring.

INTRODUCTION: Given the suggested metabolic regulatory effects of stress-responsive genes and based on the impacts of early-life stress on HPA axis development, this study aimed to characterize the maternal separation (MS) impact on the communication between glucose metabolism and HPA axis dysregulations under chronic social defeat stress (CSDS). METHODS: During the first 2 weeks of life, male Wistar rats were either exposed to MS or left undisturbed with their mothers (Std). Starting on postnatal day 50, the animals of each group were either left undisturbed in the standard group housing (Con) or underwent CSDS for 3 weeks. There were four groups (n = 10/group): Std-Con, MS-Con, Std-CSDS, and MS-CSDS. RESULTS: Early and/or adult life adversity reduced ß-cell number, muscular FK506-binding protein 51 (FKBP51) content, and BMI in adulthood. The reduction of ß-cell number and BMI in the MS-CSDS rats were more profound than MS-Con group. CSDS either alone or in combination with MS reduced locomotor activity and increased and decreased corticotropin-releasing factor type 1 receptor (CRFR1) content, respectively, in hypothalamus and pancreas. Although, under CSDS, MS intensified HPA axis overactivity and reduced isolated islets' insulin secretion, it could promote resilience to depression symptoms. No differences were observed in hypothalamic Fkbp5 gene DNA methylation and glucose tolerance among groups. CONCLUSION: MS exacerbated HPA axis overactivity and the endocrine pancreas dysfunctions under CSDS. The intensified corticosterone secretion and the diminished content of pancreatic CRFR1 protein could be involved in the reduced ß-cell number and islets' insulin secretion under CSDS. The decreased muscular FKBP51 content might be a homeostatic response to slow down insulin resistance development under chronic stress.

In vitro naive CD4+ T cell differentiation upon treatment with miR-29b-loaded exosomes from mesenchymal stem cells.

BACKGROUND: Gene regulation by microRNA (miRNA) is central in T lymphocytes differentiation processes. Here, we investigate miRNA-29b (miR-29b) roles in the reprogramming of T cell differentiation, which can be a promising therapeutic avenue for various types of inflammatory disorders such as rheumatoid arthritis and multiple sclerosis. METHODS AND RESULTS: Adipose Mesenchymal Stem Cell-derived exosomes (AMSC-Exo) enriched with miR-29b were delivered into naive CD4+ T (nCD4+) cells. The expression level of important transcription factors including RAR-related orphan receptor gamma (RORγt), GATA3 binding protein (GATA3), T-box transcription factor 21, and Forkhead box P3 was determined by quantitative Real-Time PCR. Moreover, flow cytometry and Enzyme-linked Immunosorbent Assay were respectively used to measure the frequency of T regulatory cells and the levels of cytokines production (Interleukin 17, Interleukin 4, Interferon-gamma, and transforming growth factor beta. This study indicates that the transfection of miR-29b mimics into T lymphocytes through AMSC-Exo can alter the CD4+ T cells' differentiation into other types of T cells. CONCLUSIONS: In conclusion, AMSC-Exo-based delivery of miR-29b can be considered as a new fascinating avenue for T cell differentiation inhibition and the future treatment of several inflammatory disorders.

Inhibition of the retinal orexin receptors affects the hypothalamic-pituitary-gonadal axis through retinal pituitary adenylate cyclase activating polypeptide (PACAP) in male Wistar rats.

Orexins A and B (OXA and OXB) and their receptors are expressed in the retina of both human and rodents and play a vital role in regulating signal transmission circuits in the retina. There is an anatomical-physiological relationship between the retinal ganglion cells and suprachiasmatic nucleus (SCN) through glutamate as a neurotransmitter and retinal pituitary adenylate cyclase-activating polypeptide (PACAP) as a co-transmitter. SCN is the main brain center for regulating the circadian rhythm, which governs the reproductive axis. The impact of retinal orexin receptors on the hypothalamic-pituitary-gonadal axis has not been investigated. Retinal OX1R or/and OX2R in adult male rats by 3 µl of SB-334867 (1 µg) or/and 3 µl of JNJ-10397049 (2 µg) were antagonized via intravitreal injection (IVI). Four time-periods were considered (3, 6, 12, and 24 h) for the controls without any treatment, SB-334867, JNJ-10397049, and SB-334867 + JNJ-10397049 groups. Antagonizing retinal OX1R or/and OX2R resulted in a significant elevation of retinal PACAP expression compared to control animals. In addition, expression of GnRH increased non-significantly in the hypothalamus over the 6 h of the study, and the serum concentration of LH decreased significantly in the SB-334867 group after 3 h of injection. Furthermore, testosterone serum levels declined significantly, especially within 3 h of injection; serum levels of progesterone were also exposed to a significant rise at least within 3 h of injection. However, the retinal PACAP expression changes were mediated by OX1R more effectively than by OX2R. In this study, we report the retinal orexins and their receptors as light-independent factors by which the retina affects the hypothalamic-pituitary-gonadal axis.

miR-146b-5p and miR-520h Expressions Are Upregulated in Serum of Women with Recurrent Spontaneous Abortion.

Unexplained recurrent spontaneous abortion (URSA) is characterized by two or more consecutive pregnancy losses before the 20th week of gestation with unknown etiology. Dysregulation of microRNAs (miRNAs) expression has been reported in reproductive diseases. This study aimed to compare differentially expressed miRNAs in the serum samples between URSA patients and healthy individuals. URSA cases were confirmed by a gynecologist. Peripheral blood sample was gathered from 9 URSA patients, 15 normal pregnant, and 10 non-pregnant women without abortion history. After separating serum, the expression levels of the miR-101-3p, miR-517c-3p, miR-146b-5p, miR-221-3p, and miR-520 h were measured by qRT-PCR assay. The circulating level of miR-520 h in URSA patients was significantly up-regulated compared with healthy pregnant (P < 0.01) and healthy non-pregnant (P = 0.002) women. Furthermore, miR-520 h expression was significantly different between healthy non-pregnant and pregnant women (P = 0.002). Statistical analysis indicated miR-146b-5p expression was significantly up-regulated in URSA patients compared to normal pregnant women (P = 0.018). However, the transcription level of miR-146b-5p was insignificantly different between normal non-pregnant women and the other two groups. Also, circulating levels of miR-101-3p, miR-221-3p, and miR-517c-3p were not significantly different in the studied groups. Statistical analysis showed significant correlations between both miR-221-3p and miR-517c-3p and other miRNAs (P < 0.05). The circulating levels of miR-520 h and miR-146b-5p could be considered biomarkers for URSA diagnosis. Also, miR-517c-3p and miR-221-3p might play a regulatory role in other miRNAs expressions during pregnancy. Previous work, in contrary to our findings, claims that the expression levels of miR-221-3p, miR-101-3p, and miR-517c-3p increased in plasma and tissue samples of patients with URSA. However, our research for the first time indicates that the expression level of miR-520 h and miR-146b-5p in the serum of these patients has increased. Future investigations are necessary to confirm these findings.

Nonviral siRNA delivery systems for pancreatic cancer therapy.

The serious drawbacks of the conventional treatment of pancreatic ductal adenocarcinoma (PDAC) such as nonspecific toxicity and high resistance to chemo and radiation therapy, have prompted the development and application of countless small interfering RNA (siRNA)-based therapeutics. Recent advances in drug delivery systems hold great promise for improving siRNA-based therapeutics and developing a new class of drugs, known as nano-siRNA drugs. However, many fundamental questions, regarding toxicity, immunostimulation, and poor knowledge of nano-bio interactions, need to be addressed before clinical translation. In this review, we provide recent achievements in the design and development of various nonviral delivery vehicles for pancreatic cancer therapy. More importantly, codelivery of conventional anticancer drugs with siRNA as a new revolutionary pancreatic cancer combinational therapy is completely discussed.

Antimicrobial peptides as potential therapeutics for breast cancer.

Breast cancer is the most common and deadliest cancer in women worldwide. Although notable advances have been achieved in the treatment of breast cancer, the overall survival rate of metastatic breast cancer patients is still considerably low due to the development of resistance to breast cancer chemotherapeutic agents and the non-optimal specificity of the current generation of cancer medications. Hence, there is a growing interest in the search for alternative therapeutics with novel anticancer mechanisms. Recently, antimicrobial peptides (AMPs) have gained much attention due to their cost-effectiveness, high specificity of action, and robust efficacy. However, there are no clinical data available about their efficacy. This warrants the increasing need for clinical trials to be conducted to assess the efficacy of this new class of drugs. Here, we will focus on the recent progress in the use of AMPs for breast cancer therapy and will highlight their modes of action. Finally, we will discuss the combination of AMP-based therapeutics with other breast cancer therapy strategies, including nanotherapy and chemotherapy, which may provide a potential avenue for overcoming drug resistance.

A novel natural antisense transcript at human SOX9 locus is down-regulated in cancer and stem cells.

OBJECTIVE: SOX9 is a key transcription factor with important roles in regulating proliferation and differentiation of various cell types. Dysregulation of SOX9 expression has been involved with pathogenesis of different developmental, degenerative, and neoplastic disorders. Natural antisense transcripts (NATs) are long non-coding RNAs with increasing significance in regulation of gene expression. However, the presence of a NAT at SOX9 locus has been so far unclear. RESULT: We detected a natural antisense transcript at SOX9 locus (SOX9-NAT) through strand-specific RT-PCR. In contrast to SOX9 sense RNA (mRNA), SOX9-NAT was down-regulated in cancer tissues and cell lines compared with their normal counterparts. In addition, reciprocal to SOX9 mRNA, SOX9-NAT was also down-regulated in human embryonic stem cells in comparison with human fibroblasts in vitro. CONCLUSION: The negative correlation between SOX9 mRNA and SOX9-NAT was confirmed by analyzing qPCR data, as well as RNA-Seq datasets of several human cancers. Our data suggest a functional role for SOX9-NAT in the regulation of SOX9 mRNA as a potential target in cancer treatment and regenerative medicine.

Effect of mesenchymal stem cells-derived exosomes on tumor microenvironment: Tumor progression versus tumor suppression.

Mesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into different cell types. Owing to their immunosuppressive and anti-inflammatory properties, they are widely used in regenerative medicine, but they have a dual effect on cancer progression and exert both growth-stimulatory or -inhibitory effects on different cancer types. It has been proposed that these controversial effects of MSC in tumor microenvironment (TME) are mediated by their polarization to proinflammatory or anti-inflammatory phenotype. In addition, they can polarize the immune system cells that in turn influence tumor progression. One of the mechanisms involved in the TME communications is extracellular vesicles (EVs). MSCs, as one of cell populations in TME, produce a large amount of EVs that can influence tumor development. Similar to MSC, MSC-EVs can exert both anti- or protumorigenic effects. In the current study, we will investigate the current knowledge related to MSC role in cancer progression with a focus on the MSC-EV content in limiting tumor growth, angiogenesis, and metastasis. We suppose MSC-EVs can be used as safe vehicles for delivering antitumor agents to TME.

Rn7SK small nuclear RNA is involved in cellular senescence.

Rn7SK is a conserved small nuclear noncoding RNA which its function in aging has not been studied. Recently, we have demonstrated that Rn7SK overexpression reduces cell viability and is significantly downregulated in stem cells, human tumor tissues, and cell lines. In this study, we analyzed the role of Rn7SK on senescence in adipose tissue-derived mesenchymal stem cells (AD-MSCs). For this purpose, Rn7SK expression was downregulated and upregulated via transfection and transduction, respectively, in AD-MSCs and subsequently, various distinct characteristics of senescence including cell viability, proliferation, colony formation, senescence-associated ß galactosidase activity, and differentiation potency was analyzed. Our results demonstrated the transient knockdown of Rn7SK in MSCs leads to delayed senescence, while its overexpressions shows opposite effects. When osteogenic differentiation was started, however, they exhibited a greater differentiation potential than the original MSCs, suggesting a potential tool for stem cell-based regenerative medicine.

A simple and highly efficient method for transduction of human adipose-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into a wide range of cell types and provide a potential to transfer therapeutic protein in vivo, making them valuable candidates for gene therapy and cell therapy. However, using MSCs in in vivo is limited due to the low rate of transfection and transduction efficacy. Therefore, developing methods to efficiently transfer genes into MSCs would provide a number of opportunities for using them in the clinic. Here, we introduce a simple and robust method for efficient transduction of human adipose-derived MSCs by modification under the culture condition of human embryonic kidney cells 293 (HEK293T) and MSCs. Moreover, as a transduction enhancer, polybrene was replaced with Lipofectamine, a cationic lipid. Therefore, we showed that transduction of primary cells can be increased efficiently by modifying the culture condition.

Transient induction of Cdk9 in the early stage of differentiation is critical for myogenesis.

Cdk9 is a serine-threonine protein kinase that has been recognized as a regulator of cardiac differentiation. Recently, we have reported that transient induction of Cdk9 using noncoding RNA targeting Cdk9 sequences results in efficient cardiac differentiation. Concerning Cdk9 regulatory roles, here, we proposed whether constant overexpression of Cdk9 might influence the differentiation of myoblast C2C12 cells into myotubes. We overexpressed Cdk9 in mouse myoblast C2C12 cells to investigate its regulatory roles on myogenic differentiation. Upon Cdk9 overexpression, the expression level of myogenic regulatory factors was determined. Moreover, the expression profile of three important myomiRs consist of miR 1, 133 and 206 was examined during the differentiation process. Although Cdk9 expression is necessary for inducing differentiation in the early stage of myogenesis, continuous Cdk9 expression inhibits differentiation by modulating myomiRs and myogenic gene expression. Our results indicate that the transient induction of Cdk9 in the early stage of differentiation is critical for myogenesis.

Sirt1 antisense transcript is down-regulated in human tumors.

Natural antisense transcripts (NATs) have recently been associated with the development of human cancers. Recent studies have shown that a natural antisense transcript (NAT) is present in Sirt1 gene which encodes a NAD-dependent deacetylase. Interestingly, expression of Sirt1 mRNA changes during development and progression of human cancers. However, it remains unclear to what extent Sirt1 antisense transcript (AS) may contribute to changes in the expression of Sirt1 mRNA. To determine this, we used quantitative measurement of RNA to reveal relationship between Sirt1 mRNA and Sirt1-AS across human cancer tissues, cell lines and stem cells. While Sirt1 mRNA level was increased in cancer cell lines and cancer tissues, the expression level of Sirt1-AS was lower in cancers compared to controls. This inverse correlation was observed in the expression of Sirt1 sense and antisense transcripts in normal and cancer tissues suggesting a functional role for Sirt1-AS in regulation of Sirt1 mRNA.

Cell type-dependent functions of microRNA-92a.

The role of miR-17/92 family in development and progression of various cancers has been established. The members of this miRNA family have been shown to be over expressed and target various genes within proliferation, metastasis and angiogenesis pathways. Although all members might be overexpressed in a certain cancer type, only certain members of the family may have roles in progression of that cancer. In this study, we have chosen miR-92a, a member of the miR-17/92 family to compare its function in three different cancer cell lines. HL60, MCF7, and Jurkat cell lines were transduced with miR-92a and proliferation and apoptosis was measured in these cells by cell count, MTT, and caspase assays. Although in comparison to pre-miR-17/92, the level of miR-92a is higher in Jurkat cells compared to MCF7 and HL60 cells, here we have shown that increasing miR-92a levels results in apoptosis in Jurkat cells and proliferation in MCF7 and HL60 cells. miR-92a was also microinjected into mice fertilized eggs and after dissection, apoptosis was only observed in white pulp of spleen that is mainly made up of white blood cells. Our results show that miR-92a possesses a cell-type dependent function.

Rn7SK small nuclear RNA is involved in neuronal differentiation.

Rn7SK-mediated global transcriptional regulation, key function of this small nuclear RNA (snRNA), is mediated by inhibition of the positive transcription elongation factor b (P-TEFb). Recently, we have identified a potential anti-proliferative and tumor-suppressive function of Rn7SK. However, its possible regulatory role in development and cell programming has not been investigated so far. Here, we examined transcriptional levels of Rn7SK in different mouse organs. Interestingly, an increased expression level of the RNA was observed in the brain. Furthermore, we could demonstrate that Rn7SK has a dynamic expression pattern during brain development from embryo to adult: 7SK snRNA expression was particularly high at embryonic day (E) 18.5 and adult stages, while a low level of this non-coding RNA was detected at E11.5. Moreover, a decreased transcription level was identified in proliferating progenitors whereas a strong upregulation of Rn7SK was observed during neural differentiation in vivo. Similar to the in vivo situation, in vitro neuronal differentiation experiments employing embryonic stem cells (ESCs) demonstrated the same expression pattern of 7SK with high expression levels in differentiating neurons. Neuronal differentiation of ESCs was compromised when we knocked down Rn7SK, indicating an important role of 7SK in the acquisition of a neural fate.

CDK9 Regulates Apoptosis of Myoblast Cells by Modulation of microRNA-1 Expression.

Cdk9 is the catalytic core of the positive transcription elongation factor b (P-TEFb) and regulates transcriptional elongation factors by phosphorylation of RNA pol II. Apart from its role on myogenic gene expression, Cdk9 regulation of muscle-specific microRNAs in the early stage of cardiomyogenesis is poorly understood. Here we demonstrate that Cdk9 not only regulates myogenic transcription factors, but also controls muscle-specific microRNAs. During cardiac differentiation of mouse embryonic stem cells, high Cdk9 expression preceded up-regulation of miR-1. To investigate potential regulatory roles of Cdk9 on cardiac microRNAs and myogenesis genes, we overexpressed Cdk9 in myoblast C2C12 cells, which resulted in significant induction of miR-1 and miR-206, while miR-133 was downregulated. Moreover, expression levels of MyoD and Srf, key regulators of myogenesis, also increased in cells with overexpression of Cdk9. We further observed Cdk9-mediated apoptosis in C2C12 cells corresponding to induction of miR-1 expression levels. Thus, Cdk9 plays a complex role in myocyte progenitor differentiation and apoptosis by regulating myogenic protein and muscle-specific microRNA expression. J. Cell. Biochem. 119: 547-554, 2018. © 2017 Wiley Periodicals, Inc.

The power of precise bioinformatics prediction of miRNA:mRNA interactions:miR-4699 as a potential inducer of Wnt signaling pathway.

microRNAs have attracted interest because of their regulatory effects on gene expression. Experimental detection of potential targets of miRNAs is a laborious task. Considering the expensive techniques of detection, computational approaches for miRNA target prediction can be used as the first step in miRNA research. A large number of tools and algorithms have been developed during the last two decades, led to problems such as confusion in selecting an appropriate tool and false positive or negative results. Therefore, one of the most frequent problems and critical issues of miRNA research is finding a reliable miRNA target prediction tool. In this study, we have proposed a research direction and introduced user-friendly and current databases and tools with the highest accuracy. To verify whether our proposed research direction is practical, we have provided a case example of predicting a miRNA which can target negative regulators of osteogenesis and experimentally evaluated the accuracy of the prediction results by Real-Time PCR and Luciferase assay. The results of RT-qPCR and Luciferase assay indicated a significant decline in expression of Dickkopf-related protein 1 (DKK1) and tumor necrosis factor ligand superfamily member 11 (TNFSF11) as the key negative regulators of osteogenesis upon overexpression of miR-4699-3p. The results emphasize the validity and importance of accurate in silico investigation as the first step in experimental studies. This is the first report detailing the prediction and validation of miR-4699-3p target genes. We suggest hsa-miR-4699 for further investigation as an osteogenic miRNA for therapeutics purposes.