Forkhead Box O1 Gene Research Articles

The Immunomodulatory Effects of Curcumin on Forkhead Box O1 and MicroRNA-873 in Patients with Osteoarthritis

Osteoarthritis (OA) is among the most prevalent articular disorders, whose incidence is directly related to aging. Due to the antiinflammatory potential of curcumin as the active component of turmeric, the present study evaluated the effects of curcumin on the expression of genes related to T helper 17 (Th17), including forkhead box p3 (FOXP3), forkhead box o1 (FOXO1), transforming growth factor-β (TGFB1) and microRNA-873, human (HSA-MIR-873), in OA patients. Female patients with knee OA (n=30) were randomly categorized into 2 groups, including the intervention group who received curcumin (n=15) and the placebo (n=15) in a double-blind clinical trial for 3 months. The expression of FOXO1, FOXP3, TGFB1, and HSA-MIR-873 genes was evaluated by SYBR Green real-time reverse transcription polymerase chain reaction. In the curcumin group, FOXO1 gene expression was significantly increased, while the increase in FOXP3 gene expression was not significant. Moreover, the expression level of the HSA-MIR-873 gene showed a significant increase in the curcumin group. The modulatory effects of curcumin on Th17 function might be associated with the expression of FOXO1 and HSA-MIR-873 genes.

Association of FOXO3 (rs17069665) gene polymorphism and childhood acute lymphoblastic leukemia in Egypt

BackgroundAcute lymphoblastic leukemia (ALL) is the most common malignancy in children. Genetic variations, particularly gene polymorphisms, have been closely linked to increased susceptibility to ALL. One of those genes is Forkhead box O3 (FOXO3), which is considered a potential tumor suppressor gene. AimThis study intended to examine the potential significance of the FOXO3 (rs17069665) single nucleotide polymorphism (SNP) as a risk factor for childhood ALL, in addition to its effect on the laboratory results, clinical manifestations and the clinical outcome after induction of chemotherapy. Subjects and methodsSixty-six newly diagnosed ALL children and 70 healthy children of matched age and sex as controls were recruited. FOXO3 (rs17069665) polymorphism was detected using TaqMan real time PCR. ResultsHigher frequencies of the (AG) genotype and G-allele of FOXO3 (rs17069665) variant were present in ALL patients in comparing with the controls (16.7 % vs. 4.3 %, p = 0.017 and 11.4 % vs. 2.1 %, p = 0.003, respectively). The frequencies of the FOXO3 (rs17069665) SNP reflected a noticeably higher risk of ALL under diverse genetic models, including the co-dominant model (AG vs. AA, OR = 2.55), dominant (AG + GG vs. AA, OR = 2.81), and allelic (G-allele vs. A-allele, OR = 2.9) models. The single case of c-MYC mutation was observed with the (GG) genotype. No significant association between FOXO3 (rs17069665) SNP polymorphism and response to chemotherapy was found. ConclusionOur findings showed that the FOXO3 (rs17069665) polymorphism was associated with a greater incidence of ALL in Egyptian children, which might be a potential biomarker for ALL susceptibility.

Polymorphism of the Forkhead box-O3 (FOXO3) Longevity Gene rs2802292 and senescence-associated secretory phenotype (SASP) in Indonesian Elderly Population

BACKGROUND: Forkhead box O3 (FOXO3) is a transcription factor that regulates stress resistance, metabolism, cell cycle, and apoptosis. Several studies exhibited the association of the FOXO3 polymorphism rs2802292 with human longevity and protects individuals from degenerative diseases. The emergence of degenerative diseases in the elderly is associated with the accumulation of senescent cells that secrete a secretome known as a senescence-associated secretory phenotype (SASP) consists of several cytokines, chemokines, growth factors, proteases. OBJECTIVE: The aim of this research was to analysis polymorphism of FOXO3 gene rs2802292 G-allele and its impact to the SASP by measuring IL-1α, IL-6, IL-8, and IL-10 in Indonesian elderly populations. METHODS: This study was conducted on 92 elderly subjects living at Jakarta. DNA was isolated from the whole blood then continued with PCR and sequencing for FOXO3 rs2802292 analysis. SASP was analyzed from the plasma using Luminex. Statistical analysis was performed using ANOVA and Kruskal Wallis. RESULTS: The results showed that FOXO3 rs2802292 was detected in Indonesian elderly population as follows GG, GT, TT genotype frequencies were 17.4 %, 42.4 % and 40.2 % respectively. Meanwhile, G and T allele frequencies based on the Hardy-Weinberg equilibrium test were 0.385 and 0.615 respectively. GG genotype was significantly found in subjects with longevity. Nevertheless, SASP analysis was found no significant differences both among GG, GT, TT genotypes. CONCLUSION: The frequency of FOXO3 rs2802292 G-allele in Indonesian elderly population was 0.385 and significantly detected in subjects with longevity. However, the FOXO3 rs2802292 polymorphism did not affect cytokines level as the component of SASP.

Expression of FOXO3 in the skin follicles of goose embryos during embryonic development

ABSTRACT 1. The Forkhead box O3 (FOXO3) transcription factor is a crucial regulator in controlling cell metabolism, proliferation, apoptosis, migration and response to oxidative stress. However, FOXO3 has not previously been studied much in the embryonic skin follicles of geese. 2. This study used Zhedong white geese (Anser cygnoides), Jilin white geese (Anser cygnoides) and Hungarian white geese (Anser anser). The feather follicle structure in the dorsal skin during embryonic stages was examined with haematoxylin and eosin (HE) and Pollak staining. The FOXO3 protein content in the embryonic dorsal skin from feather follicles was detected using western blotting and quantitative real-time PCR. 3. The mRNA expression level of FOXO3 in the dorsal skin of Jilin white geese was highly expressed on embryonic day 23 (E23; P < 0.01), while mRNA expression of FOXO3 was highly expressed in the feather follicle of Hungarian white geese at E28 (P < 0.01). The expression of FOXO3 protein mainly concentrated in the early embryonic phase among these goose breeds (P < 0.05). This suggested that FOXO3 plays a crucial role in the development and growth of embryonic dorsal skin of feather follicles. The location of the FOXO3 protein was determined using the IHC technique, which further verified the effect of FOXO3 in the dorsal skin for feather follicles during embryogenesis. 4. The study demonstrated the differential expression and localisation of the FOXO3 gene among different goose species. It was speculated that the gene could potentially improve goose feather follicle development and feather-related traits and provide a basis for further understanding of FOXO3 function in the dorsal tissue of goose embryos.

The forkhead box O3 (FOXO3): a key player in the regulation of ischemia and reperfusion injury.

Forkhead box O3 is a protein encoded by the FOXO3 gene expressed throughout the body. FOXO3 could play a crucial role in longevity and many other pathologies, such as Alzheimer's disease, glioblastoma, and stroke. This study is a comprehensive review of the expression of FOXO3 under ischemia and reperfusion (IR) and the molecular mechanisms of its regulation and function. We found that the expression level of FOXO3 under ischemia and IR is tissue-specific. Specifically, the expression level of FOXO3 is increased in the lung and intestinal epithelial cells after IR. However, FOXO3 is downregulated in the kidney after IR and in the skeletal muscles following ischemia. Interestingly, both increased and decreased FOXO3 expression have been reported in the brain, liver, and heart following IR. Nevertheless, these contribute to stimulating ischemia and reperfusion injury via the induction of inflammatory response, apoptosis, autophagy, mitophagy, pyroptosis, and oxidative damage. These results suggest that FOXO3 could play protective effects in some organs and detrimental effects in others against IR injury. Most importantly, these findings indicate that controlling FOXO3 expression, genetically or pharmacologically, could contribute to preventing or treating ischemia and reperfusion damage.

The impact of dietary restrictions on the expression of FOXO3 as an anti-ageing biomarker

Ageing involves destruction on the molecular, cellular, and organ level, causing disease and death. Previous research has discovered that both diet and genetics can affect ageing. Forkhead helix box (FOXO) transcription factors are found to be the major transcription factors correlated with longevity. In addition, one of the best-known signals that are also capable to affect ageing is a dietary restriction (DR) which is evolutionary conserved. The objective of this paper is to expand our knowledge regarding the FOXO3 gene expression as anti-ageing biomarker, and how DR could impact its expression. This review was written through a rigorous searching process from various databases including Google Scholar, Science Direct, Scopus, and PubMed, by utilizing keywords such as the ‘anti-ageing’, ‘dietary restriction’ along with ‘forkhead box O3 gene’. FOXO3 exerts its essential role through the protein kinase B (Akt/PKB) signaling pathway, as well as post-translational modifications, further activating downstream target genes. This would result in cell apoptosis, cell cycle arrest, DNA repair, and other important physiological processes. Relating to diet, the FOXO3 gene is expressed when organisms face low nutrient availability. It can also be downregulated by reducing the amount of circulating insulin-like growth factor 1 (IGF-1) level which can be achieved through fasting. Meanwhile, DR can enhance the FOXO3 expression, anti-ageing effects and further establish longevity. However, the research regarding the effect of DR in humans is still limited. Future studies need to investigate the optimal type of DR along with uncovering the molecular mechanism behind the FOXO3 expression and ageing.

SNPs of FOXO1 and Their Interactions Contributes to the Enhanced Risk of Diabetes Among Elderly Individuals.

We have assessed the impact of three single nucleotide polymorphisms (SNPs) of Forkhead Box O1 (FOXO1) and their interaction on susceptibility of type 2 diabetes mellitus in geriatric population from northern India. We genotyped three SNPs (rs2721068, rs17446614, and rs4581585) of FOXO1 gene in 190 elderly individuals with diabetes and 182 unrelated healthy controls of similar ethnicity by using TaqMan SNP assays. SNP-SNP and SNP-environment interactions among polymorphic loci were studied by the multifactor dimensionality reduction (MDR) method. The AA genotype carriers of rs17446614 was associated with the increased susceptibility of diabetes in both adjusted and unadjusted model, whereas rs4581585 was associated with the risk in unadjusted model only. Genotype and minor allele interaction with quantitative parameters revealed that AA genotype of rs17446614 had significantly higher fasting plasma glucose (FPG) in diabetic subjects, also minor allele (A) in patients was positively associated with FPG and glycated hemoglobin. Haplotype Trs2721068Grs17446614Trs4581585 increases the risk of diabetes, whereas carrier of haplotypes Crs2721068Grs17446614Crs4581585 and Crs2721068 Grs17446614Trs4581585 were protective. The MDR analysis revealed that interaction of rs17446614 with body mass index (BMI) increased the susceptibility of diabetes. Therefore presence of rs17446614 variant and its interaction with BMI and haplotype Trs2721068Grs17446614Trs4581585 modulates the risk of diabetes and can be used as a promising tool for identifying high-risk individuals.

MiR-221-3p regulates apoptosis of ovarian granulosa cells via targeting FOXO1 in older women with diminished ovarian reserve (DOR).

In our earlier study, we showed that the expression of microRNA‐221‐3p (miR‐221‐3p) was significantly lower in women of advanced age with diminished ovarian reserve (DOR) compared with young women with normal ovarian reserve (NOR). Therefore, in this study, we aimed to explore how miR‐221‐3p regulates apoptosis of granulosa cells and the pathogenesis of DOR. Bioinformatics prediction and dual‐luciferase reporter assay were conducted to identify the target gene of miR‐221‐3p. miR‐221‐3p expression was manipulated by transfecting KGN cells with miR‐221‐3p mimics, inhibitor, and negative control. Following transfection, apoptosis of granulosa cells was determined by flow cytometry, and the expression of the target gene was measured by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot analysis (WB). In addition, the expression of the target gene in granulosa cells of DOR patients and NOR patients was measured. miR‐221‐3p were found to directly bind the 3ʹ untranslated region of Forkhead box O1 (FOXO1). Transfection with miR‐221‐3p mimics significantly decreased the apoptosis rate of KGN cells compared with transfection with miR‐221‐3p inhibitors. The expression level of miR‐221‐3p was negatively correlated with the messenger RNA and protein levels of the FOXO1 gene. Besides, FOXO1 expression was upregulated in DOR patients. In conclusion, these results provide evidence that downregulation of miR‐221‐3p expression promotes apoptosis of granulosa cells by upregulating FOXO1 expression, thus serving an important role in DOR pathogenesis.

Fertility preservation in women with ovarian cancer: Finding new pathways: A case-control study.

Background Surgery and chemotherapy are the two most common treatments for cancers, including ovarian cancer. Although most ovarian cancers occur over the age of 45 yr, it may involve younger women and affect their reproductive ability.Objective To assess the expression of Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), Forkhead Box O1 (FOXO1), and miR-340 genes in the ovarian cancer tissues as well as ovarian cancer cell lines.Materials and Methods In this case-control study, 30 ovarian cancer samples (with the average age of 37 2.5 years) coupled with their non-tumor marginal tissue (as a control) were collected. Proliferated cell lines were treated with several concentrations of cisplatin, and the half maximal inhibitory concentration (IC50) of cisplatin was quantified by MTT-assay. After RNA extraction, cDNA synthesis and qRT-PCR were done. Finally, the results were analyzed.ResultsWhile the expression levels of miR-340 and FOXO1 genes in tumor samples displayed a significant reduction (p 0.001), the LGR5 gene presented a significant increase in expression (p 0.0001). However, conversely, the expression levels of miR-340 and FOXO1 genes in cisplatin-sensitive cell lines, after 24, 48, and 72 hr of cisplatin treatment, indicated a significant increase (p 0.001) while the expression of LGR5 gene showed a significant decrease in the cisplatin-sensitive cell line (p 0.05).ConclusionThe LGR5, FOXO1, and miR-340 genes can be targeted for early diagnosis and more accurate treatment of ovarian cancer and may prevent some of the ovarian cancer complications such as infertility.

Antineoplastic Activity of an Old Natural Antidiabetic Biguanide on the Human Thyroid Carcinoma Cell Line.

In the last decades, metformin (Met), an herbal anti-diabetic medicine, has been proposed as an anti-cancer agent. Thyroid cancers are the most common malignancy of the endocrine system. Therefore, the current study was performed to assess the effects of Met on cell proliferation and activation of the Phosphoinositide 3- Kinase (PI3K)/Protein kinase B (AKT)/Forkhead Box O1 (FOXO1) signaling pathway in the Medullary Thyroid Carcinoma (MTC) cells. The effects of Met on the expression of REarranged during Transfection (RET) proto-oncogene were also investigated. MTC cell line (TT) was treated with 0, 2.5, 5, 10, 20, 30, 40, 50, and 60 mM concentrations of Met for 24, 48, and 72h. The viability and apoptosis of the treated cells were measured by the 3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Annexin V- Propidium Iodide (PI) assays. The expression level of PI3K, AKT, FOXO1, and RET genes was investigated by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), and phosphorylation of their proteins was determined by the Enzyme-Linked Immunosorbent Assay (ELISA). Results showed that Met significantly decreased the viability of the MTC cells. Met also reduced the expression level of PI3K, AKT, and FOXO1 genes (P<0.05), whereas it elevated the expression level of RET proto-oncogene (P<0.05). It seems that the Met has a cytostatic effect on the TT cells. Our results showed that anti-tumoral effects of Met may be cell type-specific, and according to the induction of RET (as a proto-oncogene) and inhibition of FOXO1 (as a tumor suppressor gene), Met could not be an appropriate agent in the treatment of MTC. The antineoplastic activity of Met has been confirmed against several malignancies in "in vitro" and "in vivo" studies. However, its molecular mechanisms in the treatment of different carcinomas particularly in thyroid cancers are not clearly understood and more studies are required to confirm its exact effect on the MTC.

Evaluation of silent information regulator T (SIRT) 1 and Forkhead Box O (FOXO) transcription factor 1 and 3a genes in glaucoma.

Analysis of the reactive oxygen species (ROS)-detoxifying biomarkers may elucidate the mitochondrial dysfunction in glaucoma pathogenesis. Therefore, we purposed to investigate the effects of ROS-detoxifying molecules including Silent Information Regulator T1 (SIRT1) and Forkhead Box O 1 (FOXO1) and 3a (FOXO3a) transcription factors in patients with glaucoma. Our analyses included 20 eyes from patients with primary open-angle glaucoma (POAG) and 20 eyes from patients with pseudoexfoliation glaucoma (PXG) who were scheduled for trabeculectomy. After extraction of total RNA from trabecular meshwork tissue, we compared the levels of SIRT1, FOXO1and FOXO3a genes in the oxidative pathway with the level of glyceraldehyde-3 phosphate dehydrogenase (GAPDH), the reference gene, using real-time polymerase chain reaction. Relative gene expression was calculated using the threshold cycle (2-ΔΔCT) method. We observed similarly reduced expression levels of SIRT1, FOXO1, and FOXO3a genes versus GAPDH among patient groups (p = 0.40; p = 0.56; p = 0.35, respectively). This is the first study to identify the role of SIRT1 and FOXOs in human TM with glaucoma. Relative expression levels of SIRT1, FOXO1, and FOXO3a genes versus a control gene (GAPDH) were decreased in POAG and PXG groups. Our results show that SIRT1and FOXOs (1-3a) deserve special attention in the pathogenesis of glaucoma.

Astaxanthin as a Putative Geroprotector: Molecular Basis and Focus on Brain Aging.

In recent years, the scientific interest in natural compounds with geroprotective activities has grown exponentially. Among the various naturally derived molecules, astaxanthin (ASX) represents a highly promising candidate geroprotector. By virtue of the central polyene chain, ASX acts as a scavenger of free radicals in the internal membrane layer and simultaneously controls oxidation on the membrane surface. Moreover, several studies have highlighted ASX’s ability to modulate numerous biological mechanisms at the cellular level, including the modulation of transcription factors and genes directly linked to longevity-related pathways. One of the main relevant evolutionarily-conserved transcription factors modulated by astaxanthin is the forkhead box O3 gene (FOXO3), which has been recognized as a critical controller of cell fate and function. Moreover, FOXO3 is one of only two genes shown to robustly affect human longevity. Due to its tropism in the brain, ASX has recently been studied as a putative neuroprotective molecule capable of delaying or preventing brain aging in different experimental models of brain damage or neurodegenerative diseases. Astaxanthin has been observed to slow down brain aging by increasing brain-derived neurotrophic factor (BDNF) levels in the brain, attenuating oxidative damage to lipids, protein, and DNA and protecting mitochondrial functions. Emerging data now suggest that ASX can modulate Nrf2, FOXO3, Sirt1, and Klotho proteins that are linked to longevity. Together, these mechanisms provide support for a role of ASX as a potential geroneuroprotector.

Relationship of polymorphism rs3800231 in FOXO3 gene and clinical severity with oxidative stress markers in sickle cell disease

Sickle cell disease (SCD) is a hereditary disease characterized by a clinical course highly variable that is significantly affected by several modifying factors, whose majority still poorly understood. Due to the importance of the chronic inflammatory and oxidative processes in the SCD pathophysiology, the understanding of the signaling pathways that control the reactive oxygen species (ROS) levels is one of the major fields that need research. Studies involving the genetic sequence of the Forkhead box O3 (FOXO3) suggest that this gene presents a promising target of new genetic markers related to SCD clinical severity. Therefore, the aim of this study was to investigate a possible influence of the FOXO3 polymorphism (c.35-2764A > G; rs3800231) in the FOXO3 gene on the clinical severity and on the oxidative status of SCD individuals. We evaluated 313 blood samples, 196 of SCD patients (SCD) and 117 of individuals without hemoglobinopathies (CG). FOXO3 polymorphism was identified by PCR-RFLP. Biochemical parameters were measured using spectrophotometric methods. Moreover, patients were classified into clinical categories (mild, intermediate, and severe), according to their severity scores previously calculated. We found higher genotypic and allelic frequencies of the wild form (A) of the allele of the SNP rs3800231 in patients with SCD (p < .01), regardless of clinical classification. We did not find any significant involvement of FOXO3 polymorphism in the clinical severity classification, as well as in the severity scores. On the other hand, the mutant allele (G) was related to higher catalase activity (p = .01), along with no alteration on oxidized biomolecule levels (p = .65). Thus, we concluded that SNP rs3800231 did not play a significant role as a direct modifying factor in the clinical severity of SCD but it may be involved in the modulation of the oxidative profile of this genetic disorder.

Amplification-driven BCL6-suppressed cytostasis is mediated by transrepression of FOXO3 and post-translational modifications of FOXO3 in urinary bladder urothelial carcinoma

Muscle-invasive urinary bladder urothelial carcinoma (UBUC) is a lethal disease for which effective prognostic markers and potential therapy targets are still lacking. Previous array comparative genomic hybridization identified that 3q27 is frequently amplified in muscle-invasive UBUCs, one candidate proto-oncogene, B-cell CLL/lymphoma 6 (BCL6), mapped to this region. We therefore aimed to explore its downstream targets and physiological roles in UBUC progression.Methods: Specimens from UBUC patients, NOD/SCID mice and several UBUC-derived cell lines were used to perform quantitative RT-PCR, fluorescence in situ hybridization immunohistochemistry, xenograft, gene stable overexpression/knockdown and a series of in vitro experiments.Results: Amplification of the BCL6 gene lead to upregulation of BCL6 mRNA and protein levels in a substantial set of advanced UBUCs. High BCL6 protein level significantly predicted poor disease-specific and metastasis-free survivals. Knockdown of the BCL6 gene in J82 cells inhibited tumor growth and enhanced apoptosis in the NOD/SCID xenograft model. In vitro experiments demonstrated that BCL6 inhibited cytostasis, induced cell migration, invasion along with alteration of the expression levels of several related regulators. At molecular level, BCL6 inhibited forkhead box O3 (FOXO3) transcription, subsequent translation and upregulation of phosphorylated/inactive FOXO3 through phosphoinositide 3-kinase (PI3K)/AKT serine/threonine kinase (AKT) and/or epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase 1/2 (MAP2K1/2) signaling pathway(s). Two BCL6 binding sites on the proximal promoter region of the FOXO3 gene were confirmed.Conclusion: Overexpression of BCL6 served a poor prognostic factor in UBUC patients. In vivo and in vitro studies suggested that BCL6 functions as an oncogene through direct transrepression of the FOXO3 gene, downregulation and phosphorylation of the FOXO3 protein.

Role of Overexpressed Transcription Factor FOXO1 in Fatal Cardiovascular Septal Defects in Patau Syndrome: Molecular and Therapeutic Strategies.

Patau Syndrome (PS), characterized as a lethal disease, allows less than 15% survival over the first year of life. Most deaths owe to brain and heart disorders, more so due to septal defects because of altered gene regulations. We ascertained the cytogenetic basis of PS first, followed by molecular analysis and docking studies. Thirty-seven PS cases were referred from the Department of Pediatrics, King Abdulaziz University Hospital to the Center of Excellence in Genomic Medicine Research, Jeddah during 2008 to 2018. Cytogenetic analyses were performed by standard G-band method and trisomy13 were found in all the PS cases. Studies have suggested that genes of chromosome 13 and other chromosomes are associated with PS. We, therefore, did molecular pathway analysis, gene interaction, and ontology studies to identify their associations. Genomic analysis revealed important chr13 genes such as FOXO1, Col4A1, HMGBB1, FLT1, EFNB2, EDNRB, GAS6, TNFSF1, STARD13, TRPC4, TUBA3C, and TUBA3D, and their regulatory partners on other chromosomes associated with cardiovascular disorders, atrial and ventricular septal defects. There is strong indication of involving FOXO1 (Forkhead Box O1) gene—a strong transcription factor present on chr13, interacting with many septal defects link genes. The study was extended using molecular docking to find a potential drug lead for overexpressed FOXO1 inhibition. The phenothiazine and trifluoperazine showed efficiency to inhibit overexpressed FOXO1 protein, and could be potential drugs for PS/trisomy13 after validation.

Peroxiredoxin 1 silencing inhibited the growth and promoted apoptosis of pancreatic cancer cells via targeting FOXO3 gene.

ObjectiveOur study aimed to investigate the interaction between peroxiredoxin 1 (Prx1) and forkhead box O3 (FOXO3) and to explore the role of PI3K/AKT pathway in the development of pancreatic cancer.Material and methodsHuman pancreatic normal cells HPDE6-C7 and pancreatic cancer cells PANC-1 were randomly divided into control group, Prx1-silencing (si-Prx1) group, Prx1/FOXO3 dual-silencing (si-Prx1/FOXO3) group, and negative control group. Cell proliferation assay, clone formation assay, and cell apoptosis assay were performed to investigate the effects of Prx1 silencing and FOXO3 silencing on the proliferation and apoptosis ability of pancreatic cancer cells. qRT-PCR and Western blot were performed to study the Prx1 and FOXO3 mRNA in the two cells and FOXO3 protein expression in PANC-1 cells.ResultWe found Prx1 silencing could inhibit growth and promote apoptosis of PANC-1 cells. And Prx1 silencing could decrease the Prx1 mRNA level and increase FOXO3 mRNA level. To further explore the role of Prx1 in PI3K/AKT, we study the cell proliferation and apoptosis ability after adding the PI3K inhibitor and PI3K activator. We observed that PI3K inhibitor could inhibit tumor cell growth and promote cell apoptosis. And PI3K inhibitor also downregulated Prx1 protein expression.ConclusionWe concluded that the Prx1 silencing inhibited the growth and promoted apoptosis of pancreatic cancer cells via modulation of PI3K/AKT pathway by targeting FOXO3 gene.

Screening for candidate genes related with histological microstructure, meat quality and carcass characteristic in pig based on RNA-seq data.

ObjectiveThe aim of the present study was to identify genetic variants based on RNA-seq data, obtained via transcriptome sequencing of muscle tissue of pigs differing in muscle histological structure, and to verify the variants’ effect on histological microstructure and production traits in a larger pig population.MethodsRNA-seq data was used to identify the panel of single nucleotide polymorphisms (SNPs) significantly related with percentage and diameter of each fiber type (I, IIA, IIB). Detected polymorphisms were mapped to quantitative trait loci (QTLs) regions. Next, the association study was performed on 944 animals representing five breeds (Landrace, Large White, Pietrain, Duroc, and native Puławska breed) in order to evaluate the relationship of selected SNPs and histological characteristics, meat quality and carcasses traits.ResultsMapping of detected genetic variants to QTL regions showed that chromosome 14 was the most overrepresented with the identification of four QTLs related to percentage of fiber types I and IIA. The association study performed on a 293 longissimus muscle samples confirmed a significant positive effect of transforming acidic coiled-coil-containing protein 2 (TACC2) polymorphisms on fiber diameter, while SNP within forkhead box O1 (FOXO1) locus was associated with decrease of diameter of fiber types IIA and IIB. Moreover, subsequent general linear model analysis showed significant relationship of FOXO1, delta 4-desaturase, sphingolipid 1 (DEGS1), and troponin T2 (TNNT2) genes with loin ‘eye’ area, FOXO1 with loin weight, as well as FOXO1 and TACC2 with lean meat percentage. Furthermore, the intramuscular fat content was positively associated (p<0.01) with occurrence of polymorphisms within DEGS1, TNNT2 genes and negatively with occurrence of TACC2 polymorphism.ConclusionThis study’s results indicate that the SNP calling analysis based on RNA-seq data can be used to search candidate genes and establish the genetic basis of phenotypic traits. The presented results can be used for future studies evaluating the use of selected SNPs as genetic markers related to muscle histological profile and production traits in pig breeding.

Abstract 2539: MiR-96-5p functions as an oncogenic miRNA by inhibiting apoptosis in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is one of the most aggressive cancers with high mortality worldwide. MicroRNAs (miRNAs) are small non-coding RNAs that have been used as cancer-related biomarkers and expected to be therapeutic agents. We performed genome-wide miRNA expression profiling of paired HCC tumors and non-tumorous liver tissues from patients with primary HCCs using the miRNA microarray (Agilent). We found that miR-96-5p was most significantly up-regulated in HCC tumors compared to non-tumor tissues. Although miR-96-5p is suggested to be an oncogenic miRNA, the function of miR-96-5p remains largely unknown. We identified the caspase-9 gene (CASP9) as a novel target of miR-96-5p, in addition to the forkhead box O1 gene (FOXO1) which is the known target of it. Caspase-9 protein is thought to play a central role in apoptosis and to be a tumor suppressor. Overexpression of miR-96-5p decreased caspase-9 protein expression and resulted in resistance to apoptosis induced by doxorubicin and UV in HCC cells. Our results suggested that miR-96-5p functions as an oncogenic miRNA by inhibiting apoptosis through decreasing caspase-9 expression in HCC. Citation Format: Naoto Iwai, Kohichiroh Yasui, Akira Tomie, Kei Teasaki, Tomoko Kitaichi, Osamu Dohi, Yasuyuki Gen, Yoshito Ito. MiR-96-5p functions as an oncogenic miRNA by inhibiting apoptosis in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2539. doi:10.1158/1538-7445.AM2017-2539

Molecular cloning, characterisation and functional analysis of the duck Forkhead box O3 (FOXO3) gene

1. The Forkhead box O3 (FOXO3) transcription factor is a crucial regulator of cell fate that controls proliferation, apoptosis and differentiation. However, the role of FOXO3 regulation in duck myoblasts is not fully understood.2. The aim of this study was to clone and determine the complete coding sequence (CDS) of the duck FOXO3 gene and to assess its function in myoblasts.3. Primers specific for the predicted duck FOXO3 gene were designed using the public mallard duck reference sequence in GenBank. The CDS was cloned by RT-PCR and double digested to generate the expression vector pEGFP-N1-FOXO3.4. Sequence analysis showed that the full-length FOXO3 CDS is 1467 bp, encoding 488 amino acids and is highly conserved across many bird species. Amino acid sequence analysis revealed a DNA-binding domain (aa 1–77).5. Myoblast transfection with pEGFP-N1-FOXO3 showed that FOXO3 mRNA expression at 24 h was elevated in pEGFP-N1-FOXO3-transfected myoblasts compared to pEGFP-N1-transfected cells or controls. MRF4, MyoD, MyoG, Myf5 and PAX7 mRNA expression in the pEGFP-N1-FOXO3 group was lowest. However, myostatin (MSTN) and PAX3 mRNA expression did not differ.6. These results suggest that FOXO3 plays a critical role in the proliferation and differentiation of duck myoblasts.

Long‑term exposure to leptin enhances the growth of prostate cancer cells.

Obesity correlates with an increased risk of developing prostate cancer (PCa) and leptin plays an important role in PCa progression. Since leptin is produced by adipocytes, the serum leptin level is higher in obese than in non-obese individuals. However, the effects of leptin remain controversial and unclear. The aim of the present study was to investigate the effect of leptin on PCa cell aggressiveness. Three human PCa cell lines (LNCaP, DU145 and PC-3) were treated with recombinant leptin for 28 days. Cell proliferation, migration, and invasion were estimated using the WST assay, a wound-healing assay, and a BD Matrigel invasion assay, respectively. The mechanism underlying the proliferative effect of leptin was investigated by cell transfections with small interfering RNA (siRNA) against the leptin receptor (ObR) or forkhead box O1 (FOXO1), and by immunocytochemistry. Long-term exposure of PCa cells to leptin enhanced their proliferation, migration and invasion. Leptin increased ObR expression and enhanced Akt phosphorylation constitutively. Leptin also increased the phosphorylation of FOXO1 via PI3K signaling and FOXO1 gene silencing enhanced PCa cell proliferation. Leptin induced the translocation of FOXO1 from the nucleus to the cytoplasm. Furthermore, the PI3K inhibitor, LY294002 suppressed this translocation. These results suggested that leptin regulated the subcellular localization of FOXO1 and induced Akt phosphorylation. Additionally, we revealed that leptin increased the expression of cyclin D1 and decreased the expression of p21 protein. In conclusion, long-term exposure to leptin increased the cell proliferation, migration, and invasion of PCa cells through inactivation of FOXO1. This inactivation resulted from exclusion of FOXO1 from the nucleus and its restriction to the cytoplasm through PI3K/Akt signaling. Our findings contribute to an understanding of the association between obesity and PCa aggressiveness.