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Objective To investigate whether the mechanism of action of electro-acupuncture in MPTP-induced Parkinson's disease (PD) model mice is related to neuroinflammation mediated by the forkhead box protein O1 (FoXO1)/NOD-like receptor heat protein structural domain-related protein 3 (NLRP3) signaling pathway. Methods Thirty mice were randomly divided into control (Ctrl), model (MPTP), and electro-acupuncture (EA) groups, with 10 mice in each group. PD mouse model was prepared by intraperitoneal injection of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) 30 mg/(kg·d) for 7 d consecutively. Behavioral changes of mice were observed by open field and pole-climbing experiments; α-synuclein (α-syn) expression in the substantia nigra of the midbrain was observed by immunohistochemistry; FoXO1, NLRP3, ASC, and Caspase-1 were determined by Western blot; and the expression of inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β ) was detected by ELISA in the substantia nigra of the midbrain. Results Mice in the MPTP group showed significant behavioral deficits, with a significant increase in midbrain substantia nigra α-syn (P < 0.01), elevated levels of Iba-1, FoXOl, NLRP3, ASC, and Caspase-1 proteins (P < 0.05 and P < 0.01, respectively), and a significant increase in the levels of TNF-α, and IL-1β (P< 0.01). The treatment of EA was effective in improve the dyskinesia of PD mice, improve motor balance and coordination (P< 0.01), reduce the abnormal aggregation of α-syn (P < 0.01), reduce the abnormal activation of microglia, and alleviate neuroinflammation (P < 0.05). Conclusion Electro-acupuncture may play a protective role in PD by modulating FoXO1/NLRP3 pathway-mediated neuroinflammation. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of microRNA-582-5p (miR-582-5p) on neuronal damage in neonatal rats with hypoxic ischemic encephalopathy (HIE) through targeted regulation of forkhead box transcription factor O1 (FOXO1). Methods Newborn rats (n=90) were randomly grouped into the control (NC) group, the model (HIE) group, the miRNA non-specific control (LV-miRNA-NC) group, the miR-582-5p overexpression (LV-miR-582-5p) group, the miR)582-5p overexpression+mRNA control (LV-miR-582-5p+LV-NC) group and the miR-582-5p overexpression+FOXO1 overexpression (LV-miR-582-5p+LV-FOXO1) group. HIE model was established in all groups except the NC group, and neurological deficits were scored on rats. TTC staining was applied to measure the volume of cerebral infarction. Real-time PCR was applied to detect miR-582-5p and FOXO1 expression. Dual fluorescence reporter gene experiment was applied to detect the targeting relationship between miR-582-5p and FOXO1. HE staining method was applied to observe pathological changes in hippocampal tissue. TUNEL and NeuN fluorescence dual labeling co localization were applied to detect neuronal apoptosis in hippocampal tissue. Immunohistochemistry was applied to detect expressions of FOXO1 and Caspase-3 proteins. Results There was a targeting relationship between MiR-582-5p and FOXO1. Compared with the NC group, the neurological deficit score, cerebral infarction volume, FOXO1 expression, neuronal apoptosis rate, FOXO1 and Caspase-3 protein expression were increased in the HIE group, and the miR-582-5p expression obviously decreased, the hippocampal tissue showed obvious pathological damage (P<0.05). Compared with the LV-miRNA-NC group, the neurological deficit score, cerebral infarction volume, FOXO1 expression, neuronal apoptosis rate, FOXO1 and Caspase-3 protein expression obviously decreased in the LV-miR-582-5p group, and the miR-582-5p expression obviously increased, the pathological damage of hippocampus tissue was obviously improved (P<0.05). LV-FOXO1 was able to reverse the protective effect of LV-miR-582-5p on neuronal damage in HIE rats. Conclusion MiR-582-5p can directly target FOXO1, negatively regulate FOXO1 expression, reduce neuronal apoptosis in HIE neonatal rats, and have a protective effect on neural injury. [ABSTRACT FROM AUTHOR]

Objective To discuss the role of forkhead box O1 (FoxO1) in the injury and apoptosis of podocytes induced by homocysteine (Hcy). Methods Ten Cbs+/+ mice with normal cysteine β -synthase (Cbs) gene and 10 Cbs+/- mice with single gene knockout were fed a high methionine diet. After 8 weeks, the mice were sacrificed to collect kidney tissues. Morphological changes of glomerulus were observed by PAS staining. Podocytes were cultured in vitro and divided into the control group and the Hcy group (treated with cell culture medium containing 80 μmol/L Hcy for 48 h). Podocytes were transfected with Ad-FoxO1 overexpressed adenovirus and Sh-FoxO1 interfering adenovirus carrying green fluorescent protein (GFP), and they were divided into the control group, the Ad-GFP group, the Ad-FoxO1 group, the Sh-NC group, the Sh-FoxO1 group, the Ad-GFP+Hcy group, the Ad-FoxO1+Hcy group, the Sh-NC+Hcy group and the Sh-FoxO1+Hcy group. Western blot assay was used to detect the expression of Podocin and Nephrin, FoxO1 and apoptosis-related B lymphocytoma-2 (Bcl-2), Bcl-2 associated X protein (Bax) and cystine proteinase 12 (Caspase12) proteins in renal tissues and podocytes of mice. The expression of FoxO1 mRNA in mouse kidney tissue and podocytes was detected by qPCR. Results PAS staining results showed that the glomerular structure was normal, the basement membrane was clear and distributed evenly in Cbs+/+ mice, while the glomerular basement membrane presented intermittent thickening and the mesangial matrix increased in Cbs+/- mice. Compared with Cbs+/+ mice, the expression levels of Podocin, Nephrin and FoxO1 protein and FoxO1 mRNA were significantly decreased in Cbs+/- mice (P＜0.01). Compared with the control group, the expression of FoxO1 protein and mRNA in podocytes were significantly decreased in the Hcy group after treated with Hcy (P＜0.01). After overexpression of FoxO1 in podocytes, compared with the Ad-GFP+Hcy group, the protein expression of Podocin and Nephrin were significantly increased, both the ratio of Bax/Bcl-2 and the protein expression of Caspase12 were significantly decreased in the Ad-FoxO1+Hcy group (P＜0.05). After FoxO1 interference, compared with the Sh-NC+Hcy group, Podocin and Nephrin protein expressions were significantly decreased, Bax/Bcl-2 ratio and Caspase12 protein expression were significantly increased in the Sh-FoxO1+Hcy group (P＜0.05). Conclusion FoxO1 can reduce Hcy- induced renal podocyte injury and apoptosis in mice. [ABSTRACT FROM AUTHOR]

To investigate the role of silent mating type information regulator 2 homolog 3 (SIRT3) - forkhead box O1(FOXO1) pathway-mediated autophagy in myocardial ischemia-reperfusion (IR) injury. Methods SIRT3 overexpression stable strain was constructed by lentivirus infection of H9C2 cells. The cells were divided into four groups: the normal control (NC) group, the ischemia-reperfusion (IR) group, the SIRT3 overexpression +IR (S+IR) group and the SIRT3 overexpression +IR+ SIRT3 inhibitor (S+IR+3-TYP) group. The expressions of SIRT3, Ac-FOXO1, autophagy labeled protein LC3B, Beclin1 and apoptotic proteins Bcl-2 and Bax were detected by Western blot assay. The apoptosis rate was detected by flow cytometry. The level of lactate dehydrogenase (LDH) in the supernatant of cell culture was determined by automatic biochemical analyzer. Results Compared with the NC group, the expression of SIRT3 was decreased, AcFOXO1 was increased, the expressions of autophagy related proteins Beclin1 and LC3B were increased, the expression of antiapoptotic protein Bcl-2 was decreased, the expression of pro-apoptotic protein Bax was increased, LDH release and cell apoptosis rate were increased in the IR group (P＜0.05). Compared with the IR group, the expression of SIRT3 increased, Beclin1, LC3B and Bcl-2 were increased, the expressions of Ac-FOXO1, pro-apoptotic protein Bax were decreased, LDH release and cell apoptosis rate were decreased in the S+IR group (P＜0.05). After adding SIRT3 inhibitor 3-TYP, there was no significant change in SIRT3 expression compared with that of the S+IR group, but the expression of Ac-FOXO1 was increased, the expressions of Beclin1 and LC3B were decreased, the expression of anti-apoptotic protein Bcl-2 was decreased, the expression of pro-apoptotic protein Bax was increased, LDH release and cell apoptosis rate were increased (P＜0.05). Conclusion The activation of SIRT3-FOXO1 pathway can increase autophagy level of H9C2 cells, and has a protective effect on myocardial ischemia reperfusion injury. [ABSTRACT FROM AUTHOR]

To investigate the expression of microRNA (miR)-199a in gestational diabetes mellitus (GDM) and the mechanism of silent mating type information regulator 2 homolog 1/forkhead box transcription factor O1 (SIRT1/ FOXO1) pathway involved in insulin resistance (IR). Methods A total of 56 GDM pregnant women were selected as the research objects (GDM group), and 60 normal pregnant women were selected as the control group. Real-time fluorescence quantitative PCR (qPCR) was used to detect the level of miR-199a in placenta. The insulin resistance index (HOMA-IR) was calculated. Pearson method was used to analyze the correlation between miR-199a and HOMA-IR. Human beings chorial trophcytes HTR-8/SVneo cells were cultured in vitro, and IR model was made. The function of miR-199a was verified. Cells were divided into the model group, the miRNA inhibitor NC group, the miR-199a inhibitor group, the miRNA mimic NC group and the miR-199a mimic group. The expression levels of miR-199a were detected by qPCR in each group. The intracellular glucose uptake was measured by anthrone method. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by kit. The protein levels of SIRT1, FOXO1 and acely (Ac)-FOXO1 were detected by Western blot assay. The double luciferase reporter gene assay was used to identify the targeting relationship between miR199a and SIRT1. Results Compared with the normal pregnant women, the levels of miR-199a and HOMA-IR in placenta were higher in GDM patients (P＜0.05), and there was a positively correlation between miR-199a level and HOMA-IR (P＜ 0.05). The combined action of palmitic acid and insulin on cells for 24 h could establish an IR model. Compared with the model group and the miRNA inhibitor NC group, the levels of miR-199a and MDA were decreased in the miR-199a inhibitor group, while the levels of glucose uptake, SOD and SIRT1 protein were increased (P＜0.05). Compared with the model group and the miRNA mimic NC group, the levels of miR-199a, Ac-FOXO1 protein and MDA were increased in the miR-199a mimic group, while the levels of glucose uptake and SIRT1 protein were decreased (P＜0.05). Tarbase database analysis showed that miR-199a and SIRT1 had complementary binding sites, which were verified by double luciferase. Conclusion The level of miR-199a in placenta is highly expressed in GDM patients. The increased miR-199a can promote FOXO1 acetylation by targeting down-regulation of SIRT1, relulting in IR. [ABSTRACT FROM AUTHOR]

The present study aimed to explore the effects of different exercise modes on neuromuscular junction (NMJ) and metabolism of skeletal muscle-related proteins in aging rats. Ten from 38 male Sprague-Dawley (SD) rats (3-month-old) were randomly selected into young (Y) group, while the rest were raised to 21 months old and randomly divided into elderly control (O), endurance exercise (EN) and resistance exercise (R) groups. After 8 weeks of corresponding exercises training, the gastrocnemius muscles of rats were collected, and the expression of S100B in Schwann cells was detected by immunofluorescence staining. Western blot was used to detect the protein expression levels of agglutinate protein (Agrin), low-density lipoprotein receptor-related protein 4 (Lrp4), muscle- specific kinase protein (MuSK), downstream tyrosine kinase 7 (Dok7), phosphorylated protein kinase B (p-Akt), phosphorylated mammalian target rapamycin (p-mTOR), and phosphorylated forkhead box O1 (p-FoxO1) in rat gastrocnemius muscles. The results showed that, endurance and resistance exercises increased the wet weight ratio of gastrocnemius muscle in the aging rats. The protein expression of S100B in the R group was significantly higher than those in the O and EN groups. Proteins related to NMJ function, including Agrin, Lrp4, MuSK, and Dok7 were significantly decreased in the O group compared with those in the Y group. Resistance exercise up-regulated these four proteins in the aging rats, whereas endurance exercise could not reverse the protein expression levels of Lrp4, MuSK and Dok7. Regarding skeletal muscle-related proteins, the O group showed down-regulated p-Akt, and p-mTOR protein expression levels and up-regulated p-FoxO1 protein expression level, compared to the Y group. Resistance and endurance exercises reversed the changes in p-mTOR and p-FoxO1 protein expression in the aging rats. These findings demonstrate that both exercise modes can enhance NMJ function, increase protein synthesis and reduce the catabolism of skeletal muscle-related proteins in aging rats, with resistance exercise showing a more pronounced effect.

Objective: To investigate the protective effect of Xuebijing injection on acute lung injury (ALI) associated with cardiopulmonary bypass (CPB) by regulating the apoptosis of polymorphonuclear neutrophils (PMN)., Methods: Thirty male Sprague-Dawley (SD) rats were randomly divided into sham operation group (Sham group), CPB model group (CPB group) and Xuebijing pretreatment group (XBJ group) according to the random number table method, with 10 rats in each group. Rats in the CPB group and XBJ group undergoing CPB procedures for 60 minutes. Rats in the Sham group did not undergo CPB. Rats in the XBJ group received intraperitoneal injection of 4 mL/kg Xuebijing injection 2 hours before CPB. Rats in the Sham group and CPB group were injected with an equal amount of normal saline. 4 hours after CPB, arterial blood was collected for blood gas analysis to calculate respiratory index (RI), and lung tissue of rats was collected for determination of lung index (LI) and pulmonary water containing rate. PMN in bronchoalveolar lavage fluid (BALF) were collected and the activity of caspase-3 was detected. The apoptosis rate was detected by flow cytometry. The expressions of microRNA-142-3p (miR-142-3p) and FoxO1 mRNA were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). The protein expression of FoxO1 was detected by Western blotting. In addition, HL-60 cells were divided into control oligonucleotide transfection group, miR-142-3p mimics transfection group, and miR-142-3p inhibitor transfection group. After 48 hours of transfection, the activity of miR-142-3p binding to FoxO1 was detected using dual luciferase reporter genes., Results: Compared with Sham group, RI, LI and pulmonary water containing rate were significantly increased in CPB group. The caspase-3 activity and apoptosis rate of PMN obtained from BALF were significantly decreased, the expression of miR-142-3p was decreased, and the expression of FoxO1 protein was increased. However, compared with CPB group, RI, LI and pulmonary water containing rate were significantly decreased in XBJ group [RI: 0.281±0.066 vs. 0.379±0.071, LI: 4.50±0.26 vs. 5.71±0.42, pulmonary water containing rate: (80.31±32.50)% vs. (84.59±3.41)%, all P < 0.01]. The caspase-3 activity and apoptosis rate of PMN obtained from BALF were significantly increased [caspase-3 activity: 0.350±0.021 vs. 0.210±0.014, apoptosis rate: (15.490±1.382)% vs. (8.700±0.701)%, both P < 0.01], the expression of miR-142-3p was significantly up-regulated (2 -ΔΔCt : 2.61±0.17 vs. 0.62±0.05, P < 0.01), and the protein expression of FoxO1 was decreased [FoxO1/GAPDH (relative expression level): 0.81±0.04 vs. 1.22±0.06, P < 0.01]. However, there was no statistically significant difference in FoxO1 mRNA expression among the three groups. The bioinformatics analysis results showed that miR-142-3p can bind to the FoxO1 3'untranslated region (3'UTR). In HL-60 cells, compared with control oligonucleotide transfection group, the transfection of miR-142-3p mimics could reduce the expression of FoxO1 protein [FoxO1/GAPDH (relative expression level): 0.48±0.06 vs. 1.00±0.05, P < 0.01], however, the transfection of miR-142-3p inhibitor increased the expression of FoxO1 protein [FoxO1/GAPDH (relative expression level): 1.37±0.21 vs. 1.00±0.05, P < 0.05]. But, transfection with miR-142-3p mimics or inhibitor had no effect on FoxO1 mRNA expression. The luciferase reporter gene showed that miR-142-3p could bind to the FoxO1 3'UTR to inhibit FoxO1 expression., Conclusions: Xuebijing injection may promote the apoptosis of pulmonary alveolar PMN through the miR-142-3p/FoxO1 axis, and play a role in the prevention and treatment of CPB-induced ALI.

Objective: To investigate the expression of WTAP, a m 6 A methylase, in a mouse model of renal ischemia-reperfusion (I/R) injury and the effect of WTAP knockdown on biological behavior of renal tubular epithelial cells exposed to I/R injury., Methods: Sixteen C57BL/6 mice with renal I/R injury or sham operation ( n =8) were examined for blood urea nitrogen (BUN) and creatinine (Scr) levels to assess renal function, and renal pathologies were observed with HE staining. The expressions of WTAP and FOXO1 proteins in the kidneys of the mice were detected using immunohistochemistry. Human renal tubular epithelial cells (HK-2) were transfected with si-WTAP or si-NC followed by hypoxia-reoxygenation (H/R) exposure, Protein and mRNA expression were assessed by Western blot and qRT-PCR, and changes and changes in cell viability and apoptosis were assessed using CCK8 assay and TUNEL staining, respectively; LDH release level and caspase-3 activity of the cells were measured using commercial assay kits. FOXO1 m 6 A modification sites were predicted using SRAMP website (http://www.cuilab.cn/sramp/), and the interaction between WTAP and FOXO1 mRNA was analyzed with RIP experiment; the level of FOXO1 modified by m 6 A was detected by MeRIP-qPCR., Results: Compared with sham-operated mice, the mice with renal I/R injury showed significantly increased Scr and BUN levels ( P < 0.001) and renal expressions of WTAP mRNA and protein ( P < 0.001). In cultured HK-2 cells, H/R exposure significantly decreased the cell viability ( P < 0.001) and increased cellular LDH release ( P < 0.001) and expressions of WTAP mRNA and protein ( P < 0.001). WTAP knockdown obviously reduced the cell damage induced by I/R injury and significantly decreased the mRNA and protein levels of FOXO1 in the cells ( P < 0.001). RIP experiment confirmed WTAP binding to FOXO1 mRNA, and inhibition of WTAP expression significantly reduced FOXO1 m 6 A level in HK-2 cells ( P < 0.001)., Conclusion: WTAP expression is up-regulated in the kidneys of mice with renal I/R injury and in HK-2 cells with H/R exposure. Inhibition of WTAP alleviates H/R-induced apoptotic damage in HK-2 cells possibly by inhibiting FOXO1 expression.

Objective To investigate whether glucagon-like peptide-1 (GLP-1) can regulate lipid metabolism in hepatocytes by modifying forkhead box protein O1 (FoxO1) phosphorylation sites. Methods The hepatic carcinoma cell line HepG2 cells were used to construct the hepatic steatosis model. Measuring the change of intracellular triglyceride (TG) of the GLP-1 treated cells fatty liver model, Western blot was used to detect changes in phosphorylation of Thr24, Ser256, and Ser319 in FoxO1; construction and transfection of the FoxO1-Ser256 mutant plasmid, RT-PCR was used to detect the changes of ACC and Fasn genes regulated by FoxO1. Results Compared with model group, ACC and Fasn mRNA level were lower than in GLP-1 treated model group, the difference was statistically significant (1.33±0.30 vs 4.21±0.66, 1.41±0.48 vs 3.05±0.67, t=5.31,4.16, all P<0.05). Compared with model group, the phosphorylation levels of ser256 and ser319 significantly elevated in GLP-1 group (t=7.34, 8.79, all P<0.05). After transfected with FoxO1-WT, the expression of ACC and Fasn mRNA were reduced in GLP-1 group (t=5.28, 4.33, all P<0.05). After transfected with FoxO1-MT, the expression of ACC and Fasn mRNA were not reduced in GLP-1 group (t=1.16, 2.51, all P>0.05). Conclusions GLP-1 may affect the expression of Foxol downstream gene ACC and Fasn by modifying the phosphorylation sites of FoxO1, and reduce lipid formation in the fatty liver cell model. [ABSTRACT FROM AUTHOR]

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Objective: To investigate the expression rules of FOXO1a, FOXO3a, FOXO4 and FOXO6 proteins in the human testis, and explore their roles in the development and progression of testicular aging., Methods: We collected the para-carcinoma testis tissue from 4 testis cancer patients aged 28, 31, 32 and 46 years, and the testis tissue from another 2 PCa patients aged 66 and 81 years after castration surgery from January 2018 to December 2020. We detected the expressions of FOXO1a, FOXO3a, FOXO4 and FOXO6 proteins in the testis tissue by Western blot, determined the locations of FOXO1a, FOXO3a, FOXO4 and FOXO6 in the testis cells by immunofluorescence staining, and performed semi-quantitative and statistical analyses using image J and SPSS 23.0 software, respectively., Results: The expression levels of FOXO1a and FOXO3a proteins were significantly decreased in the testis tissue of the elderly patients (P < 0.01), with an age-dependent reduction in the proportion of the positive cells. No statistically significant difference was observed in the expression levels of FOXO4 and FOXO6 between different age groups. FOXO1a was mainly expressed at the base of the seminiferous tubules, FOXO3a and FOXO4 in the Leydig cells, and FOXO6 in the seminiferous tubules. In addition, FOXO4 underwent age-related nuclear translocation in the senescent Leydig cells, suggesting its involvement in the aging of Leydig cells., Conclusion: FOXO1a/3a/4 may be closely related to human testicular aging and corresponding pathological changes, but its underlying mechanism remains to be further explored.

Objective: To explore whether tumor suppressor gene Foxo1 and PTEN play a critical role in the tumorigenesis of mouse natural killer-cell lymphoma., Methods: NKp46-iCre mice were crossed with mice carrying floxed Foxo1 alleles (Foxo1 fl/fl ) as well as floxed PTEN alleles (PTEN fl/fl ) to generate mice in which Foxo1 and PTEN in NK cells were knock-out, referred as Foxo1 △NK PTEN △NK . The growth and development of the mice and tumor formation were observed. The flow cytometry was used to detect the percentages of NK cells in main lymphatic organs. B16F10 metanoma model of tumor metastasis was utilized to investigate NK cell-mediated tumor surveillance in vivo after NK cells special deletion of Foxol and PTEN., Results: The mouse model with NK cell-special Foxo1 and PTEN double knockout was established. Compared with control group (Foxo1 fl/fl PTEN fl/fl mice), Foxo1 △NK PTEN △NK mice were born alive and appeared to be healthy over a period of 46 weeks. No spontaneous tumor formation was observed at this stage. There were no significant differences in NK cell percentages of gated lymphocytes from various organs including blood, bone marrow, peripheral lymph node and spleen between Foxo1 △NK PTEN △NK mice and Foxo1 fl/fl PTEN fl/fl mice [PB: 4.76%±0.46% vs 4.17%±0.64% (P＞0.05, n=8); BM: 1.13%±0.23% vs 1.31%±0.10% (P＞0.05, n=8) ; LN: 0.50%±0.10% vs 0.85%±0.20% (P＞0.05, n=8); SP: 4.41%±0.65% vs 3.50%±0.24% (P＞0.05, n=8)]. B16F10 melanoma metastasis model of tumor was established, No differences in median survival time were observed in the 2 types of mice (P＞0.05, n=13)., Conclusion: The simultaneous deletion of the Foxo1 and PTEN genes may not plays significant role in the tumorigenesis of mouse natural killer-cell lymphoma and NK cell-mediated tumor surveillance in vivo.

Long-term alcohol stimulation may inhibit the expression of silent information regulator 1 in hepatocytes, which increases the acetylation level of forkhead box transcription factor O1, reduces nuclear localization, and reduces the binding capacity of DNA sequence. This further downregulates the expression of downstream adiponectin receptor 2 and microsomal triglyceride transfer protein, causes lipid metabolism disorders and triglyceride deposition in hepatocytes by affecting adiponectin signal transduction and synthesis of very-low-density lipoprotein, and finally promotes the development of alcoholic fatty liver disease.

Recent studies found that forkhead box protein O1 (FoxO1) does not only demonstrate important biological functions in cell proliferation, gluconeogenesis, energy metabolism, and oxidative stress, but it also plays a vital role in the remodeling process of bones. FoxO1 can regulate bone mass by affecting osteoblasts, osteoclasts, and precursor cells. In this article, we review the role of FoxO1 in bone metabolism and elucidate its underlying mechanism.

Objective: To explore the expression of miR-135b in endometrial carcinoma and the mechanism by which miR-135b promotes the proliferation of endometrial cancer cells., Methods: The expressions of miR-135b and FOXO1 were using RT-PCR detected in 22 fresh endometrial cancer tissues and paired adjacent tissues and also in endometrial cancer cell lines JEC, Ishikawa, HEC-1-B, and RL-952. The RL-952 and Ishikawa cell lines were transfected with miR-135b mimics or inhibitors, and the changes in their proliferative activity were detected with MTT assay; the expressions of FOXO1 mRNA and protein were detected by RT-PCR and Western blotting, respectively., Results: The expression of miRNA135b was significantly up-regulated and FOXO1 expression was down-regulated in endometrial carcinoma tissues as compared with the adjacent tissues (P<0.05). The mRNA expression of miR-135b was negatively correlated with the expression of FOXO1 in endometrial carcinoma. In RL-952 and Ishikawa cell lines, transfection with miR-135b mimics obviously promoted the cell proliferation (P<0.05). Up-regulation of miR-135b significantly decreased the expressions of FOXO1 protein and mRNA (P<0.05), and down- regulation of miR-135b increased FOXO1 expressions (P<0.05)., Conclusions: MiR-135b plays an important role in the occurrence and development of endometrial carcinoma partially by regulating its target gene FOXO1.

Objective: To investigate the effect of Metformin on the proliferation and collagen synthesis of the human keloids fibroblasts as well as the effect on phosphorylation of Akt/FoxO1 signal transduction pathway., Methods: Fibroblasts of keloid were divided into control group treated with medium solution and experimental groups treated with different concentrations of Metformin. 48 h later CCK-8 assay was adopted to evaluate cell survival; Western blot was performed to detect the Akt and FoxO1 phosphorylation; and Hydroxyproline reagent kit was used to detect the collagen synthesis., Results: With different concentrations (30, 60, 90, 120 mmol/L) of Metformin, the absorbance of cultured keloid fibroblasts detected by CCK8 assay decreased by (13.30 ± 2.04)%, (22.64 ± 4.70)%, (54.00 ± 5.34)% and (63.12 ± 3.48)%. The growth of fibroblasts was suppressed by Metformin in a dose-dependent manner. It showed that the level of phoshpo-akt and phoshpo-foxOl in keloids fibroblasts in experimental groups was lower than that in the control group and the collagen synthesis were also decreased in experimental groups, all in a dose-dependent manner (P < 0.05, P < 0.01)., Conclusions: Metformin can effectively inhibit the proliferation and collagen synthesis of the human keloids fibroblasts in vitro, which may be associated with the suppression of phosphorylation of Akt/FoxO1 signaling pathway

Objective: To identity whether there is muscle atrophy phenomenon in end-stage kidney disease patients and to detect the level of transcription factor Foxo1 and the activity of ubiquitin-proteasome system., Methods: Twenty-two patients in chronic kidney disease (CKD) stage 5 were selected and their mean muscle cross sectional area was measured. mRNA and protein levels of Foxo1, Atrogin-1, MuRF1 in rectus abdominis biopsies obtained from consecutive patients were detected. Control biopsies were obtained from 8 healthy subjects during elective surgery for abdominal wall hernias and 6 subjects during elective surgery for adenomyosis., Results: Compared with the control group, cross sectional area of muscle fibers decreased and the transcription and protein levels of Foxo1, Atrogin-1, MuRF1 were upregulated in CKD group (P < 0.05). Protein level of p-Foxo1 decreased in CKD group (P < 0.05)., Conclusion: There exist muscle atrophy phenomenon in CKD patients, which may associate with the upregulation of Foxo1 and activation of ubiquitin-proteasome system.

Silent information regulator factor 2-related enzyme 1 (Sirtuins 1, SIRT1) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, which can deacetylate histone and non-histone proteins and other transcription factors, and is involved in the regulation of many physiological functions, including gene transcription, energy metabolism, cell senescence and oxidative stress. Recent studies show that through adjusting the activity of endothelial nitric oxide syntheses (eNOS), p53, forkhead box class O (FOXO) and nuclear factor kappa B (NF-kappaB), SIRT1 can protect the functions of vascular endothelia and nerves in a variety of pathological conditions. Therefore, SIRT1 may be used as a potential therapeutic target of these diseases, particularly erectile dysfunction, which are associated with endothelial dysfunction.

This study was aimed to analyze the expression profiles of PI3K/AKT signaling pathway genes from bone marrow samples of AML and ALL patients and normal samples. AML, ALL and normal bone marrow samples were collected from 6 AML, 6 ALL patients and 4 normal persons. The expression of PI3K/AKT signaling pathway genes including PTEN, CCND1, mTOR, RICTOR, FOXO1 were detected by real-time fluorescent quantification RT-PCR while GAPDH gene expression was used as an internal reference. The relative gene expression level was calculated by the method of the 2(-ΔΔCt). The results showed that the gene expression profiles were different between normal and leukemic groups. PTEN, mTOR and RICTOR expression levels were down-regulated, while FOXO1 and CCND1 levels were up-regulated in AML and ALL. PTEN was down-regulated in 10 out of the 12 samples; mTOR was down-regulated in 9 out of the 12 samples; RICTOR was down-regulated in 7 out of the 12 samples; FOXO1 was up-regulated in 9 out of the 12 samples and CCND1 was up-regulated in 7 out of the 12 samples. It is concluded that PI3K/AKT signal pathway is activated in both AML and ALL leukemic cells.

Objective: We analyzed the effect of the stable expressed Hepatitis C virus core protein on PCK1 mRNA expression level and the molecular mechanisms involved in Huh7-lunet cells., Methods: A retroviral vector mediated mammalian cell expression cell line of the HCV core protein was constructed. The mRNA and protein levels of PCK1, FOXO1 and PGC-1alpha were analyzed by Real-time PCR and luciferase assay in Huh7-lunet-core cells., Results: HCV CORE upregulated the mRNA levels of PCK1 significantly. Both the mRNA and protein levels of FOXO1 were not affected in Huh7-lunet-core cells, whereas a decreased phosphorylation status of FOXO1 was exhibited. Moreover, activation of FOXO1 by HCV CORE was detected. Further, the mRNA level of PGC-1alpha was found to be significantly elevated in Huh7-lunet-core cells., Conclusion: Our results revealed for the first time that HCV core protein expression-mediated FOXO1 activation and the increased PGC-1alpha leaded to the elevation of PCK1 at the mRNA level, which suggesting the immoderate gluconeogenesis in HCV-infected hepatocytes. Our findings contributed to the understanding of the molecular mechanisms of HCV-related insulin resistance and provided potential new clues for the prevention and therapy of diabetes.

Objective: To establish a model of ER stress-induced apoptosis with tunicamycin and to examine whether Bim is dysregulated and its potential role in resistance of melanoma cells to apoptosis under endoplasmic reticulum (ER) stress., Methods: A model of ER stress-induced apoptosis was established with tunicamycin. Apoptotic cells were quantitated using the annexin V/propidium iodide method by flow cytometry. Hoechst staining was also used to confirm the apoptotic cell death. Western blotting was used to measure the activation of caspase-3 and -9, and the expression of Bim, GRP78, CHOP, and Foxo1 at the protein level. The expression of Bim, CHOP and Foxo1 at the mRNA level was quantitated by qPCR. The siRNA technique was used to inhibit the expression of Bim., Results: Treatment of the melanoma cells with tunicamycin did not induce significant apoptosis and activation of caspase cascade, whereas it caused marked activation of caspase-3 and -9, and apoptosis in HEK293 cells which were used as a control. With exposure to tunicamycin (3 µmol/L) for 12, 24, 36 hours the Bim protein levels were not increased in Mel-RM and MM200 cells. Its mRNA levels were 0.37 ± 0.05, 0.13 ± 0.02 and 0.02 ± 0.01 in Mel-RM cells, while 0.41 ± 0.06, 0.16 ± 0.04 and 0.21 ± 0.03 in MM200 cells, respectively. The expression of Bim mRNA was significantly reduced compared with that in the control groups of the two cell lines (P < 0.01). siRNA knockdown of Bim protected HEK293 cells against activation of caspase-3. The cell apoptosis of Bim siRNA group was (5.69 ± 0.38)%, significantly lower than that of the siRNA control group (40.32 ± 1.64)% and blank control group (35.46 ± 2.01)% (P < 0.01). In the melanoma cells after exposure to tunicamycin (3 µmol/L) for 6, 12, 24, and 36 hours the transcription factor CHOP at mRNA level were significantly increased and the expressions at protein level were also up-regulated. The expressions of another transcription factor Foxo1 at mRNA level significantly decreased and the expressions at protein level were down-regulated, too., Conclusions: The lack of Bim up-regulation contributes to the resistance of melanoma cells to ER stress-induced apoptosis and may be a mechanism by which melanoma cells adapt to ER stress conditions. Transcription factors CHOP and Foxo1 may be responsible for the dysregulation of Bim in melanoma cells upon ER stress.

Adiponectin is an adipokine mainly secreted by adipose tissue, which exerts insulin-sensitivity, anti-inflammation, anti-atherosclerosis and cardio-protective effects. These biological effects are mediated by adiponectin receptor 1 and adiponectin receptor 2. Expression levels of adiponectin receptor affect activation of downstream signaling pathway and biological effects. Research about regulation factors of adiponectin receptor expression contributes discovering the molecular mechanism and provides new ideas for the prevention and treatment of the metabolism disorder and cardiovascular diseases.

Aging-related oxidative stress and free radical theory has become accepted increasingly as explaination, at least in part of the aging process. In murine models of aging, a genetic deficiency of the p66(Shc) (66-kilodalton isoform of Shc gene products) gene, which encodes a phosphotyrosine signal adapter protein, extends life span by 30%, and confers resistance to oxidative stress. Upon oxidative stress, p66(Shc) is phosphorylated at Ser36, contributing to inactivation of the forkhead-type transcription factors (FKHR/ FoxO1), which regulates the gene expression of cellular antioxidants. The p66(Shc) has a direct connection with the life span related signaling, which is conserved evolutionarily. Shc is basically not expressed in mature neurons of the adult brain and spinal cord. Instead, two Shc homologues, Sck/ShcB and N-Shc/ ShcC, which have been proved to be effective on oxidative stress and aging, are expressed in neural system. The expression of Shc-related genes is affected in the aging process, which may be relevant to cellular dysfunction, stress response and/or cognitive decline during aging.

Objective: To detect the PAX3/PAX7-FKHR fusion transcripts to identify genetic alteration in embryonal rhabdomyosarcoma (ERMS) and alveolar rhabdomyosarcoma (ARMS) tissues., Methods: One-step reverse transcription- polymerase chain reaction (RT-PCR) were used to detect the expression of the PAX3/PAX7-FKHR fusion transcrips in 16 cases of rhabdomyosarcoma (7 cases of ARMS, 9 cases of ERMS) and 16 specimens were compared to the surrounding normal tissue. Comparative genomic hybridization (CGH) was employed to detect the genomic imbalance (DNA loss or amplification) in 16 RMS cases., Results: PAX3-FKHR fusion transcripts were positive in 3/7 and PAX 7-FKHR fusion transcripts were positive in 2/7 of ARMS patients, respectively, and were all negative in ERMS and Control tumors. There were different chromosome variations for each RMS, chromosome amplification was frequently seen in 1p36 (69%), 5q32 (56%), 8q21 (63%), 13q14 (69%), 19q (63%), 20q (56%). Chromosome loss was frequently seen in 3p21-pter (56%), 9p23-pter (50%), 10q (69%), 16/16q24 (56%)., Conclusion: One-step RT-PCR assay for detection specific fusion gene provides a useful tool for confirmation of the diagnosis of RMS in diagnostically difficult cases and in retrospective studies. Chimeric gene transcript resulting from specific chromosomal translocations is a reliable index for the molecular diagnosis of RMS.

Objective: To detect the PAX3-FKHR and PAX7-FKHR fusion transcripts in formalin-fixed, paraffin-embedded rhabdomyosarcoma tissues by one-step RT-PCR and discuss its diagnostic potential., Methods: One-step RT-PCR were used to detect the expression of the PAX3-FKHR and PAX7-FKHR fusion transcripts in 15 cases of rhabdomyosarcoma (6 cases of ARMS, 9 cases of ERMS and 1 case of PRMS) and 15 cases of non-rhabdomyosarcomous small round cell tumor., Results: PAX3-FKHR and PAX7-FKHR fusion transcripts were positive in 3/6 and 1/6 of ARMS patients, respectively, and were all negative in ERMS, PRMS and Control tumors including 4 cases of synovial sarcoma,4 cases of Ewing's sarcoma,4 cases of lymphoma and 3 cases of neuroblastoma., Conclusion: Expression of PAX3-FKHR and PAX7-FKHR fusion transcripts detected by one-step RT-PCR is useful in diagnosis and classification of rhabdomyosarcoma.

Background & Objective: There were a few reports about the role of PAX3-FKHR fusion gene in the oncogenesis of alveolar rhabdomyosarcoma (ARMS); however, its effect on muscle-producing factor (MPF) is not clear. This study was designed to observe the effects of fusion gene PAX3-FKHR transfection on expression of MRF4 and differentiation of cultured human rhabdomyosarcoma RD cells., Methods: To construct an eukaryotic expression vector of PAX3-FKHR gene; the plasmids of MRF4 cDNA and PAX3-FKHR cDNA were transfected into cultured RD cells with lipofectin methods. The changes of cells growth, morphologic and the expression of MHC and alpha-actin were observed., Results: There was no significant changes in RD cells after transfection with PAX3-FKHR gene in growth rate, growth manner and the expression of MHC and alpha-actin. In MRF4 expressing RD cells after transfected with PAX3-FKHR, although the expression of MRF4 was not changed, morphologic appearance showed a low differentiation; expression of MHC and alpha-actin significantly decreased., Conclusion: There is no obvious influence on biological character and differentiation in RD cells with transfection of PAX3-FKHR gene; PAX3-FKHR does not inhibit the expression of MRF4, but can reverse the effect of MRF4 on promoting the differentiation of RD cell.