Akt2 inhibitor promotes M2 macrophage polarization in rats with periapical inflammation by reducing miR-155-5p expression / 南方医科大学学报

OBJECTIVE@#To investigate the effect of Akt2 inhibitor on macrophage polarization in the periapical tissue in a rat model of periapical inflammation.@*METHODS@#Rat models of periapical inflammation were established in 28 normal SD rats by opening the pulp cavity of the mandibular first molars, followed by injection of normal saline and Akt2 inhibitor into the left and right medullary cavities, respectively. Four rats without any treatment served as the healthy control group. At 7, 14, 21 and 28 days after modeling, 7 rat models and 1 control rat were randomly selected for observation of inflammatory infiltration in the periapical tissues by X-ray and HE staining. Immunohistochemistry was used to detect the expression and localization of Akt2, macrophages and the inflammatory mediators. RT-PCR was performed to detect the mRNA expressions of Akt2, CD86, CD163, inflammatory mediators, miR-155-5p and C/EBPβ to analyze the changes in macrophage polarization.@*RESULTS@#X-ray and HE staining showed that periapical inflammation was the most obvious at 21 days after modeling in the rats. Immunohistochemistry and RT-PCR showed that compared with those in the control rats, the expressions of Akt2, CD86, CD163, miR-155-5p, C/EBPβ, and IL-10 increased significantly in the rat models at 21 days (P < 0.05). Compared with saline treatment, treatment with the Akt2 inhibitor significantly decreased the expression levels of Akt2, CD86, miR-155-5p and IL-6 and the ratio of CD86+M1/CD163+M2 macrophages (P < 0.05) and increased the expression levels of CD163, C/EBPβ and IL-10 in the rat models (P < 0.05).@*CONCLUSION@#Inhibition of Akt2 can delay the progression of periapical inflammation in rats and promote M2 macrophage polarization in the periapical inflammatory microenvironment possibly by reducing miR-155-5p expression and activating the expression of C/EBPβ in the Akt signaling pathway.

Systematic Effect of Modified Renshen Wumeitang in Regulation of GABA Signaling Pathway in Rats with Diarrhea / 中国实验方剂学杂志

ObjectiveTo investigate the effect of modified Renshen Wumeitang（MRWT） on the related regulatory factors of the γ-aminobutyric acid （GABA） signaling pathway in colon tissues of rats with diarrhea， and reveal the mechanism of MRWT in invigorating Qi， generating fluid， and checking diarrhea. MethodForty-eight SD immature rats were randomly divided into a blank group （n=12） and an experimental group （n=36）. The diarrhea model was induced in the experimental group by Sennae Folium combined with overstrain and improper diet for 14 days. Subsequently， the model rats were randomly divided into a model group （normal saline， 20 mL·kg-1）， a western medicine group （Medilac-Vita， 0.7 g·kg-1）， and a Chinese medicine group （MRWT， 35 g·kg-1）， with 12 rats in each group. The rats in the blank group received normal saline at 20 mL·kg-1， and those in the other groups were treated correspondingly， once a day for 7 days. The general condition， loose stool rate， and diarrhea index of the rats were observed daily. Immunohistochemistry was used to detect the optical density expression of GABA protein in the colon of rats. The content of phosphatidylinositol-3 kinase （PI3K）， protein kinase B2 （Akt2）， phosphorylated Akt （p-Akt）， and interleukin-1β （IL-1β） was determined by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expression levels of PI3K， Akt2， and GABA type A receptor subunit β2 （GABRB2） in the colon of rats were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. ResultCompared with the blank group， the model group showed worsened general condition， The difference was not statistically significant of loose stool rate and diarrhea index， increased expression of GABA protein （P<0.05）， elevated expression of PI3K， Akt2， p-Akt， and IL-1β （P<0.05， P<0.01）， and up-regulated PI3K， Akt2， and GABRB2 mRNA and protein expression （P<0.01）. Compared with the model group， the western medicine group and the Chinese medicine group showed the improved general condition， decreased loose stool rate and diarrhea index （P<0.01）， and decreased content of PI3K， Akt2， p-Akt， and IL-1β （P<0.05）. The Chinese medicine group displayed decreased mRNA expression of PI3K， Akt2， and GABRB2 （P<0.05， P<0.01） and down-regulated protein expression of GABA， PI3K， and GABRB2 （P<0.05， P<0.01）. The western medicine group exhibited down-regulated mRNA expression of PI3K，Akt2，and protein of PI3K （P<0.05）. ConclusionMRWT can regulate the GABA signaling pathway， reduce Cl- flow in intestinal epithelial cells to the intestinal lumen， and improve the imbalance of colonic fluid metabolism in the colon of diarrhea rats， thereby exerting its effects of invigorating qi， generating fluid， and checking diarrhea.

Ablation of Akt2 and AMPKα2 rescues high fat diet-induced obesity and hepatic steatosis through Parkin-mediated mitophagy

@#Given the opposing effects of Akt and AMP-activated protein kinase (AMPK) on metabolic homeostasis, this study examined the effects of deletion of Akt2 and AMPKα2 on fat diet-induced hepatic steatosis. Akt2–Ampkα2 double knockout (DKO) mice were placed on high fat diet for 5 months. Glucose metabolism, energy homeostasis, cardiac function, lipid accumulation, and hepatic steatosis were examined. DKO mice were lean without anthropometric defects. High fat intake led to adiposity and decreased respiratory exchange ratio (RER) in wild-type (WT) mice, which were ablated in DKO but not Akt2−/− and Ampkα2−/− mice. High fat intake increased blood and hepatic triglycerides and cholesterol, promoted hepatic steatosis and injury in WT mice. These effects were eliminated in DKO but not Akt2−/− and Ampkα2−/− mice. Fat diet promoted fat accumulation, and enlarged adipocyte size, the effect was negated in DKO mice. Fat intake elevated fatty acid synthase (FAS), carbohydrate-responsive element-binding protein (CHREBP), sterol regulatory element-binding protein 1 (SREBP1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), peroxisome proliferator-activated receptor-α (PPARα), PPARγ, stearoyl-CoA desaturase 1 (SCD-1), phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6Pase), and diglyceride O-acyltransferase 1 (DGAT1), the effect was absent in DKO but not Akt2−/− and Ampkα2−/− mice. Fat diet dampened mitophagy, promoted inflammation and phosphorylation of forkhead box protein O1 (FoxO1) and AMPKα1 (Ser485), the effects were eradicated by DKO. Deletion of Parkin effectively nullified DKO-induced metabolic benefits against high fat intake. Liver samples from obese humans displayed lowered microtubule-associated proteins 1A/1B light chain 3B (LC3B), Pink1, Parkin, as well as enhanced phosphorylation of Akt, AMPK (Ser485), and FoxO1, which were consolidated by RNA sequencing (RNAseq) and mass spectrometry analyses from rodent and human livers. These data suggest that concurrent deletion of Akt2 and AMPKα2 offers resilience to fat diet-induced obesity and hepatic steatosis, possibly through preservation of Parkin-mediated mitophagy and lipid metabolism.

Hypoglycemia caused by AKT2 gene mutation:a case report and literature review / 中华全科医师杂志

A 15-month-old baby girl presenting with hypoglycemia was admitted in Children′s Hospital of Capital Institute of Pediatrics in October 2019. The blood glucose level was 2.4 mmol/L at admission, she showed asymmetry of left and right limbs. The levels of D-3-hydroxybutyric acid, urinary ketone body and free fatty acid were all decreased during hypoglycemia attack, the hyperglycemic hormone was increased, but insulin level was<0.2 μIU/ml. The whole exon gene testing showed that the patient had heterozygous mutation of AKT2 gene c.49G>A (p.E17K), which was mosaicism; then the patients was diagnosed as hypoinsulinemic hypoketotic hypo-fatty-acidemic hypoglycemia due to mutation of AKT2 gene. Blood glucose levels were dynamically monitored, high carbohydrate diet was administered and raw corn starch supplementation was given before bedtime. After 18 months of treatment, the growth and development of the patient was normal, the frequency of hypoglycemia attacks decreased, and bilateral limb asymmetry improved. The relevant literature was searched from Wanfang Database, CNKI and PubMed from January 1980 to March 2021 by using search term"hypoglycemia"and"AKT2 gene". Five cases of hypoglycemia caused by AKT2 mutation were retrieved, all were reported from other countries, no one case from China. The clinical manifestation of this disease is similar to hyperinsulinemic hypoglycemia, but insulin could not be detected during the attack of hypoglycemia, and the patients may have hemihypertrophy. The study suggests that if the patient has hypoglycemia accompanied by hypoinsulinemia and hemihypertrophy, we should consider the possibility of AKT2 gene mutation, and genetic testing should be recommended.

Effect of miR-137 on the proliferation and apoptosis of endothelial cells regulated by AKT2 gene / 中华医学遗传学杂志

Objective@#To assess the effect of miR-137 on the proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) induced by high glucose and its mechanism.@*Methods@#HUVECs cells were divided into low-glucose group (5.5 mmol/L glucose-treated cells), high-glucose group (33.36 mmol/L glucose-treated cells), anti-NC group (cells treated with 33.36 mmol/L glucose after anti-NC transfection) and anti-miR-137 group (cells treated with 33.36 mmol/L glucose after anti-miR-137 transfection). After 48 hours, qRT-PCR was used to determine the expression of miR-137. CCK-8 assay and flow cytometry were used to detect cell proliferation and apoptosis rate, respectively. The targeting relationship between miR-137 and AKT2 was validated by dual fluorescence reporter gene detection system and AKT2 protein expression after overexpression or inhibition of miR-137.@*Results@#High glucose could significantly up-regulate the expression of miR-137 in HUVECs cells, and the expression of miR-137 in HUVECs cells transfected with miR-137 inhibitor was significantly decreased (P<0.05). High glucose can significantly inhibit HUVECs cell proliferation and induce apoptosis, while inhibition of miR-137 expression can weaken the effect of high glucose on HUVECs cell proliferation inhibition and apoptosis promotion (P<0.05). Inhibiting AKT2 expression could weaken the inhibitory effect of miR-137 inhibitor on HUVECs cell proliferation and apoptosis (P<0.05).@*Conclusion@#Inhibiting the expression of miR-137 gene can attenuate the proliferation inhibition and apoptosis promotion of HUVECs induced by high glucose, and the mechanism is related to activating the expression of AKT2.

Overexpressing miR-29c targeting AKT2 enhances the radiosensitivity of human hepatocellular carcinoma cell line HepG2 / 中华放射医学与防护杂志

Objective To investigate the effect of miR-29c on radiosensitivity of hepatoma HepG2 cells by targeting AKT2 gene.Methods The expression of miR-29c in human normal hepatocytes THLE-3 and hepatoma cell HepG2 was detected by RT-PCR.The relationship between miR-29c and AKT2 were predicted by predicted by informative analysis and verified by dual luciferase reporter gene test and Western blot.miR-29c mimic/AKT2 gene recombinant plasmid and miR-29c inhibitor/ lentivirus vector AKT2 shRNA were transfected into HepG2 cells by Liposome 2000.The cells were irradiated with different doses (0,2,4,6 and 8 Gy) of X-rays,and the effects of miR-29/AKT2 on the survival and cell viability of HepG2 cells were detected by cloning and MTT assays.Results Compared with THLE-3 cells,the expression of miR-29c in HepG2 cells was significantly lower (t=17.816,P＜0.05).After 2,4,6 and 8 Gy X-ray irradiation,the survival of HepG2 cells was significantly lower than that of THLE-3 cells (t =4.541,6.823,7.218,9.363,P＜0.05),and the expression of miR-29c in HepG2 cells was significantly decreased (t =5.599,9.262,10.470,10.873,P＜0.05).The survival and viability of HepG2 cells were decreased by miR-29c overexpression (tsurvival rate =4.307,7.668,7.668,6.894,P＜0.05;tcell viability =3.443,8.116,13.434,P ＜ 0.05) but they were increased by miR-29c inhibition (tsurvival rate =4.003,6.713,7.141,P＜0.05;tcell viability =4.282,5.113,P＜0.05).Double luciferase reporter gene experiments showed that AKT2 was the target gene of miR-29c since the expression of AKT2 was negatively regulated by miR-29c.After the silence of AKT2 or overexpression of AKT2,the survival and viability of HepG2 cells were consistent with the overexpression of miR-29c or the inhibition of miR-29c,respectively.Conclusions MiR-29c increases the radiosensitivity of hepatoma cell HepG2 by targeting AKT2.

Pathogenesis and treatment of insulin resistance in chronic kidney disease and interventional effects of Chinese herbal medicine / 中国中药杂志

The kidney is the target organ of insulin with abundant insulin receptors. Thereinto,the renal intrinsic cells including glomerular podocytes,endothelial cells,mesangial cells,renal tubular epitheliums and collecting duct epithelial cells are all highly sensitive to insulin as the effector cells. Furthermore,the structural and functional abnormalities of these cells are closely related to insulin and its receptors activity. It is reported that the chronic kidney disease（CKD）patients have systemic or renal insulin resistance（IR）. IR is not only the pathogenic factor of CKD but also one of the mechanisms of CKD progression. The pathogenic factors of IR in the CKD patients include the systemic factors and the local factors in muscles and fat cells. The pathogenesis of IR is related to glomeruli,proximal tubules,collecting ducts and corresponding renal intrinsic cells such as podocytes,mesangial cells,renal tubular epitheliums and collecting duct epithelial cells. IR-related signaling pathways include insulin receptor substrate（IRS）/phosphatidylinositol 3 kinase（PI3K）/serine threonine kinase（Akt）pathway,adenosine monophosphate activated protein kinase（AMPK）pathway,glucose transporter4（GLUT4）pathway,nuclear factor（NF）-κB pathway and mitogen activated protein kinase（MAPK）pathway. Among them,IRS1/PI3K/Akt2 is the main signaling pathway of IR in podocytes of glomeruli, thus intervening its activity can improve podocyte injury. In clinic,some classical oral hypoglycemic agents and diuretic including metformin,rosiglitazone,glibenclamide,thiazolidinedione and spironolactone,as well as some extracts from Chinese herbal medicines including astragalus polysaccharides,quercetin,puerarin,emodin,berberine,curcumin and geniposide can both affect insulin and its receptor activity,and regulate IR-related signaling pathways,thereby ameliorating IR and CKD progression. Overall,the pharmacological studies based on IR-related signaling pathways in the renal intrinsic cells of CKD will become one of the developmental directions in the future.

Regulatory effects of PI3K/AKT2/mTOR signaling pathway on autophagy activation in cardiac tis-sues of mice with vial myocarditis / 中华微生物学和免疫学杂志

Objective To study the phosphorylation of AKT2 protein and autophagy activation in cardiac tissues of mice infected with coxsackievirus B3 (CVB3) for further analyzing the regulatory mecha-nism of PI3K/AKT2/mTOR signaling pathway on autophagy activation in viral myocarditis. Methods Thir-ty BALB/c mice were randomly divided into three groups (n=10): control group, myocarditis group and AKT activator-treated group. Those in the latter two groups were intraperitoneally injected with CVB3 to es-tablish the mouse model of acute viral myocarditis. Daily intraperitoneal injection of 0.04 mg/g of Akt acti-vator (SC79) was given to each mouse in the AKT activator-treated group 24 hours after CVB3 infection for 7 consecutive days,while the mice in the other two groups were given the same dose of normal saline. HE staining was used to observe the infiltration of inflammatory cells and tissue necrosis. Expression of CVB3 and inflammatory cytokines such as IL-1β and IL-6 in cardiac tissues at mRNA level was detected by q-PCR. Brain natriuretic peptide(BNP) and cardiac troponin I(cTnI) were measured by ELISA to evaluate myocardial injury. Changes in the expression of autophagy-related protein LC3 and Beclin1 at protein level as well as PI3K/AKT2/mTOR pathway were analyzed by Western blot assay. Results Compared with the con-trol group,massive inflammatory cell infiltration was observed in cardiac specimens of mice with myocarditis, but no obvious tissue necrosis was detected. Moreover,expression of CVB3 and inflammatory factors in car-diac tissues at mRNA level,levels of BNP and cTnI in blood,LC3Ⅱto LC3Ⅰratio as well as Beclin1 pro-tein level in cardiac tissues were significantly increased after CVB3 infection(P<0.05),whereas the activi-ty of PI3K/AKT2/mTOR signaling pathway was decreased. AKT activator not only down-regulated the LC3Ⅱ to LC3Ⅰratio and the expression of Beclin1 protein, but also enhanced the activation of PI3K/AKT2/mTOR signaling pathway in cardiac tissues of mice with myocarditis (P<0.05). Conclusion Enhanced autophagy and suppressed PI3K/AKT2/mTOR signaling pathway are observed in cardiac tissues of mice with myocarditis,indicating that the activation of autophagy may be regulated by PI3K/AKT2/mTOR signaling pathway.

Effects of dihydromyricetin on insulin resistance in 3 T3-L1 adipocytes and its mechanism / 中国药理学通报

Aim To investigate the effect of dihydro-myricetin (DMY) on the insulin resistance in 3T3-L1 adipocytes and its mechanism. Methods The differ-entiated adipocytes were treated with 1 μmol · L-1 dexamethasone ( dex ) for 7 days to induce insulin re-sistance. With or without insulin, DMY (1 × 10 -6 ~ 1 × 10 -8 mol·L-1 ) was exposed to the normal and in-sulin-resistant 3 T3-L1 adipocytes for 48 hour and 72 hour, respectively. Rosiglitazone ( 1 × 10 -6 mol · L-1 ) was used as a positive control. The glucose up-take was evaluated by glucose consumption. The mR-NA expressions of glucose transporter 4 ( GLUT4 ) , protein kinase B ( PKB/Akt) and adiponectin were de-termined by RT-PCR analysis. Results DMY ( 5 × 10 -7 ~ 1 × 10 -8 mol·L-1 ) concentration-dependently increased the glucose uptake in insulin-resistant 3 T3-L1 adipocytes, similar to rosiglitazone. However, DMY did not affect the glucose consumption in normal 3T3-L1 cells. After treatment of DMY to insulin-resist-ant 3T3-L1 adipocytes for 72 hours, the expressions of GLUT4 , Akt2 and adiponectin mRNA were markedly increased, compared with the dexamethasone-treated group. Conclusion DMY could improve the insulin resistance in 3T3-L1 adipocytes, which is related to in-creasing the mRNA expression of GLUT4 , Akt2 and adiponectin.

Role of miR-184 in oxygen-glucose-deprivation induced SK-N-SH cell ischemic injury and its regulation on AKT2 level / 实用医学杂志

Objective To explore the role of miR-184 in Oxygen-Glucose-Deprivation (OGD) induced SK-N-SH cell ischemic injury and its regulation on AKT2 level. Method We used a combination of oxygen and glucose deprivation to imitate ischemic conditions in vivo. MiR-184 mimic and inhibitor were transfected into SK-N-SH cell to alter miR-184 levels. The expression of miR-184 and AKT2 were determined by using Real-time PCR. The extent of SK-N-SH cell survival rate was assessed by thiazolyl blue tetrazolium bromide (MTT) assay. Result Here, we observed that miR-184 was significantly inhibited in SK-N-SH cell after OGD (P<0.05). The changes of miR-184 level altered the expression of AKT2 mRNA. In addition, alteration of miR-184expressionsignificantly affected cell survival rate after OGD. Conclusion miR-184 plays an important role in ischemic injury through negatively regulating AKT2 level, which may provide a potential therapeutic target for ischemic stroke in miRNA levels.

Expression of AKT2 gene in human liver cancer and its clinical significance / 中华肝胆外科杂志

ObjectiveTo study the expression of AKT2 gene in liver cancer and its relationship to tumor progression.MethodsThe expression of AKT2 in liver cancer was detected by SP immunohistochemical stainin and reverse transcription polymerase chain reaction (RT-PCR).Four patients with benign liver tumors were used as control.ResultsThe positive rates of AKT2 in liver cancer tissue and benign control tissue were 62.5％ (28/32) and 0％ (0/4),respectively.The difference was significant.In addition,a positive expression of AKT2 correlated significantly with poor differentiation,positive lymph node and distant metastasis.The median survival after surgery was significantly shorter in patients with positive than with negative AKT2 (76d vs 463d).ConclusionThe detection of AKT2 was useful in assessing the progression of liver cancer,in determining prognosis and eventually in rendering a possible target for novel therapeutic strategies.

The effects of inhibiting AKT2 by siRNA on the proliferation and invasion of liver cancer cells / 中华肝胆外科杂志

Objective To study the effects of inhibing AKT2 by siRNA on SMMC7721 liver cancer cells proliferation,apoptosis,migration and invasion.Methods The siRNA targeting AKT2 was designedandthe SMMC7721AKT2- siRNAplasmidwasconstructed andtransfected into SMMC7721 cells.The stable cell lines were screened by G418.The effects of AKT2 by siRNA on SMMC7721 liver cancer cells,growth inhibition was evaluated by MTT assay.Cell cycle was analyzed by flowcytometry (FCM).Protein of P27 and CyclinD1 was evaluated by Western-blot.The ability of migration and invasion was evaluated by wound healing and Transwell assay.ResultsThe growth of SMMC7721 cells was significantly inhibited by siRNA (P＜0.05).Flow cytometry display that AKT2 by siRNA can induce G1 phase arrest,the ratio of G1 phase increased homologously and S phase declined homologously.The protein of CyclinD1 was declined and the protein of P27 was increased by Western-blot.Wound healing and Transwell assay show that the ability of cells,migration and invasion was inhibited by AKT2 by siRNA.Conclusion AKT2 by siRNA can significantly inhibit the growth of SMMC7721 cells,arrest cell cycle.AKT2 by siRNA can inhibit the ability of invasion and migration of SMMC7721 cells.

Detection of gene amplification in MYCN, C-MYC, MYCL1, ERBB2, EGFR, AKT2, and human papilloma virus in samples from cervical smear normal cytology, intraepithelial cervical neoplasia (CIN I, II, III), and cervical cancer / Detección de amplificación en los genes MYCN, C-MYC, MYCL1, ERBB2, EGFR y AKT2 y del virus de papiloma humano en muestras de cepillado cervical en mujeres con citología normal y con neoplasia intraepitelial cervical (NIC) I, II, III y cáncer de cuello uterino

Introduction: Cervical cancer is the second most common cancer among women worldwide and the second cause of cancer mortality in women. It has been demonstrated that the process of cervical carcinogenesis displays genetic and environmental epigenetic components. Currently, research is focused on new prognosis markers like oncogene amplification.Objectives: To perform detection of MYCN, C-MYC, MYCL1, ERBB2, EGFR, and AKT2 amplification. Additionally, to detect human papillomavirus in samples from normal cytology smear, cervical intraepithelial neoplasia (CIN) I, II, and III and cervical cancer patients.Methods: Papillomavirus (HPV) genotyping by reverse line blot (RLB) performed and gene amplification by detection with real-time PCR with Taqman probes.Results: HPV was present in 4% of the patients with normal cytology, 48% in CIN I, 63.6% in CIN II, 64% in CIN III, and 70.8% in cervical cancer. Genes amplified in cervical cancer were MYCN (39.1%), ERBB2 (34.7%), and MYCL1 (30.4%); showed higher amplification in high-grade lesions and cervical cancer in relation to low-grade lesions and normal cytology with statistically significant differences. Besides the genes, C-MYC, EGFR, and AKT2 were amplified in samples from patients with cervical cancer by 12%, 18%, and 13%, respectively; we did not find statistical differences.Conclusion: Higher prevalence of gene amplification and HPV was found in high-grade cervical lesions and cervical cancer.

Introducción: El cáncer cervical es el segundo cáncer más importante en mujeres a nivel mundial y es la segunda causa de muerte por cáncer en mujeres. Se ha demostrado que el proceso de carcinogénesis cervical presenta componentes tanto genéticos como epigenéticos y medio ambientales. En la actualidad, hay gran interés en la búsqueda de marcadores moleculares asociados con la progresión de esta enfermedad, uno de los posibles mecanismos y que además está poco estudiado en cáncer cervical es la amplificación génica de algunos oncogenes como la familia MYC, EGFR y AKT entre otros.Objetivos: Detectar la amplificación génica de MYCN, C-MYC, MYCL1, ERBB2, EGFR y AKT2 además de la presencia del virus de papiloma humano en cepillados cervicales en mujeres con citología normal o con neoplasia intraepitelial cervical (NIC) I, II y III o con cáncer cervical.Métodos: Se genotipificó mediante reverse line blot (RLB) el virus de papiloma humano (VPH) y se determinó el estado de amplificación génica de los genes mencionados mediante PCR en tiempo real utilizando sondas taqman.Resultados: El VPH se encontró presente en 4% de las pacientes con citología normal, en 48% en NIC I, 63.6% en NIC II, 64% en NIC III y 70.8% en cáncer cervical. Los genes MYCN, MYCL1 y ERBB2 mostraron mayor amplificación en lesiones de alto grado y cáncer con diferencias estadísticamente significativas a las lesiones de bajo grado y citología normal, en 39.1%, 34.7% y 30.4% respectivamente. Además, se encontraron amplificados los genes C-MYC, EGFR y AKT2, en muestras de pacientes con cáncer cervical, en 12%, 18% y 13% respectivamente. Sin embargo, no se observaron diferencias estadísticamente significativas con respecto a las lesiones de alto y bajo grado y citología normal.Conclusión: En las lesiones de alto grado como en cáncer cervical, se encuentra mayor prevalencia del virus al igual que se detectan mayor cantidad de alteraciones genéticas como la amplificación génica.

Effect of Spindle Checkpoint on Akt2-mediated Paclitaxel-resistance in A2780 Ovarian Cancer Cells / 华中科技大学学报（医学）（英德文版）

Recent evidence has suggested that Akt2 plays an important role in the protection of cells from paclitaxel(PTX)-induced apoptosis and control of the cell cycle.In addition,some scholars suggested that the PTX sensitivity depends on a functional spindle assembly checkpoint.In the present study,we investigated the role of the Akt2/Bub1 cross-talking in apoptosis and cell cycle after exposure of the A2780 ovarian cancer cells to paclitaxel(PTX).Recombinant expression plasmid WT-Akt2 was transfected into A2780 cells by lipofectamine2000,and then the expression level of Akt2 gene was detected by using RT-PCR and Western blotting.Cell apoptosis and cell cycle were detected by flow cytometry and Hoechst 33342 staining after treatment with PTX.Moreover,we compared the expression level of Bubl in different groups by Western blotting.Our study showed that up-regulation of Akt2 contributed to A2780 ovarian cancer cells overriding PTX-induced G2/M arrest,and inhibited Bub1 expression.Our findings might shed light on the molecular mechanism of PTX-induced resistance in ovarian cancer and help develop novel anti-neoplastic strategies.

The Effect of RNA Interference Suppressing the Expression of AKT2 on Sensitivity of Human Pancreatic Cancer Cell Line Xenograft in Nude Mice to Gemcitabine / 华中科技大学学报(医学版)

Objective To study the effect of RNA interference suppressing the expression of AKT2 on sensitivity of human pancreatic cancer cell line xenograft in nude mice to gemcitabine.Methods The human pancreatic cancer cell implanted tumor mode1 in the nude mice was established.By abdominal cavity administration and intratumoral injection,the mice bearing tumor were treated with gemcitabine in combination with vector of pAKT2-siRNA.Tumor growth of tumor tissues was observed,the AKT2 mRNA and protein expression levels in tumor tissues detected by RT-PCR and immunohistochemistry respectively,and tumor apoptosis measured by Tdt-mediated dUTP nick end labeling(TUNEL).Results In chemotherapy+AKT2-siRNA group the expression of AKT2 mRNA and protein was significantly lower than in control group,chemotherapy group and chemotherapy+blank plasmid group.The tumor weight and tumor volume in chemotherapy+AKT2-siRNA group were significantly reduced as compared with those in other three groups.The inhibition rate and apoptosis rate in chemotherapy+AKT2-siRNA group were significantly higher than those in other three groups.Conclusion Sensitivity of human pancreatic cancer cell line to gemcitabine could be significantly improved by RNA interference suppressing the expression of AKT2.

Effect on the Akt2 in Skeletal Muscle of Rats with Insulin Resistance Treated by Acupuncture / 针灸推拿医学（英文版）

Objective: To observe the effect of acupuncture on Akt2 mRNA expression in skeletal muscle of insulin resistant rat models. Methods: Twenty-four SD rats were randomly divided into a control group, a model group and an acupuncture group. Fasting plasma glucose (FPG), fasting insulin (FINS), insulin sensitivity index (ISI), C peptide (C-P), as well as Akt2 mRNA expression in skeletal muscle of rats were detected by glucose oxidase method, ELISA and real-time PCR. Results: Compared with the control group, the level of FPG, FINS and C-P increased significantly (P<0.01) while ISI and Akt2 mRNA expression decreased markedly in the model group (P<0.01, P<0.05). In the acupuncture group, the levels of FPG, FINS and C-P were much lower than in the model group (P<0.01, P<0.01,P<0.05) and ISI and Akt2 mRNA expression increased markedly (P<0.01). Conclusion: The mechanism of acupuncture in treating insulin resistance may relate to the up-regulated of the Akt2 mRNA expression and to the improvement of the signal transduction of PI3K pathway.

O-GlcNAc modification on IRS-1 and Akt2 by PUGNAc inhibits their phosphorylation and induces insulin resistance in rat primary adipocytes

It has been known that O-linked beta-N-acetylglucosamine (O-GlcNAc) modification of proteins plays an important role in transcription, translation, nuclear transport and signal transduction. The increased flux of glucose through the hexosamine biosynthetic pathway (HBP) and increased O-GlcNAc modification of protein have been suggested as one of the causes in the development of insulin resistance. However, it is not clear at the molecular level, how O-GlcNAc protein modification results in substantial impairment of insulin signaling. To clarify the association of O-GlcNAc protein modification and insulin resistance in rat primary adipocytes, we treated the adipocytes with O-(2-acetamido-2deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), a potent inhibitor of O-GlcNAcase that catalyzes removal of O-GlcNAc from proteins. Prolonged treatment of PUGNAc (100 micrometer for 12 h) increased O-GlcNAc modification on proteins in adipocytes. PUGNAc also drastically decreased insulin-stimulated 2-deoxyglucose (2DG) uptake and GLUT4 translocation in adipocytes, indicating that PUGNAc developed impaired glucose utilization and insulin resistance in adipocytes. Interestingly, the O-GlcNAc modification of IRS-1 and Akt2 was increased by PUGNAc, accompanied by a partial reduction of insulin-stimulated phosphorylations of IRS-1 and Akt2. The PUGNAc treatment has no effect on the expression level of GLUT4, whereas O-GlcNAc modification of GLUT4 was increased. These results suggest that the increase of O-GlcNAc modification on insulin signal pathway intermediates, such as IRS-1 and Akt2, reduces the insulin-stimulated phosphorylation of IRS-1 and Akt2, subsequently leading to insulin resistance in rat primary adipocytes.

Significance of AKT2 and PTEN protein expressions and their correlation with human colon cancer / 西安交通大学学报(医学版)

Objective To detect the expression of AKT2 in human colon cancer and understand its relationship with PTEN.Methods The expressions of AKT2 and PTEN were detected in 30 patients with normal colonic tissues,30 patients with colon adenoma tissues,and 64 patients with colon carcinoma tissues by immunohistochemical SP staining method.Results The positive expressive rate of PTEN presented a trend of progressive decrease from normal tissues,adenoma tissues to colon carcinoma tissues,while the positive expression rate of AKT2 presented a trend of progressive increase.PTEN expression was obviously higher in normal colon tissues than in colon adenoma tissues and colon cancer tissues(?2=68.855,P

Expression of Protein Kinase B Isoforms in the Gastrocnemius Muscle of Rats at Different Ages / 中国微创外科杂志

Objective To study the expression of protein kinase B (Akt/PKB) isoforms in the gastrocnemius muscle of rats at different ages. Methods The expression levels of mRNA and protein of three Akt isoforms in the gastrocnemius muscle of 30-month-old and 6-month-old rats were detected respectively by RT-PCR and Western blotting. Data were analyzed statistically. Results (1) The levels of Akt1 and Akt2 mRNA in the 30-month-old rats was significantly higher than those in the 6-month-old rats (t=7.124, P=0.000; t=2.598, P=0.021), however, no significant difference was found in the level of Akt3 mRNA between the two groups (t=0.460, P=0.653) . (2) The level of Akt-Thr308 phosphorylation in the 30-month-old rats was significantly lower than that in the 6-month-old rats (t=-9.861, P=0.000), while the level of Akt2 protein in the 30-month group was significantly higher than that in the younger rats (t=7.522, P=0.000). No significant differences were detected in the levels of Akt1 and Akt3 proteins between the two groups (t=0.469, P=0.646; t=0.058, P=0.955). Conclusion The expression levels of three Akt isoforms in the gastrocnemius muscle of rats change with age, suggesting that the three isoforms of Akt have different functions in the gastrocnemius muscle metabolism of rats at different ages.