Micro RNA-21 promotes phosphatase gene and protein kinase B/phosphatidylinositol 3-kinase expression in colorectal cancer

AIM: To explore the regulatory mechanism of the target gene of micro RNA-21(mi R-21), phosphatase gene(p TEN), and its downstream proteins, protein kinase B(AKT) and phosphatidylinositol 3-kinase(p I3K), in colorectal cancer(CRC) cells. METHODS: Quantitative real-time p CR(q RT-p CR) and Western blot were used to detect the expression levels of mi R-21 and p TEN in HCT116, HT29, Colo32 and SW480 CRC cell lines. Also, the expression levels of p TEN m RNA and its downstream proteins AKT and p I3 K in HCT116 cells after downregulating mi R-21 were investigated. RESULTS: Comparing the mi R-21 expression in CRC cells, the expression levels of mi R-21 were highest in HCT116 cells, and the expression levels of mi R-21 were lowest in SW480 cells. In comparing mi R-21 and p TEN expression in CRC cells, we found that the protein expression levels of mi R-21 and p TEN were inversely correlated(p < 0.05); when mi R-21 expression was reduced, m RNA expression levels of p TEN did not significantly change(p > 0.05), but the expression levels of its protein significantly increased(p < 0.05). In comparing the levels of p TEN protein and downstream AKT and p I3 K in HCT116 cells after downregulation of mi R-21 expression, the levels of AKT and p I3 K protein expression significantly decreased(p < 0.05). CONCLUSION: p TEN is one of the direct target genesof mi R-21. Thus, phosphatase gene and its downstream AKT and p I3 K expression levels can be regulated by regulating the expression levels of mi R-21, which in turn regulates the development of CRC.

Telencephalin protects PAJU cells from amyloid beta protein-induced apoptosis by activating the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway

Telencephalin is a neural glycoprotein that reduces apoptosis induced by amyloid beta protein in the human neural tumor cell line PAJU. In this study, we examined the role of the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway in this process. Western blot analysis demonstrated that telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B were not expressed in PAJU cells transfected with empty plasmid, while they were expressed in PAJU cells transfected with a telencephalin expression plasmid. After treatment with 1.0 nM amyloid beta protein 42, expression of telencephalin and phosphorylated phosphatidylinositol-3-kinase/protein kinase B in the transfected cells gradually diminished, while levels of phosphorylated ezrin/radixin/moesin increased. In addition, the high levels of telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B expression in PAJU cells transfected with a telencephalin expression plasmid could be suppressed by the phosphatidylinositol-3-kinase inhibitor LY294002. These findings indicate that telencephalin activates the ezrin/radixin/moesin family/phosphatidylinositol-3-kinase/protein kinase B pathway and protects PAJU cells from amyloid beta protein-induced apoptosis.

Response of Subcutaneous Xenografts of Endometrial Cancer in Nude Mice to Inhibitors of Phosphatidylinositol 3-Kinase/Akt and Mitogen-Activated Protein Kinase (MAPK) Pathways: An Effective Therapeutic Strategy for Endometrial Cancer

Objective: This study was designed to explore whether inhibition of the extracellular-regulated kinase (ERK) and phosphatidylinositol-3-kinase (PI3K) signaling pathways can inhibit the growth of xenografts of endometrial cancer cell lines with different estrogen receptors (ER) profiles in vivo and to provide preliminary laboratory basis for the probability of endometrial adenocarcinoma treatment with blockage of the two pathways, especially to endometrial cancer with low ER status. Methods: Human endometrial cancer Ishikawa bearing ER and HEC-1Awith low ER status cells were subcutaneously injected into BALB/c nude mice to establish endometrial cancer xenograft tumor models. The effects of PI3K/Akt inhibitor LY294002, MAPK/ERK1/2 inhibitor PD-98059 and their combinations on the growth of the xenograft tumors and apoptotic state of Ishikawa and HEC-1Acells were tested in vivo using the inhibitory rate, the terminal deoxynucleotidyl transferase-mediated nick-end labeling assay, H/E-stain. Western blot analysis was used to detect the alterations of activated ERK (P-ERK) and AKT (P-AKT) during this process. Results: LY294002, a PI3K/Akt pathway inhibitor, induced significant suppression in the growth of both Ishikawa and HEC-1Acell xenograft tumors, concomitant with increased apoptosis in xenografts as evidenced by TUNEL. A similar effect was also observed when the MAPK/ERK1/2 signaling pathway was inhibited by PD98059. Concurrent inhibition of the PI3K/Akt and MAPK/ERK1/2 pathways showed enhanced anti-tumor effects in vivo as indicated by increased apoptosis. At the same time, the levels of P-ERK and P-AKT in both xenograft tumors decreased, and their levels in combination group was the lowest. Conclusions: PD98059, LY294002 and their combinations showed remarkable inhibitory effects on xenograft tumors of endometrial carcinoma cell lines with different expression status of ER in vivo through blockage of PI3K/Akt and MAPK/ERK1/2 signaling pathways. This suggests that targeting these pathways may be an effective therapeutic strategy against endometrial carcinomas, especially for ER-negative cancers which show poor response to endocrinal therapy.

Expression of phosphatidylinositol-3 kinase and effects of inhibitor Wortmannin on expression of tumor necrosis factor-α in severe acute pancreatitis associated with acute lung injury

BACKGROUND: Acute lung injury(ALI) is a common and serious complication of severe acute pancreatitis(SAP). The study aimed to investigate the protective effect and mechanism of phosphatidylinositol-3 kinase(PI3K) inhibitor Wortmannin in SAP associated with ALI.METHODS: Ninety rats were randomly divided into three groups: sham operation(SO) group(n=30), SAP group(n=30), and SAP+Wortmannin(SAP+W) group(n=30). SAP model was induced by retrograde injection of 4% sodium taurocholate into the biliopancreatic duct of rats. The rate of lung water content, myeloperoxidase(MPO), matrix metalloproteinase 9(MMP-9), protein kinase B(PKB), abdphosphorylation of protein kinase B(P-PKB) activity in the lung tissue were evaluated.RESULTS: In the SAP group, the p-PKB expression in the lung tissue began to rise at 3 hours after modeling, and peaked at 12 hours(P<0.05); the rate of lung water content, MPO and TNF-α activity were also gradually increased, and the degree of lung lesion gradually increased(P<0.05). In the SAP+Wortmannin group, the p-PKB expression in the lung tissue began to rise at 3 hours after modeling, and peaked at 12 hours; it was higher than that in the SO group(P<0.05), but significantly lower than that in the SAP group(P<0.05). The rest indicators in the SAP+Wortmannin group were also significantly decreased as compared with the SAP group(P<0.05).CONCLUSIONS: The expression of phosphatidylinositol-3 kinase/protein kinase B was elevated in severe pancreatitis rats with lung injury. This suggested that PI3 K signal transduction pathway is involved in the control and release of proinfl ammatory cytokines TNF-α, which may play an important role in the pathogenesis of severe acute pancreatitis associated with lung injury. This finding indicated that Wortmannin can block the PI3 K signal transduction pathway, and inhibit the release of infl ammatory factor TNF-α.

PI3K-like Kinases Restrain Pim Gene Expression in Endothelial Cells

Pim kinases contribute to tumor formation and development of lymphoma,which shows enhanced DNA replication,DNA recombination and repair.Endothelial cells (ECs) express all the three members of Pim kinase gene family.We hypothesized that DNA repair gene would regulate Pim ex-pression in ECs.Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in M199 culture medium.The cellular distribution of Pim-3 in ECs was determined by immunofluorescent staining.The siRNA fragments were synthesized and transfected by using Lipofectamine LTX.The total cellular RNA was extracted from the cells by using Trizol reagent.cDNAs were quantified by semi-quantity PCR.The effects of LY294002 and wortmannin on RNA stability in ECs were also ex-amined.Our data showed that LY294002 and wortmannin,phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase inhibitors,increased Pim mRNA expression in ECs without altering the mRNA stabil-ity.RNA interference (RNAi) targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) increased mRNA expression of Pim-3 and Pim-1,respectively.Silencing of Akt decreased Pim-1 instead of Pm-2 and Pim-3 gene expression in ECs.But etoposide,a nucleoside analogue,which could activate DNA-PKcs and ATM,increased Pim expression in ECs.Our study indicates that the expression of Pim kinases is physiologically related to DNA-PKcs and ATM in ECs.

Phosphoprotein Phosphatase 1 Isoforms Alpha and Gamma Respond Differently to Prodigiosin Treatment and Present Alternative Kinase Targets in Melanoma Cells

Reversible protein phosphorylation is a central regulatory mechanism of cell function. Deregulation of the balanced actions of protein kinases and phosphatases has been frequently associated with several pathological conditions, including cancer. Many studies have already addressed the role of protein kinases misregulation in cancer. However, much less is known about protein phosphatases influence. Phosphoprotein Phosphatase 1 (PPP1) is one of the major serine/threonine protein phosphatases who has three catalytic isoforms: PPP1CA, PPP1CB, and PPP1CC. Its function is achieved by binding to regulatory subunits, known as PPP1-interacting proteins (PIPs), which may prefer a catalytic isoform. Also, some inhibitors/enhancers may exhibit isoform specificity. Here we show that, prodigiosin (PG), a molecule with anticancer properties, promotes the formation of PPP1CA-AKT complex and not of PPP1CC-MAPK complex. Both, AKT and MAPK, are well-known PIPs from two pathways that crosstalk and regulate melanoma cells survival. In addition, the analysis performed using surface plasmon resonance (SPR) technology indicates that PPP1 interacts with obatoclax (OBX), a drug that belongs to the same family of PG. Overall, these results suggest that PG might, at least in part, act through PPP1C/PIPs. Also, this study is pioneer in demonstrating PPP1 isoform-specific modulation by small molecules.

Salvianolic acid B promotes the invasion and migration of H_(2)O_(2)-induced HTR-8/Svneo trophoblast cells by upregulating matrix metalloproteinase-9 via the phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B pathway

OBJECTIVE:To elucidate the regulatory effects of salvianolic acid B(Sal B)on trophoblast cells in preeclampsia(PE).METHODS:3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide(MTT)assays were used to detect the viability of human extravillous trophoblast HTR-8/Svneo cells induced by H_(2)O_(2)following treatment with different concentrations of Sal B.The levels of oxidative stressrelated molecules,including superoxide dismutase,glutathione-Px and malondialdehyde were detected using corresponding kits.Cell apoptosis was detected using a Terminal deoxynucleotidyl transferase(Td T)d UTP NickEnd Labeling(TUNEL)assay,and the expression of apoptosis-related proteins was detected using western blot analysis.In the present study,wound healing and Transwell assays were performed to measure the levels of cell invasion and migration.Western blot analysis was also used to detect the expression levels of epithelialmesenchymal transition-related proteins.The mechanisms underlying Sal B were further investigated using reverse transcription-quantitative real-time polymerase chain reaction(RT-q PCR)and western blot analysis,to determine the expression levels of matrix metallopeptidase 9(MMP-9)and phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)/protein kinase B(Akt).RESULTS:Sal B increased the activity of HTR-8/Svneo cells,inhibited oxidative damage and promoted the invasion and migration of trophoblast cells induced by H_(2)O_(2).Furthermore,the expression levels of MMP-9 and members of the PI3K/Akt signaling pathway were significantly decreased.The pathway agonist,LY294002,and MMP-9 inhibitor,GM6001,reversed the effects of Sal B on H_(2)O_(2)-induced cells.CONCLUSIONS:Sal B promoted the invasion and migration of H_(2)O_(2)-induced HTR-8/Svneo trophoblast cells by upregulating MMP-9 via the PI3K/Akt signaling pathway.

Bornyl acetate extracted from Sharen(Fructus Amomi)inhibits proliferation,invasion and induces apoptosis by suppressing phosphatidylinositol-3-kinase/protein kinase B signaling in colorectal cancer

OBJECTIVE:To investigate the antitumor effects of bornyl acetate(BA)isolated from Sharen(Fructus Amomi)in colorectal cancer(CRC)and the underlying mechanisms.METHODS:SW480 and HT29 cells were treated with increasing doses of BA in order to determine its antitumor effects in vitro.Cell viability,colony formation,cell cycle,and apoptosis as well as migration and invasion were assessed using various assays.In addition,the in vivo antitumor effects of BA were assessed using a xenograft mouse model.We then assessed the mechanism of action of BA by conducting pathway activator-mediated rescue experiments and assessed the protein levels by Western blot analysis.RESULTS:BA showed anti-CRC tumor activities in vitro by suppressing cell proliferation and colony formation,inducing apoptosis,blocking cell cycle,and inhibiting migration and invasion.These effects were mediated via suppression of the phosphatidylinositol-3-kinase/protein kinase B(PI3K/AKT)pathway.In the tumor xenograft experiment,BA was found to repress tumor growth in vivo with low toxicity.CONCLUSIONS:The results demonstrated that BA exerts antitumor effects by suppressing the PI3K/AKT pathway,with low toxicity.Thus,BA might be a potential novel therapeutic agent for CRC.

黄芪阳和汤调控PI3K/AKT/NF-κB信号通路促进糖尿病足溃疡大鼠创面愈合

目的基于磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/核因子-κB(NF-κB)信号通路探究黄芪阳和汤对糖尿病足溃疡(DFU)大鼠创面愈合的影响。方法构建DFU大鼠模型,将建模成功的48只大鼠随机分为模型组,黄芪阳和汤低(8.5 g/kg)、高(17 g/kg)剂量组,黄芪阳和汤高剂量(17 g/kg)+LY294002(PI3K/AKT通路抑制剂,0.3 mg/kg)组;每组12只;另取12只大鼠为对照组。各组大鼠给予对应药物干预,连续4周。第14、28天给药后,观察大鼠一般状态及创面变化,计算创面愈合率,检测大鼠空腹血糖(FBG)水平和大鼠创面周围组织经皮氧分压(TcpO2);酶联免疫吸附试验检测大鼠血清血管内皮生长因子(VEGF)、缺氧诱导因子-1α(HIF-1α)、C反应蛋白(CRP)、白细胞介素(IL)-6水平;苏木素-伊红染色观察大鼠创面组织病理学变化;免疫组织化学染色测定大鼠创面组织微血管密度;蛋白免疫印迹法检测大鼠创面组织中PI3K、磷酸化PI3K(p-PI3K)、AKT、磷酸化AKT(p-AKT)、NF-κB p65、磷酸化NF-κB p65(p-NF-κB p65)、NF-κB抑制蛋白α(IκB-α)蛋白表达。结果对照组大鼠毛色光滑,饮食、饮水、排泄均正常,较活跃,创面愈合快,创面组织炎症反应较轻,新生血管较多,肉芽组织中成纤维细胞及胶原基质丰富;模型组大鼠毛色暗淡无光泽,活动减少,且出现多饮、多食、多尿症状,创面颜色较深,且周围组织出现水肿、溃疡,创面组织可见大量炎性细胞浸润,伴组织坏死、渗出,新生血管及成纤维细胞较少,创面愈合率、创面周围组织TcpO2、血清VEGF、HIF-1α、创面组织微血管密度、p-PI3K、p-AKT、IκB-α蛋白表达水平降低,FBG、血清CRP、IL-6、创面组织p-NF-κB p65蛋白表达升高(P<0.05);与模型组相比,黄芪阳和汤低、高剂量组大鼠状态逐渐改善,创面组织病变程度依次减轻,创面愈合率、创面周围组织TcpO2、血清VEGF、HIF-1α、创面组织微血管密度、p-PI3K、p-AKT、IκB-α蛋白表达水平依次升高,FBG、血清CRP、IL-6、创面组织p-NF-κB p65蛋白表达依次降低(P<0.05);LY294002能部分逆转高剂量黄芪阳和汤对DFU大鼠的治疗作用(P<0.05)。结论黄芪阳和汤能调控PI3K/AKT/NF-κB信号通路,抑制DFU大鼠炎症反应,促进血管新生,从而促进创面愈合。

栀子苷调节PI3K/AKT/mTOR信号通路在动脉粥样硬化形成过程中对Th17/Treg功能的影响

目的:观察栀子苷对载脂蛋白E缺乏(ApoE^(-/-))小鼠Th17/调节性T(Treg)细胞失衡的影响及其作用机制。方法:将50只纯合子ApoE^(-/-)雌性小鼠随机分为对照组、模型组和栀子苷低剂量组、栀子苷中剂量组、栀子苷高剂量组。对照组小鼠喂养普通饲料,模型组和栀子苷组小鼠喂养高脂饲料。从第8周开始,栀子苷各剂量组每日灌胃栀子苷(25、50、100 mg/kg),连续8周。试验结束时,采用油红O染色评估主动脉及其根部动脉粥样硬化(AS)病变面积比。采用定量逆转录聚合酶链式反应(RT-PCR)分析主动脉组织肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6、IL-17A和IL-10 mRNA表达;采用流式细胞仪分析脾脏中Th17和Treg细胞百分比;蛋白免疫印迹法(Western Blot)检测主动脉组织磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关蛋白表达。结果:油红O染色病变显示,栀子苷中剂量组、栀子苷高剂量组病变百分比低于模型组(P<0.05)。与对照组比较,模型组主动脉TNF-α、IL-6和IL-17A mRNA表达水平升高(P<0.05);栀子苷各剂量组主动脉TNF-α、IL-6和IL-17A mRNA表达水平降低(P<0.05)。与对照组比较,模型组主动脉抗炎细胞因子IL-10 mRNA表达水平降低(P<0.05);栀子苷各剂量组主动脉抗炎细胞因子IL-10 mRNA表达水平升高(P<0.05)。与对照组比较,模型组小鼠脾脏中Th17细胞百分比升高,Treg细胞百分比降低(P<0.05)。栀子苷处理恢复了AS小鼠Th17和Treg细胞的平衡。栀子苷抑制PI3K的表达及AKT和mTOR的磷酸化,MHY1485(mTOR活化剂)减弱了栀子苷对T细胞分化的影响。结论:栀子苷抗AS作用机制可能与抑制PI3K/AKT/mTOR信号引起的Treg细胞增多和Th17细胞减少有关。

基于PI3K/AKT/GSK-3β信号通路探讨EA改善APP/PS1双转基因小鼠认知功能障碍的内在机制

目的探讨鞣花酸(ellagicacid,EA)对APP/PS1双转基因小鼠认知功能的影响,并基于磷脂酰肌醇3-激酶/蛋白激酶B/糖原合成酶激酶-3(PI3K/AKT/GSK-3β)信号通路探讨鞣花酸对双转基因小鼠海马氧化应激水平的调节机制。方法将32只SPF级6月龄APP/PS1双转基因小鼠随机分为4组,即APP/PS1组、APP/PS1+EA组、APP/PS1+LY294002组、APP/PS1+EA+LY294002组,每组8只,另外选取8只SPF级C57BL/6J野生型小鼠(Wildtype)作为空白对照组,即WT组。APP/PS1+EA组给予50mg·kg^(-1)·d^(-1)灌胃EA;APP/PS1+LY294002组予以1.5mg·kg^(-1)·d^(-1)腹腔注射PI3K抑制剂LY294002;APP/PS1+EA+LY294002组予以50mg·kg^(-1)·d^(-1)灌胃EA,同时按1.5mg·kg^(-1)·d^(-1)腹腔注射LY294002;WT组和APP/PS1组于相同时间点灌胃等体积10%二甲基亚砜(DMSO)。每日给药1次,连续给药60天。Morris水迷宫检测小鼠学习和记忆能力,免疫组化、蛋白免疫印迹法检测PI3K、AKT、GSK-3β相关蛋白的表达,透射电镜观察小鼠海马组织超微结构变化。结果与WT组相比,其他四组的逃避潜伏期均增长(P<0.05),穿越平台次数明显减少(P<0.01);APP/PS1组、APP/PS1+LY294002组和APP/PS1+EA+LY294002组中的PI3K、AKT蛋白表达量显著降低(P<0.01),GSK-3β表达量显著升高(P<0.01);APP/PS1+EA组的PI3K表达量降低(P<0.05),AKT表达量显著降低(P<0.01),GSK-3β表达量升高(P<0.05);与WT组相比,APP/PS1组海马神经元细胞数目较少,线粒体结构破坏,大部分线粒体出现肿胀,线粒体的内膜和外模不完整,部分线粒体嵴消失,微管、微丝缠结,排列紊乱,而APP/PS1+EA组神经元细胞数较APP/PS1组增多,线粒体结构较清晰,可见清楚的线粒体嵴,线粒体轻度水肿。微管、微丝排列较整齐有序。结论鞣花酸改善AD模型小鼠的学习和记忆能力、减少海马神经元细胞损伤和凋亡,其作用机制可能是通过调节PI3K、AKT、GSK-3β等相关蛋白降低AD模型小鼠海马氧化应激水平。

基于转录组学分析PI3K/AKT通路对冷却滩羊肉贮藏期间肌肉代谢稳态的影响

为揭示磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,AKT)信号通路在冷却滩羊肉贮藏期间代谢调控中的作用,采用高通量测序技术研究冷却滩羊肉经PI3K抑制剂LY294002注射处理后分别在贮藏期为0、4、8 d时转录组的变化。在对照组和LY294002处理组之间,0、4、8 d时被鉴定为显著差异表达基因(differentially expressed genes,DEGs)数分别为474、96、13。功能富集分析表明,DEGs主要与代谢稳态有关,如氨基酸的生物合成、糖酵解/糖异生、甘油脂类代谢过程。此外,组织切片分析显示,与对照组相比,LY294002组肌纤维间距增加,肌纤维直径显著减小,表明肌肉发生萎缩。结果表明,PI3K/AKT信号通路在维持滩羊肉贮藏过程中代谢平衡方面起着至关重要的作用,反过来又确保了肌肉的正常生长。研究为进一步阐明冷却滩羊肉贮藏中PI3K/AKT信号代谢稳态的分子机制提供了方向。

Yes相关蛋白(YAP)通过激活PI3K/AKT通路促进皮肤鳞状细胞癌细胞侵袭和迁移

目的探讨Yes相关蛋白(YAP)在皮肤鳞状细胞癌(cSCC)中表达及与cSCC侵袭和迁移中的作用。方法通过免疫组织化学染色法检测cSCC、鲍温病(BD)、癌旁正常皮肤组织中YAP的表达水平,并分析与临床病理参数之间的关系;利用慢病毒转染构建YAP基因敲低的A431稳定细胞株,利用四甲基罗丹明标记的鬼笔环肽检测A431细胞微丝分布和数量,Transwell TM实验检测细胞侵袭能力,划痕实验检测A431细胞的迁移能力;免疫荧光细胞化学染色法观察敲低YAP后上皮间质转化(EMT)相关标志物上皮钙黏素(E-cadherin)、锌指转录因子Snail的表达;Western blot法检测E-cadherin、Snail、β-catenin、磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(AKT)、磷酸化的蛋白激酶B(p-AKT)、核糖体蛋白S6(S6)、磷酸化S6(p-S6)、4E结合蛋白1(4EBP1)、磷酸化的4EBP1(p-4EBP1)的表达。结果YAP在cSCC和BD中表达显著高于癌旁正常皮肤组织;cSCC中YAP高表达与肿瘤大小、分化程度、侵袭程度密切相关,与患者的性别、年龄、发病部位、形态类型、是否神经脉管侵犯不相关;敲低A431细胞中YAP后,肿瘤细胞的侵袭、迁移能力降低,细胞微丝变细、伪足变少;E-cadherin表达增加,Snail和β-catenin蛋白表达降低,p-AKT、p-S6及p-4EBP1蛋白表达降低。结论YAP在cSCC中高表达,YAP激活PI3K/AKT信号通路促进cSCC的侵袭、迁移及EMT过程。

miR-93-5p通过PI3K/AKT通路调控HepG2细胞自噬

目的:探究miR-93-5p对HepG2细胞增殖、自噬、葡萄糖消耗以及磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinases,PI3K)/蛋白激酶B(protein kinase B,AKT)通路的影响。方法:高浓度葡萄糖诱导构建胰岛素抵抗(insulin resistance,IR)细胞模型,设计合成miR-93-5p inhibitor和NC,结合自噬抑制剂3-MA,将细胞分为Control组、IR组、IR+inhibitor NC组、IR+inhibitor组、IR+3-MA+inhibitor组。CCK8法检测各组细胞增殖活力,试剂盒检测各组细胞葡萄糖消耗量和细胞糖原合成情况,Western-Blot法检测细胞自噬基因微管相关蛋白1轻链3(autophagy genes microtubule-associated protein 1 light chain 3,LC3)-I、LC3-II、肝细胞生长因子(hepatocyte growth factor,HGF)蛋白、PI3K/AKT通路蛋白(AKT、p-AKT)的表达。结果:与对照组相比,IR组细胞增殖活力、葡萄糖消耗量和细胞糖原合成量、HGF蛋白、LC3-II蛋白表达下降(P<0.01),LC3-I蛋白和p-AKT/AKT值表达均增加(P<0.01)。与IR组和IR+inhibitor NC组相比,IR+inhibitor组细胞增殖活力、葡萄糖消耗量和细胞糖原合成量、HGF蛋白、LC3-II蛋白表达增加(P<0.01),LC3-I蛋白和p-AKT/AKT值表达均下降(P<0.01)。与IR+inhibitor组相比,3-MA能逆转miR-93-5p inhibitor的作用。结论:miR-93-5p通过PI3K/AKT通路促进HepG2细胞自噬,进而抑制胰岛素抵抗,缓解糖尿病造成的机体损伤。

秋水仙碱通过激动PI3K/AKT/eNOS信号通路对急性心肌梗死大鼠心功能的影响

目的:探究秋水仙碱通过调节磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/内皮型一氧化氮合酶(eNOS)信号通路对急性心肌梗死(AMI)大鼠心功能的影响。方法:78只大鼠中随机选取12只作为假手术组,剩余大鼠构建AMI模型,造模成功大鼠分为模型组、秋水仙碱组[4 mg/(kg·d)]、秋水仙碱[4 mg/(kg·d)]+LY294002(20 mg/mL)组、秋水仙碱[4 mg/(kg·d)]+MK-2206(60μg/mL)组、秋水仙碱[4 mg/(kg·d)]+L-NAME(1.6 mg/mL)组,每组12只。超声心动图检测大鼠左心室舒张末期内径(LVEDD)、左心室收缩末期内径(LVESD)、射血分数(EF)及短轴缩短率(FS)。处死大鼠,苏木素-伊红(HE)染色检测大鼠心肌组织石蜡切片病理学变化;末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法检测心肌细胞凋亡率;酶联免疫吸附实验(ELISA)测定大鼠血清肿瘤坏死因子α(TNF-α)、白细胞介素-6(IL-6)、肌酸激酶同工酶(CK-MB)以及心肌组织匀浆中超氧化物歧化酶(SOD)、丙二醛(MDA)、过氧化氢酶(CAT)水平;免疫印迹法(Western Blot)测定大鼠心肌组织PI3K/AKT/eNOS通路蛋白表达。结果:与假手术组相比,模型组大鼠LVESD、LVEDD,血清CK-MB、TNF-α、IL-6水平,心肌匀浆MDA、心肌细胞凋亡率升高,EF、FS水平,心肌匀浆SOD、CAT,心肌组织磷酸化PI3K(p-PI3K)/PI3K、磷酸化AKT(p-AKT)/AKT、磷酸化eNOS(p-eNOS)/eNOS降低(P<0.05);与模型组相比,秋水仙碱组大鼠LVESD、LVEDD,血清CK-MB、TNF-α、IL-6水平,心肌匀浆MDA、心肌细胞凋亡率降低,EF、FS水平,心肌匀浆SOD、CAT,心肌组织p-PI3K/PI3K、p-AKT/AKT、p-eNOS/eNOS升高(P<0.05);与秋水仙碱组相比,秋水仙碱+LY294002组、秋水仙碱+MK-2206组、秋水仙碱+L-NAME组大鼠LVESD、LVEDD,血清CK-MB、TNF-α、IL-6水平,心肌匀浆MDA、心肌细胞凋亡率升高,EF、FS水平,心肌匀浆SOD、CAT,心肌组织p-PI3K/PI3K、p-AKT/AKT、p-eNOS/eNOS降低(P<0.05)。结论:秋水仙碱可能通过激活PI3K/AKT/eNOS信号通路对AMI大鼠发挥心功能保护作用。

PI3K/Akt信号通路通过上调HKDC1促进人肝癌HepG2细胞糖酵解、增殖及迁移

目的探讨磷脂酰肌醇-3-激酶/蛋白激酶B(PI3K/Akt)通过调节含己糖激酶结构域的蛋白1(HKDC1)对人肝癌HepG2细胞糖酵解、增殖和迁移的影响及机制。方法将体外培养的对数期人肝癌HepG2细胞,分为对照组、LY294002组和MK-2206组,RT-qPCR法检测各组细胞HKDC1 mRNA的表达水平,western blotting法分析各组细胞HKDC1蛋白的表达水平。将细胞分为si-NC组(转染si-NC)、si-HKDC1组(转染si-HKDC1),western blotting法分析各组细胞HKDC1蛋白的表达水平,CCK-8、5-乙炔基-2′-脱氧尿苷(EdU)实验检测各组细胞增殖活力和划痕,Transwell实验检测各组细胞迁移率,细胞外酸化率(ECAR)实验检测分析各组细胞的糖酵解和糖酵解能力,葡萄糖及乳酸含量测定实验检测各组细胞内葡萄糖及乳酸含量。将细胞分为对照组、LY294002组、过表达HKDC1+LY294002组、MK-2206组、过表达HKDC1+MK-2206组,葡萄糖及乳酸含量测定实验检测各组细胞内葡萄糖及乳酸含量。结果与对照组比较,LY294002组细胞HKDC1 mRNA表达明显降低(P<0.001),HKDC1蛋白表达降低(P<0.01);MK-2206组细胞HKDC1 mRNA表达降低(P<0.01),HKDC1蛋白表达明显降低(P<0.001)。与si-NC组相比,si-HKDC1组细胞HKDC1蛋白表达降低(P<0.001);与si-NC组相比,si-HKDC1组细胞增殖活力降低(P<0.05),EdU阳性细胞数明显减少(P<0.01);与si-NC组相比,si-HKDC1组细胞划痕愈合率明显降低(P<0.001),迁移细胞数明显减少(P<0.001);与si-NC组相比,si-HKDC1组细胞糖酵解和糖酵解能力明显减弱(P<0.01);与si-NC组相比,si-HKDC1组细胞内葡萄糖和乳酸含量明显降低(P<0.05)。与LY294002组相比,过表达HKDC1+LY294002组细胞内葡萄糖和乳酸含量明显升高(P<0.01);与MK-2206组相比,过表达HKDC1+MK-2206组细胞内葡萄糖和乳酸含量明显升高(P<0.001,P<0.01)。结论PI3K/Akt信号通路通过HKDC1促进人肝癌HepG2细胞糖酵解、增殖和迁移。

葛根芩连汤通过IRS-1/PI3K/AKT通路对胃肠湿热型2型糖尿病大鼠糖脂代谢的影响

目的探究葛根芩连汤通过胰岛素受体底物-1(IRS-1)/磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)通路对胃肠湿热型2型糖尿病大鼠糖脂代谢的影响。方法将40只SD大鼠随机分为正常组(2 mL生理盐水灌胃)、造模组(2 mL生理盐水灌胃)、二甲双胍组(4.17 mg/100 g二甲双胍灌胃)和葛根芩连汤组(1 g/100 g葛根芩连汤灌胃),每组10只。采用高脂高糖饲料加腹腔注射链脲佐菌素(STZ)构建2型糖尿病大鼠模型,随后喂食油脂、42°白酒及蜂蜜水构建胃肠湿热型2型糖尿病大鼠模型。测量各组大鼠不同时间节点体质量,血糖仪测定空腹血糖(FBG);ELISA检测空腹胰岛素(FINS)、三酰甘油(TG)、总胆固醇(TC)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平变化、计算胰岛素抵抗指数(HOMA-IR);HE染色检测肝组织病理学变化;检测肝组织过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)及丙二醛(MDA)含量变化。Western blot检测肝组织IRS-1、PI3K、p-PI3K、AKT及p-AKT蛋白变化。结果与正常组比较,造模组大鼠体质量、FBG、FINS及HOMA-IR、GSH-Px、CAT、SOD、IRS-1、p-PI3K/PI3K及p-AKT/AKT水平均明显下降(P<0.05)、TG、TC、IL-6、TNF-α及MDA含量均显著升高(P<0.05),可见局灶性肝实质损失。与造模组比较,二甲双胍组及葛根芩连汤组大鼠体质量、FBG、FINS及HOMA-IR、GSH-Px、CAT、SOD、IRS-1、p-PI3K/PI3K及p-AKT/AKT水平均明显升高(P<0.05)、TG、TC、IL-6、TNF-α及MDA含量均显著降低(P<0.05),显示正常的肝实质。结论葛根芩连汤可明显改善胃肠湿热型2型糖尿病糖脂紊乱,可能是通过IRS-1/PI3K/AKT通路发挥作用。

积雪草酸调节PI3K/AKT信号通路对七氟烷诱导的海马神经元HT-22细胞凋亡的影响

目的探讨积雪草酸(AA)通过调节磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/AKT)信号通路对七氟烷(SEVO)诱导的HT-22细胞凋亡的影响。方法使用不同浓度(0、5、10、15、20、30μmol/L)AA处理七氟烷诱导HT-22细胞24 h,CCK-8检测HT-22细胞活力;将HT-22细胞分为对照(Control)组、七氟烷(SEVO)组、AA低浓度(AA-L,10μmol/L)组、AA中浓度(AA-M,15μmol/L)组、AA高浓度(AA-H,20μmol/L)组和AA高浓度+PI13K通路抑制剂LY294002(AA-H+LY294002,20μmol/L AA+5μmol/L LY294002)组。显微镜下观察各组细胞形态变化,ELISA检测炎性因子肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)及氧化应激指标超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)水平,活性氧(ROS)试剂盒检测ROS水平,TUNEL试剂盒检测HT-22凋亡,JC-1法检测线粒体膜电位,三磷酸腺苷(ATP)含量检测试剂盒检测ATP含量,Western blot检测B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、p-PI3K、PI3K、p-AKT和AKT蛋白表达。结果与0μmol/L相比,5~30μmol/L AA处理的HT-22细胞活力呈浓度依赖性升高(P<0.05),选择浓度为10、15、20μmol/L的AA用于后续实验。与SEVO组相比,AA-L组、AA-M组和AA-H组TNF-α、IL-6、MDA和ROS水平、细胞凋亡率、Bax和Caspase-3蛋白表达降低,SOD和GSH-Px水平、红/绿JC-1荧光比、ATP含量、Bcl-2蛋白表达、PI3K和AKT磷酸化水平增加(P<0.05),且呈浓度依赖性。LY294002可逆转AA对七氟烷诱导的HT-22细胞损伤的保护作用(P<0.05)。结论AA通过激活PI3K/AKT信号通路保护七氟烷诱导的HT-22细胞损伤,为新型减轻七氟烷诱导的神经毒性的药物开发提供了一定的理论参考。

艾司氯胺酮通过糖原合成酶激酶-3β/NOD样受体热蛋白结构域蛋白3通路改善新生鼠缺氧缺血性心肌损伤的研究

目的基于糖原合成酶激酶-3β/NOD样受体热蛋白结构域蛋白3(GSK-3β/NLRP3)通路探讨艾司氯胺酮对新生鼠缺氧缺血性心肌损伤的作用。方法将30只新生大鼠随机分为假手术组、模型组及艾司氯胺酮组,每组10只。假手术组新生鼠行颈部正中切口,暴露双侧颈总动脉;模型组和艾司氯胺酮组新生鼠采用结扎颈总动脉联合低氧环境建立缺血缺氧模型;艾司氯胺酮组新生鼠给予艾司氯胺酮干预(50 mg/kg)。检测各组新生鼠左心室射血分数(LVEF)、左心室短轴缩短率(LVFS)、左心室舒张末期内径(LVEDD)、左心室收缩末期内径(LVESD)、血清肌酸激酶同工酶(CK-MB)、肌钙蛋白I(cTnI)、乳酸脱氢酶(LDH)、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6、IL-1β水平,心肌损伤、心肌细胞凋亡及凋亡蛋白半胱氨酸蛋白酶1/3/9(caspase 1/3/9)水平,心肌组织中性粒细胞浸润情况,以及心肌组织中GSK-3β、NLRP3蛋白水平变化。结果相比于假手术组,模型组新生鼠LVEF、LVFS降低,LVEDD、LVESD增高,且艾司氯胺酮组新生鼠LVEF、LVFS高于模型组,LVEDD、LVESD低于模型组,差异均有统计学意义(P<0.05)。模型组血清CK-MB、cTnI、LDH、TNF-α、IL-6、IL-1β水平,心肌损伤及凋亡,心肌组织切割的caspase 1/3/9蛋白水平、中性粒细胞数量、GSK-3β、NLRP3蛋白水平增加,且艾司氯胺酮组上述指标低于模型组,差异有统计学意义(P<0.05)。结论艾司氯胺酮通过抑制炎症反应改善新生鼠缺氧缺血性心肌损伤,其作用机制与GSK-3β/NLRP3通路有关。

LncRNA MALAT1通过靶向miR-146a调节PI3K/Akt信号通路影响胃癌细胞的增殖、迁移和侵袭

目的 探究长链非编码RNA(long non-coding RNA,LncRNA)肺腺癌转移相关转录子1(metastasis-associated lung adenocarcinoma transcript 1,MALAT1)通过调节miR-146a对胃癌(gastric cancer, GC)细胞的增殖、迁移和侵袭的影响及其机制。方法 收集GC组织和配对正常胃上皮组织,将GC细胞MNK-45分为空白对照(blank control, BC)组(未转染)、MALAT1 siRNA-NC组(转染MALAT1 siRNA-NC)、MALAT1 siRNA组(转染MALAT1 siRNA)、miR-146a mimics-NC组(转染miR-146a mimics-NC)、miR-146a mimics组(转染miR-146a mimics)、MALAT1 siRNA+miR-146a inhibitor-NC组(共转染MALAT1 siRNA+miR-146a inhibitor-NC)、MALAT1 siRNA+miR-146a inhibitor组(共转染MALAT1 siRNA+miR-146a inhibitor)。定量荧光PCR(qRT-PCR)检测胃组织或细胞中MALAT1、miR-146a表达量;CCK-8法和克隆形成实验检测细胞增殖能力;Transwell法检测细胞侵袭和迁移能力;RNA pull down实验、双荧光素酶报告实验分析MALAT1和miR-146a的结合情况;Western blot检测磷脂酰肌醇3激酶(phosphatidylinositol 3-kinase, PI3K)/蛋白激酶B(protein kinase B,Akt)通路蛋白及c-Myc、基质金属蛋白酶9(matrix metalloproteinase 9,MMP9)蛋白表达量。结果 转染MALAT1 siRNA可明显降低MNK-45细胞的MALAT1表达量,敲低MALAT1或过表达miR-146a可降低细胞活力、克隆能力、迁移和侵袭,增加miR-146a表达,降低PI3Kp85α、PI3Kp85β、c-Myc、MMP9蛋白表达量及p-Akt/Akt水平;MALAT1可结合并靶向下调miR-146a表达;低表达miR-146a可逆转敲低MALAT1对MNK-45细胞增殖、迁移和侵袭的抑制效应。结论 MALAT1可能作为ceRNA吸附并降解miR-146a,敲低MALAT1可上调miR-146a表达,并通过PI3K/Akt通路抑制GC细胞增殖、迁移和侵袭。