PTEN/PI3K/AKT蛋白、miR-21在青海藏族及汉族胃癌患者癌组织中的表达差异

目的探讨PTEN/PI3K/AKT蛋白、miR-21在青海藏族及汉族胃癌患者癌组织中的表达差异。方法选取2016年12月至2018年12月我院胃肠外科进行手术的78例胃癌患者为研究对象(藏族38例,汉族40例)。采用实时荧光定量PCR法检测癌组织标本及癌旁组织中miR-21水平,采用免疫组化染色法检测PTEN/PI3K/AKT蛋白,分析不同民族胃癌患者不同组织PTEN/PI3K/AKT蛋白、miR-21表达情况,同时分析其与临床病理特征之间的关系。结果汉族、藏族胃癌患者癌组织中miR-21表达水平高于癌旁组织(P<0.05),且汉族癌组织中miR-21表达水平明显高于藏族(P<0.05)。汉族、藏族胃癌患者癌组织中PTEN、PI3K、AKT蛋白阳性率比较,差异均有统计学意义(P<0.05),其中汉族胃癌组织中PTEN蛋白阳性率低于藏族,但PI3K、AKT蛋白阳性率高于藏族(P<0.05)。两民族胃癌组织中的PTEN、AKT蛋白、miR-21表达与TNM分期、分化程度、淋巴转移相关(P<0.05),PI3K蛋白表达与TNM分期、淋巴转移相关(P<0.05)。在汉族、藏族胃癌患者中,miR-21与PTEN蛋白均呈负相关,与PI3K、AKT蛋白均呈正相关(P<0.05)。结论PTEN在藏族、汉族胃癌患者中表达水平降低,且汉族表达低于藏族,且PTEN与PI3K、AKT呈负相关,miR-21可能通过抑制PTEN,激活PI3K、AKT信号通路从而参与胃癌患者的发生、发展。     

PI3K/Akt signaling requires spatial compartmentalization in plasma membrane microdomains

Spatial compartmentalization of signaling pathway components generally defines the specificity and enhances the efficiency of signal transduction. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is known to be compartmentalized within plasma membrane microdomains; however, the underlying mechanisms and functional impact of this compartmentalization are not well understood. Here, we show that phosphoinositide-dependent kinase 1 is activated in membrane rafts in response to growth factors, whereas the negative regulator of the pathway, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), is primarily localized in nonraft regions. Alteration of this compartmentalization, either by genetic targeting or ceramide-induced recruitment of PTEN to rafts, abolishes the activity of the entire pathway. These findings reveal critical steps in raft-mediated PI3K/Akt activation and demonstrate the essential role of membrane microdomain compartmentalization in enabling PI3K/Akt signaling. They further suggest that dysregulation of this compartmentalization may underlie pathological complications such as insulin resistance.     

The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway

The PTEN/MMAC1 phosphatase is a tumor suppressor gene implicated in a wide range of human cancers. Here we provide biochemical and functional evidence that PTEN/MMAC1 acts a negative regulator of the phosphoinositide 3-kinase (PI3-kinase)/Akt pathway. PTEN/MMAC1 impairs activation of endogenous Akt in cells and inhibits phosphorylation of 4E-BP1, a downstream target of the PI3-kinase/Akt pathway involved in protein translation, whereas a catalytically inactive, dominant negative PTEN/MMAC1 mutant enhances 4E-BP1 phosphorylation. In addition, PTEN/MMAC1 represses gene expression in a manner that is rescued by Akt but not PI3-kinase. Finally, higher levels of Akt activation are observed in human prostate cancer cell lines and xenografts lacking PTEN/MMAC1 expression when compared with PTEN/MMAC1-positive prostate tumors or normal prostate tissue. Because constitutive activation of either PI3-kinase or Akt is known to induce cellular transformation, an increase in the activation of this pathway caused by mutations in PTEN/MMAC1 provides a potential mechanism for its tumor suppressor function.     

Acute ethanol exposure inhibits insulin signaling in the liver

Chronic ethanol consumption may produce hepatic injury and impair the ability of the liver to regenerate principally through its action on insulin signaling. These effects are mediated by insulin receptor substrate-1 (IRS-1) via the mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/Erk) pathway and by survival signals through phosphatidylinositol-3 kinase (PI3K) and protein kinase B (Akt). Because a protein phosphatase, phosphatase tensin homolog deleted on chromosome 10 (PTEN), has been reported to block insulin signaling through PI3K, we explored acute ethanol effects on signaling in the context of PTEN function. We measured upstream components of the insulin signal transduction pathway and Akt phosphorylation as an indicator of signaling through PI3K, including the generation of survival signals via glycogen synthase kinase 3beta (GSK3beta) and Bcl-2-associated death promoter (BAD). In addition, the physical association between PTEN and PI3K regulatory (p85alpha) and catalytic (p110alpha) subunits was evaluated both in vitro and in vivo. In Huh-7 cells, there was no effect of acute ethanol exposure on tyrosyl phosphorylation of the insulin receptor, IRS-1, and the association of IRS-1 with PI3K. However, Akt phosphorylation was impaired. The association of PTEN with the PI3K p85alpha subunit was substantially increased and led to the inhibition of downstream insulin-mediated survival signals through Akt, GSK3beta, and BAD; the ethanol effect was reversed by PTEN knockdown with small interfering RNA. These results were confirmed in the liver. Conclusion: Short-term ethanol exposure rapidly attenuates insulin signaling. The major cellular mechanism involves the increased association of PTEN with the PI3K p85alpha subunit, which results in reduced phospho-Akt formation and impaired downstream survival signaling. These findings may have relevance to acute toxic effects of ethanol on the liver.     

Apoptosis regulator through modulating IAP expression (ARIA) controls the PI3K/Akt pathway in endothelial and endothelial progenitor cells

Endothelial and endothelial progenitor cells (ECs and EPCs) play a fundamental role in angiogenesis that is essential for numerous physiological and pathological processes. The phosphatase and tensin homolog (PTEN)/ phosphoinositide 3-kinase (PI3K) pathway has been implicated in angiogenesis, but the mechanism in the regulation of this pathway in ECs and EPCs is poorly understood. Here we show that ARIA (apoptosis regulator through modulating IAP expression), a transmembrane protein that we recently identified, regulates the PTEN/PI3K pathway in ECs and EPCs and controls developmental and postnatal angiogenesis in vivo. We found that ARIA is abundantly expressed in EPCs and regulates their angiogenic functions by modulating PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling. Genetic deletion of ARIA caused nonfatal bleeding during embryogenesis, in association with increased small vessel density and altered expression of various vascular growth factors including angiopoietins and VEGF receptors. Postnatal neovascularization induced by critical limb ischemia was substantially enhanced in ARIA-null mice, in conjunction with more bone marrow (BM)-derived ECs detected in ischemic muscles. Administration of PI3K or NO synthase inhibitor completely abolished the enhanced neovascularization in ARIA(-/-) mice. Mechanistically, we identified that ARIA interacts with PTEN at the intracellular domain independently of the PTEN phosphorylation in its C-terminal tail. Overexpressed ARIA increased PTEN in the membrane fraction, whereas ARIA-silencing reduced the membrane-associated PTEN, resulting in modified PI3K/Akt signaling. Taken together, our findings establish a previously undescribed mode of regulation of the PTEN/PI3K/Akt pathway by ARIA, and reveal a unique mechanism in the control of angiogenesis. These functions of ARIA might offer a unique therapeutic potential.     

MicroRNA-7 inhibits tumor growth and metastasis by targeting the phosphoinositide 3-kinase/Akt pathway in hepatocellular carcinoma

MicroRNAs (miRNAs) are known to be involved in carcinogenesis and tumor progression in hepatocellular carcinoma (HCC). Recently, microRNA-7 (miR-7) has been proven to play a substantial role in glioblastoma and breast cancer, but its functions in the context of HCC remain unknown. Here, we demonstrate that miR-7 inhibits HCC cell growth and metastasis in vitro and in vivo. We first screened and identified a novel miR-7 target, phosphoinositide 3-kinase catalytic subunit delta (PIK3CD). Overexpression of miR-7 would specifically and markedly down-regulate its expression. miR-7-overexpressing subclones showed significant cell growth inhibition by G(0) /G(1) -phase cell-cycle arrest and significant impairment of cell migration in vitro. To identify the mechanisms, we investigated the phosphoinositide 3-kinase (PI3K)/Akt pathway and found that Akt, mammalian target of rapamycin (mTOR), and p70S6K were down-regulated, whereas 4EBP1 was up-regulated in miR-7-overexpressing subclones. We also identified two novel, putative miR-7 target genes, mTOR and p70S6K, which further suggests that miR-7 may be a key regulator of the PI3K/Akt pathway. In xenograft animal experiments, we found that overexpressed miR-7 effectively repressed tumor growth (3.5-fold decrease in mean tumor volume; n = 5) and abolished extrahepatic migration from liver to lung in a nude mouse model of metastasis (n = 5). The number of visible nodules on the lung surface was reduced by 32-fold. A correlation between miR-7 and PIK3CD expression was also confirmed in clinical samples of HCC. CONCLUSION: These findings indicate that miR-7 functions as a tumor suppressor and plays a substantial role in inhibiting the tumorigenesis and reversing the metastasis of HCC through the PI3K/Akt/mTOR-signaling pathway in vitro and in vivo. By targeting PIK3CD, mTOR, and p70S6K, miR-7 efficiently regulates the PI3K/Akt pathway. Given these results, miR-7 may be a potential therapeutic or diagnostic/prognostic target for treating HCC.     

miR-21/PTEN/Akt参与白藜芦醇促EPC的体外成血管能力

目的探讨miR-21及其下游PTEN/Akt信号通路在白藜芦醇促进大鼠骨髓源性内皮祖细胞(EPC)体外成血管能力中的作用。方法采用密度梯度离心法分离大鼠四肢骨髓的单个核细胞,培养于含10%胎牛血清的EGM-2MV完全培养基中诱导分化成EPC,实验用3~5代的EPC。白藜芦醇(20μmol/L)干预12 h后,采用Matrigel胶检测EPC的体外成血管能力;实时荧光定量PCR检测miR-21及PTEN基因的表达情况;干扰miR-21表达后检测EPC的体外成血管能力;双荧光素酶报告基因检测miR-21对PTEN的靶向调控;Western blot检测PTEN蛋白表达情况以及Akt磷酸化水平。结果白藜芦醇(20μmol/L)明显促进EPC的体外成血管能力(P<0.05),抑制了miR-21(P<0.01)的表达,然而却促进了miR-21下游靶基因PTEN的基因(P<0.01)及蛋白表达(P<0.05),进而抑制PTEN下游信号分子Akt的磷酸化水平。双荧光素酶报告基因检测结果显示,miR-21可与PTEN mRNA的3′UTR靶向结合(P<0.01)。结论miR-21调控了白藜芦醇促EPC的体外成血管能力,其机制可能是通过PTEN/Akt信号通路来发挥的,该研究结果揭示了白藜芦醇调控EPC功能一种新的细胞内信号机制。     

Lifelong accumulation of bone in mice lacking Pten in osteoblasts

Bone formation is carried out by the osteoblast, a mesenchymal cell whose lifespan and activity are regulated by growth factor signaling networks. Growth factors activate phosphatidylinositol 3-kinase (PI3K), which enhances cell survival and antagonizes apoptosis through activation of Akt/PKB. This process is negatively regulated by the Pten phosphatase, which inhibits the activity of PI3K. In this study, we investigated the effects of Akt activation in bone in vivo by conditionally disrupting the Pten gene in osteoblasts by using Cre-mediated recombination. Mice deficient in Pten in osteoblasts were of normal size but demonstrated a dramatic and progressively increasing bone mineral density throughout life. In vitro osteoblasts lacking Pten differentiated more rapidly than controls and exhibited greatly reduced apoptosis in association with markedly increased levels of phosphorylated Akt and activation of signaling pathways downstream of activated Akt. These findings support a critical role for this tumor-suppressor gene in regulating osteoblast lifespan and likely explain the skeletal abnormalities in patients carrying germ-line mutations of PTEN.     

MicroRNA-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 microRNA-21 (miR-21), phosphatase gene (PTEN), and its downstream proteins, protein kinase B (AKT) and phosphatidylinositol 3-kinase (PI3K), in colorectal cancer (CRC) cells.METHODS: Quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the expression levels of miR-21 and PTEN in HCT116, HT29, Colo32 and SW480 CRC cell lines. Also, the expression levels of PTEN mRNA and its downstream proteins AKT and PI3K in HCT116 cells after downregulating miR-21 were investigated.RESULTS: Comparing the miR-21 expression in CRC cells, the expression levels of miR-21 were highest in HCT116 cells, and the expression levels of miR-21 were lowest in SW480 cells. In comparing miR-21 and PTEN expression in CRC cells, we found that the protein expression levels of miR-21 and PTEN were inversely correlated (P < 0.05); when miR-21 expression was reduced, mRNA expression levels of PTEN did not significantly change (P > 0.05), but the expression levels of its protein significantly increased (P < 0.05). In comparing the levels of PTEN protein and downstream AKT and PI3K in HCT116 cells after downregulation of miR-21 expression, the levels of AKT and PI3K protein expression significantly decreased (P < 0.05).CONCLUSION: PTEN is one of the direct target genes of miR-21. Thus, phosphatase gene and its downstream AKT and PI3K expression levels can be regulated by regulating the expression levels of miR-21, which in turn regulates the development of CRC.     

An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/- mice

PTEN phosphatase acts as a tumor suppressor by negatively regulating the phosphoinositide 3-kinase (PI3K) signaling pathway. It is unclear which downstream components of this pathway are necessary for oncogenic transformation. In this report we show that transformed cells of PTEN^+/− mice have elevated levels of phosphorylated Akt and activated p70/S6 kinase associated with an increase in proliferation. Pharmacological inactivation of mTOR/RAFT/FRAP reduced neoplastic proliferation, tumor size, and p70/S6 kinase activity, but did not affect the status of Akt. These data suggest that p70/S6K and possibly other targets of mTOR contribute significantly to tumor development and that inhibition of these proteins may be therapeutic for cancer patients with deranged PI3K signaling.     

HES1 opposes a PTEN-dependent check on survival, differentiation, and proliferation of TCRβ-selected mouse thymocytes

The developmental progression of immature thymocytes requires cooperative input from several pathways, with Notch signals playing an indispensable role at the T-cell receptor (TCR)-β selection checkpoint. Notch signals affect the activation of the PI3K/Akt pathway, which is required for pTα/TCRβ (pre-TCR)-induced survival, differentiation, and proliferation of developing αβ-lineage thymocytes. However, the molecular players responsible for the interaction between the Notch and PI3K pathways at this critical developmental stage are unknown. Here, we show that Notch induction of Hes1 is necessary to repress the PI3K/Akt pathway inhibitor, PTEN (phosphatase and tensin homolog), which in turn facilitates pre-TCR-induced differentiation. In support of this mechanism, deletion or down-regulation of Pten overcomes the Notch signaling requirement for survival and differentiation during β-selection. In addition, c-Myc is a critical target of Notch at this stage, as c-Myc expression overcomes the Notch signaling requirement for proliferation during β-selection. Collectively, our results point to HES1, via repression of PTEN, and c-Myc as critical mediators of Notch function at the β-selection checkpoint.     

The role of phosphoinositide-3 kinase and PTEN in cardiovascular physiology and disease

Phosphoinositide-3 kinases (PI3Ks) are a family of evolutionary conserved lipid kinases that mediate many cellular responses in both physiologic and pathophysiologic states. Class I PI3K can be activated by either receptor tyrosine kinase (RTK)/cytokine receptor activation (class I(A)) or G-protein-coupled receptors (GPCR) (class I(B)). Once activated PI3Ks generate phosphatidylinositols (PtdIns) (3,4,5)P(3) leading to the recruitment and activation of Akt/protein kinase B (PKB), PDK1 and monomeric G-proteins (e.g. Rac-GTPases), which then activate a range of downstream targets including glycogen synthase kinase-3beta (GSK-3beta), mammalian target of rapamycin (mTOR), p70S6 kinase, endothelial nitric oxide synthase (eNOS) and several anti-apoptotic effectors. Class I(A) (PI3Kalpha, beta and delta) and class I(B) (PI3Kgamma) PI3Ks mediate distinct phenotypes in the heart and under negative control by the 3'-lipid phosphatase, phosphatase and tensin homolog on chromosome ten (PTEN) which dephosphorylate PtdIns(3,4,5)P(3) into PtdIns(4,5)P(2). PI3Kalpha, gamma and PTEN are expressed in cardiomyocytes, fibroblasts, endothelial cells and vascular smooth muscle cells where they modulate cell survival/apoptosis, hypertrophy, contractility, metabolism and mechanotransduction. Several transgenic and knockout models support a fundamental role of PI3K/PTEN signaling in the regulation of myocardial contractility and hypertrophy. Consequently the PI3K/PTEN signaling pathways are involved in a wide variety of diseases including cardiac hypertrophy, heart failure, preconditioning and hypertension. In this review, we discuss the biochemistry and molecular biology of PI3K (class I isoforms) and PTEN and their critical role in cardiovascular physiology and diseases.     

Molecular aspects of gefitinib antiproliferative and pro-apoptotic effects in PTEN-positive and PTEN-negative prostate cancer cell lines

To date, no effective therapeutic treatment allows abrogation of the progression of prostate cancer (PCa) to more invasive forms. One of the major targets for the therapy in PCa can be epidermal growth factor receptor (EGFR), which signals via the phosphoinositide 3'-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways, among others. Despite multiple reports of overexpression in PCa, the reliance on activated EGFR and its downstream signalling to the PI3K and/or MAPK/extracellular signal-regulated kinase (ERK) pathways has not been fully elucidated. We reported that the EGFR-selective tyrosine kinase inhibitor gefitinib (ZD1839; Iressa) is able to induce growth inhibition, G(1) arrest and apoptosis in PCa cells and that its effectiveness is associated primarily with phosphatase and tensin homologue deleted from chromosome 10 (PTEN) expression (and thus Akt activity). In fact PTEN-negative PCa cells are slowly sensitive to gefitinib treatment, because this molecule is unable to downregulate PI3K/Akt activity. PI3K inhibition, by LY294002 or after PTEN transfection, restores EGFR-stimulated Akt signalling and sensitizes the cells to pro-apoptotic action of gefitinib. The MAPK pathway seems to be involved primarily on cell-growth modulation because dual blockade of EGFR and ERK1/2 phosphorylation potentiates growth inhibition (both not cell apoptosis) in PTEN-positive PCa cells and reduced EGF-mediated growth in PTEN-negative cells. Thus the effectiveness of gefitinib requires growth factor receptor-stimulated PI3K/Akt and MAPK signalling to be intact and functional. The loss of the PTEN activity leads to uncoupling of this signalling pathway, determining a partial gefitinib resistance. Moreover, gefitinib sensitivity may be maintained in these cells through its inhibitory potential in MAPK/ERK pathway activity, modulating proliferative EGFR-triggered events. Therefore, our data suggest that the inhibition of EGFR signalling can result in a significant growth reduction and in increased apoptosis in EGFR-overexpressing PCa cells with different modalities, which are regulated by PTEN status, and this may have relevance in the clinical setting of PCa.     

miR-29a levels are elevated in the db/db mice liver and its overexpression leads to attenuation of insulin action on PEPCK gene expression in HepG2 cells

MicroRNAs comprise a class of small (～22 nucleotide) non-coding RNA species and they bind to their complementary sequence on the 3'UTR of target genes and cause translational repression. In the present study, we report that miR-29a levels are significantly elevated in the diabetic db/db mice liver. Further, we report the effects of such elevation on insulin action in HepG2 cells. Overexpression of miR-29a narrowed down insulin mediated Akt phosphorylation without altering the total Akt levels presumably due to another upstream mediator being directly targeted by miR-29a. This hunt led us to the discovery that the p85α subunit of PI3K (phosphoionositide-3-kinase), the upstream molecule in the insulin signaling cascade harbors the miR-29a binding site on its 3'UTR and a marked inhibition of PI3Kp85α was observed by this microRNA. This was consequently accompanied by attenuation of insulin inhibition of PEPCK gene expression. All these events could be significantly prevented in the presence of the miR-29a inhibitor. Our results, for the first time, show the effect of miR-29a in counteracting insulin action on PEPCK gene expression by primarily targeting PI3K and abrogating downstream insulin signaling in HepG2 cells.