Overexpression Of Insulin Receptor Substrate-1 Research Articles

Endothelial Cell Insulin Signaling Regulates CXCR4 (C-X-C Motif Chemokine Receptor 4) and Limits Leukocyte Adhesion to Endothelium.

An activated, proinflammatory endothelium is a key feature in the development of complications of obesity and type 2 diabetes and can be caused by insulin resistance in endothelial cells. We analyzed primary human endothelial cells by RNA sequencing to discover novel insulin-regulated genes and used endothelial cell culture and animal models to characterize signaling through CXCR4 (C-X-C motif chemokine receptor 4) in endothelial cells. CXCR4 was one of the genes most potently regulated by insulin, and this was mediated by PI3K (phosphatidylinositol 3-kinase), likely through FoxO1, which bound to the CXCR4 promoter. CXCR4 mRNA in CD31+ cells was 77% higher in mice with diet-induced obesity compared with lean controls and 37% higher in db/db mice than db/+ controls, consistent with upregulation of CXCR4 in endothelial cell insulin resistance. SDF-1 (stromal cell-derived factor-1)-the ligand for CXCR4-increased leukocyte adhesion to cultured endothelial cells. This effect was lost after deletion of CXCR4 by gene editing while 80% of the increase was prevented by treatment of endothelial cells with insulin. In vivo microscopy of mesenteric venules showed an increase in leukocyte rolling after intravenous injection of SDF-1, but most of this response was prevented in transgenic mice with endothelial overexpression of IRS-1 (insulin receptor substrate-1). Endothelial cell insulin signaling limits leukocyte/endothelial cell interaction induced by SDF-1 through downregulation of CXCR4. Improving insulin signaling in endothelial cells or inhibiting endothelial CXCR4 may reduce immune cell recruitment to the vascular wall or tissue parenchyma in insulin resistance and thereby help prevent several vascular complications.

Insulin receptor substrate 1(IRS1) is related with lymph node metastases and prognosis in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the leading causes of cancer-related mortality globally with a high risk of lymph node metastasis (LNM). In this study, weighted gene co-expression network analysis (WGCNA) showed the identification of 10 modules among which the significant module (turquoise), including 1352 genes, was correlated with LNM. A group 52 overlapping differentially expressed genes (DEGs) was identified based on the comparison of turquoise module with GSE23400 and GSE20347 datasets. Using Ctyohubba plugin, we identified 7 hub genes (ACTG2, SORBS1, MYH11, CXCL12, CNN1, IRS1 and CXCL8). IRS1 displayed significant correlation with metastasis. The decreased expression of IRS1 was also a predictor of poor OS of ESCC patients whereas the hub genes namely ACTG2, MYH11, CXCL8, CXCL12, IRS1 and CNN1 were associated with RFS of ESCC patients. These findings suggest that the altered expression of these hub genes are associated with prognosis and thus can be used as potential biomarkers for ESCC. Moreover, immunohistochemical staining and cell functional experiments displayed that the overexpression of IRS1 was negatively associated with metastasis in ESCC. In general, our research revealed several novel genes in ESCC especially the association of IRS1 with LNM in ESCC, which could provide novel insights into the initiation and progression of ESCC.

Long noncoding RNA ADIRF antisense RNA 1 upregulates insulin receptor substrate 1 to decrease the aggressiveness of osteosarcoma by sponging microRNA-761

ABSTRACT An increasing number of studies have supported the critical regulatory actions of long noncoding RNAs (lncRNAs) in osteosarcoma (OS). However, the detailed roles of adipogenesis regulatory factor-antisense RNA 1 (ADIRF-AS1) in OS have not been comprehensively described. Hence, we first detected ADIRF-AS1 expression in OS and evaluated its clinical significance. Functional experiments were then performed to determine the modulatory role of ADIRF-AS1 in OS progression. ADIRF-AS1 was found to be overexpressed in OS, and the overall survival of patients with OS who had high ADIRF-AS1 levels was shorter than that of those with low levels. ADIRF-AS1 knockdown led to restricted proliferation, migration, and invasiveness of OS cells and increased apoptosis. Additionally, ADIRF-AS1 downregulation impeded tumor growth in vivo. Mechanistically, ADIRF-AS1 acted as a competitive endogenous RNA for microRNA-761 (miR-761) that siphoned miR-761 away from its target, namely insulin receptor substrate 1 (IRS1), leading to IRS1 overexpression. Rescue experiments showed that low levels of miR-761 or restoration of IRS1 could neutralize the effects of ADIRF-AS1 ablation in OS cells. In summary, ADIRF-AS1 exacerbates the oncogenicity of the OS cells by targeting the miR-761/IRS1 axis. Our findings may aid in the advancement of lncRNA-directed therapeutics for OS.

Insulin Receptor Substrate 1 Is Involved in the Phycocyanin-Mediated Antineoplastic Function of Non-Small Cell Lung Cancer Cells.

Phycocyanin, derived from marine algae, is known to have noteworthy antineoplastic properties. However, the underlying mechanism involved in phycocyanin-mediated anti-growth function on non-small cell lung cancer (NSCLC) cells is still ambiguous. Here, we investigated the mechanism of action of phycocyanin on H1299, A549, and LTEP-a2 cells. According to the results obtained, insulin receptor substrate 1 (IRS-1) expression was reduced by phycocyanin. Cell phenotype tests showed that siRNA knockdown of IRS-1 expression significantly inhibited the growth, migration, colony formation, but promoted the apoptosis of NSCLC cells. Meanwhile, phycocyanin and IRS-1 siRNA treatment both reduced the PI3K-AKT activities in NSCLC cells. Moreover, overexpression of IRS-1 accelerated the proliferation, colony formation, and migration rate of H1299, A549, and LTEP-a2 cells, which was contradicting to the knockdown results. Overall, this study uncovered a regulatory mechanism by which phycocyanin inhibited the growth of NSCLC cells via IRS-1/AKT pathway, laying the foundation for the potential target treatment of NSCLC.

Endothelial-specific insulin receptor substrate-1 overexpression worsens neonatal hypoxic-ischemic brain injury via mTOR-mediated tight junction disassembly

Hypoxic-ischemic (HI) encephalopathy is the major cause of mortality and disability in newborns. The neurovascular unit is a major target of acute and chronic brain injury, and therapies that protect simultaneously both neurons and vascular endothelial cells from neonatal HI injury are in demand. Insulin receptors and its key downstream molecule-insulin receptor substrate −1 (IRS-1) are potential neuroprotective targets and expressed both in neuron and endothelial cells. To investigate whether IRS-1 can act similarly in neurons and vascular endothelial cells in protecting neurovascular units and brain form HI injury, we found that neuron-specific IRS-1 transgenic rats showed reduced neurovascular injury and infarct volumes, whereas endothelial-specific IRS-1 transgenic rats showed increased blood-brain barrier (BBB) disruption and exaggerated neurovascular injury after neonatal HI brain injury. Endothelial-specific IRS-1 overexpression increased vascular permeability and disassembled the tight junction protein (zonula occludens-1) complex. Inhibition of mammalian target of rapamycin (mTOR) by rapamycin preserved tight junction proteins and attenuated BBB leakage and neuronal apoptosis after HI in the endothelial-specific IRS-1 transgenic pups. Together, our findings suggested that neuronal and endothelial IRS-1 had opposite effects on the neurovascular integrity and damage after neonatal HI brain injury and that endothelial IRS-1 worsens neurovascular integrity after HI via mTOR-mediated tight junction protein disassembly.

Palmitic Acid Regulates miRNA-3148 via Insulin Receptor Substrate-1 and is Involved in Insulin Resistance

Previous studies reported that saturated fatty acid palmitic acid (PA) is closely related to insulin resistance. miR-3148 regulates insulin receptor substrate-1 (IRS1) predicted by MiRDB analysis. However, whether PA regulates IRS1 via miR-3148 remains to be elucidated. Therefore, in this work, we assessed whether PA regulates miRNA-3148 via IRS1 in insulin resistance. We cultured HepG2 cells in vitro and classified them into control group (NC group), miR-3148 Mimics group, and miR-3148 Mimics+ pFBD-IRS1 group. We used qRT-PCR to detect miR-3148 and IRS1 mRNA; used Dual-Luciferase Reporter Assays to detect miR-3148 with 3′-UTR region of IRS1 mRNA; and utilized Western blot (WB) to detect IRS1, p-AKT, AKT and Tubulin. Our results showed that PA could increase miR-3148 and decrease IRS1 which is a target protein of miR-3148, as shown by Dual-Luciferase Reporter assays. miR-3148 significantly inhibited the impact of insulin on p-AKT level (P < 0.01) and over-expression of IRS1 by pFBD-IRS1 can partially alleviate the inhibitory effect of miR-3148 mimics on p-AKT. In HepG2 cells, PA regulates miR-3148. Via targeting IRS1 mRNA, miR-3148 impairs insulin signaling pathway, leading to insulin resistance. Over-expression of IRS1 by pFBD-IRS1 alleviates miR-3148-induced insulin resistance.

Adenosine monophosphate activated protein kinase (AMPK) is essential for the memory improving effect of adiponectin

Adiponectin (APN) plays a major role in the regulation of insulin sensitivity and glucose homeostasis. Insulin and APN have a positive effect on memory. In this study, we examined whether the inhibition of AMPK could block the memory improving effect of APN or affect the IRS1 expression.Animal model of AD was developed by intracerebroventricular (icv) injection of 3 mg/kg streptozotocin (STZ), in 12 weeks old Wistar rats, on days 1 and 3 after cannulation. Dorsomorphin (DM) and APN (600 nM) were injected 30 and 20 min before the acquisition phase, respectively. DM was applied in 3 different doses (0.2, 2 and 20 μM). All behavioral tests were performed on days 15 and 16; the Preference Index (PI) was calculated for novel object recognition (NOR) test, while the step through latency (STL) and total time in dark compartment (TDC) were recorded and analyzed for the passive avoidance task. Relative expression of insulin receptor substrate-1 (IRS-1) protein in the hippocampus was measured by western blotting.In early retrieval test, STZ + APN treatment increased STL (P < 0.0001) and decreased TDC (P < 0.05) in comparison to STZ group, while STZ + APN + DM (2μM) caused a decrease in STL (P < 0.05) and increase in TDC (0.2μM and 2μM DM; P < 0.05). Icv injection of DM (0.2μM and 2μM) before APN decreased the PI significantly (P < 0.05) in comparison to STZ + APN group. APN treatment raised the IRS-1 expression and DM reversed this increment, significantly (P < 0.0001).It is concluded that the memory improving effect of APN is mediated, at least in part, by the AMPK pathway. APN is also able to boost insulin signaling by overexpression of IRS-1 in the hippocampus.

Linagliptin affects IRS1/Akt signaling and prevents high glucose-induced apoptosis in podocytes

Diabetes-induced podocyte apoptosis is considered to play a critical role in the pathogenesis of diabetic kidney disease (DKD). We proposed that hyperglycaemia can induce podocyte apoptosis by inhibiting the action of podocyte survival factors, thus inactivating the cellular effects of insulin signalling. In this study, we aimed to determine the effects of linagliptin on high glucose-induced podocyte apoptosis. Linagliptin reduced the increase in DNA fragmentation as well as the increase in TUNEL-positive cells in podocytes induced by high-glucose condition. Furthermore, linagliptin improved insulin-induced phosphorylation of insulin receptor substrate 1 (IRS1) and Akt, which was inhibited in high-glucose conditions. Adenoviral vector-mediated IRS1 overexpression in podocytes partially normalised DNA fragmentation in high-glucose conditions, while downregulation of IRS1 expression using small interfering RNA increased DNA fragmentation even in low-glucose conditions. Because reactive oxygen species inhibit glomerular insulin signalling in diabetes and Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is one of the most important intrinsic antioxidative systems, we evaluated whether linagliptin increased Nrf2 in podocytes. High-glucose condition and linagliptin addition increased Nrf2 levels compared to low-glucose conditions. In summary, linagliptin offers protection against DKD by enhancing IRS1/Akt insulin signalling in podocytes and partially via the Keap1/Nrf2 pathway. Our findings suggest that linagliptin may induce protective effects in patients with DKD, and increasing IRS1 levels could be a potential therapeutic target in DKD.

Insulin receptor substrate 1 overexpression promotes survival of glioblastoma cells through AKT1 activation.

Glioblastoma multiforme (GBM) is the most common and malignant type of central nervous system tumours in adults. Strict regulation of glucose homeostasis has a significant role in GBM pathogenesis. Insulin receptor substrate 1 (IRS1) protein is the most important adaptor molecule involved in the regulation of glucose metabolism. It interacts with many cancer-related receptors and its overexpression is strongly associated with cell proliferation and survival. Our study was aimed to understand the role of IRS1 proteins in GBM cell viability. U-87 MG cells were transfected with pcDNA3.1-flagtagged-human IRS1 expression vector. Insulin induced phosphorylation levels of IRS1, AKT1 and ERK1/2 and Grb2 expression were examined to determine the effects of ectopic IRS1 overexpression on insulin signalling and the viability levels of U-87 MG cells were determined by MTT analysis. Overexpression of IRS1 in U-87 MG cells led to an increase in cell viability. Its overexpression also increased Grb2 expression and phosphorylation of AKT1 through elevation of IRS1 tyrosine phosphorylation in IRS1-transfected U-87 MG cells compared to control and mock transfected groups. Our study showed that increased IRS1 expression and activation may promote the cell viability via AKT1 activation. IRS1 signalling may be considered as a therapeutic target for further studies.

273-LB: Leukocyte-Endothelial Interaction Is Reduced by Insulin-Induced Downregulation of Endothelial CXCR4

An activated, pro-inflammatory endothelium is a key feature of obesity and type 2 diabetes and may contribute to adipose tissue inflammation, atherosclerosis and tumor formation. We have previously published a striking 4-fold increase in leukocyte adhesion to endothelium in mice with endothelial cell knockout of the insulin receptor. Here, we searched for insulin-regulated genes involved in leukocyte-endothelial cell interaction by performing RNA sequencing in primary human endothelial cells. Among genes with the largest change in expression after insulin treatment was C-X-C motif chemokine receptor 4 (CXCR4), which was downregulated by 54%. This reduction was completely prevented by pretreatment with wortmannin but not by U0126, an inhibitor of MAPK signaling. In CD31+ cells isolated by fluorescence-activated cell sorting of enzymatically dissociated tissue, CXCR4 mRNA in was 77% higher in mice with diet-induced obesity compared to lean controls and 37% higher in db/db mice compared to db/+ controls, consistent with upregulation of CXCR4 in endothelial cell insulin resistance. Stromal-derived factor-1 (SDF-1), the ligand for CXCR4, increased leukocyte adhesion to cultured endothelial cells. Insulin treatment of endothelial cells prevented 80% of this increase which in turn was abrogated by plerixafor, a clinically approved CXCR4 antagonist, or deletion of CXCR4 by gene editing. To study gain of endothelial cell insulin signaling in vivo, we examined transgenic mice with endothelial overexpression of insulin receptor substrate-1 (IRS-1). In vivo microscopy of mesenteric venules showed an increase in leukocyte rolling and adhesion after intravenous injection of SDF-1 in wildtype mice which was reduced by 83% in transgenic mice. We conclude that endothelial cell insulin signaling limits leukocyte-endothelial cell interaction through downregulation of CXCR4 and that targeting endothelial CXCR4 may protect against inflammation in insulin resistant states. Disclosure B. Kunkemoeller: None. T. Rathjen: Employee; Self; Bayer AG. J.C. Patti: None. Q. Li: None. K. Park: None. G.L. King: Research Support; Self; Sanofi. C. Rask-Madsen: None.

MicroRNA‑466 inhibits cell proliferation and invasion in osteosarcoma by directly targeting insulin receptor substrate 1.

Accumulating evidence has demonstrated that microRNAs (miRNAs) are frequently dysregulated in osteosarcoma (OS), and the aberrant expression of miRNAs is associated with OS initiation and progression. Previous studies demonstrated that miRNA‑466 (miR‑466) is dysregulated, and serves important roles in various types of human cancer. However, the role of miR‑466 in the formation and progression of OS remains unclear. In the present study, the expression level of miR‑466 was identified to be markedly downregulated in OS tissues and cell lines. Additionally, miR‑466 overexpression inhibited the proliferative and invasive abilities of OS cells. In the present study, bioinformatics analyses and luciferase assays were employed to show that miR‑466 was able to directly target the 3'‑untranslated region of insulin receptor substrate 1 (IRS1) gene, negatively regulating the mRNA and the protein expression levels of IRS1 in OS cells. Furthermore, IRS1 was upregulated in OS tissues, and the increased expression level of IRS1 exhibited an inverse correlation with the expression level of miR‑466. Furthermore, IRS1 overexpression was able to partially reverse the suppressive effects of miR‑466 overexpression in OS cells. To the best of the authors' knowledge, the present study is the first to suggest that miR‑466 is downregulated in OS and inhibits the progression of OS by directly targeting IRS1. The present results suggested that miR‑466 may represent a novel potential therapeutic target for the treatment of patients with OS.

Hyperglycemia induces vascular smooth muscle cell dedifferentiation by suppressing insulin receptor substrate-1–mediated p53/KLF4 complex stabilization

Hyperglycemia and insulin resistance accelerate atherosclerosis by an unclear mechanism. The two factors down-regulate insulin receptor substrate-1 (IRS-1), an intermediary of the insulin/IGF-I signaling system. We previously reported that IRS-1 down-regulation leads to vascular smooth muscle cell (VSMC) dedifferentiation and that IRS-1 deletion from VSMCs in normoglycemic mice replicates this response. However, we did not determine IRS-1's role in mediating differentiation. Here, we sought to define the mechanism by which IRS-1 maintains VSMC differentiation. High glucose or IRS-1 knockdown decreased p53 levels by enhancing MDM2 proto-oncogene (MDM2)-mediated ubiquitination, resulting in decreased binding of p53 to Krüppel-like factor 4 (KLF4). Exposure to nutlin-3, which dissociates MDM2/p53, decreased p53 ubiquitination and enhanced the p53/KLF4 association and differentiation marker protein expression. IRS-1 overexpression in high glucose inhibited the MDM2/p53 association, leading to increased p53 and p53/KLF4 levels, thereby increasing differentiation. Nutlin-3 treatment of diabetic or Irs1-/- mice enhanced p53/KLF4 and the expression of p21, smooth muscle protein 22 (SM22), and myocardin and inhibited aortic VSMC proliferation. Injecting normoglycemic mice with a peptide disrupting the IRS-1/p53 association reduced p53, p53/KLF4, and differentiation. Analyzing atherosclerotic lesions in hypercholesterolemic, diabetic pigs, we found that p53, IRS-1, SM22, myocardin, and KLF4/p53 levels are significantly decreased compared with their expression in nondiabetic pigs. We conclude that IRS-1 is critical for maintaining VSMC differentiation. Hyperglycemia- or insulin resistance-induced IRS-1 down-regulation decreases the p53/KLF4 association and enhances dedifferentiation and proliferation. Our results suggest that enhancing IRS-1-dependent p53 stabilization could attenuate the progression of atherosclerotic lesions in hyperglycemia and insulin-resistance states.

IRS-1 targets TAZ to inhibit adipogenesis of rat bone marrow mesenchymal stem cells through PI3K-Akt and MEK-ERK pathways

Gene modification of mesenchymal stem cells (MSCs) offers a promising approach for clinical stem cell therapy. Transcriptional co-activator with PDZ-binding motif (TAZ) plays a vital role in MSCs’ differentiation. We aim to explore the interaction of insulin receptor substrate-1 (IRS-1) with TAZ to regulate MSCs’ adipogenesis in this study. Initially, IRS-1 and TAZ followed similar decreasing expression pattern at the early stage of adipogenesis. And, overexpression of IRS-1 decreased the CCAAT/enhancer binding protein β (C/EBPβ) and peroxi-some proliferator-activated receptor gamma (PPARγ) expression with TAZ upregulation. Accordingly, knockdown of IRS-1 induced the upexpression of C/EBPβ and PPARγ with TAZ downregulation. Indeed, IRS-1 targeted TAZ to downregulate the C/EBPβ and PPARγ expression, while knockdown of TAZ attenuated the IRS-1 inhibited adipogenesis. Furthermore, both LY294002 (the PI3K-Akt inhibitor) and U0126 (the MEK-ERK inhibitor) blocked the regulation of IRS-1 on TAZ during adipogenesis. Additionally, IRS-1 and TAZ influenced the cell proliferation in the above process. Taken together, this study suggests for the first time that IRS-1 is a key regulator of the MSCs’ adipogenesis and may serve as a potential therapeutic target for differential alterations in bone marrow.

TAZ enhances mammary cell proliferation in 3D culture through transcriptional regulation of IRS1

WW domain-containing transcriptional regulator 1 (TAZ) is a transcriptional co-activator and effector of the Hippo signaling pathway. In certain breast cancer subtypes, Hippo signaling is dysregulated leading to activation of TAZ and altered expression of TAZ transcriptional targets. Over the past decade, we and others have found that TAZ transcriptionally regulates genes that affect multiple aspects of breast cancer cell behaviour. However, while cancer cell-intrinsic oncogenic functions of TAZ have emerged, less is known about whether TAZ might also contribute to tumourigenesis by sensitizing tumour cells to factors present in the tumour microenvironment or in systemic circulation. Here, we show that TAZ directly regulates the expression of insulin receptor substrate 1 (IRS1) in breast cancer cells. TAZ or IRS1 overexpression induces a similar proliferative transformation phenotype in MCF10A mammary epithelial cells. TAZ enhances IRS1 mRNA, protein levels and downstream signaling in MCF10A. Mechanistically, TAZ interacts with the IRS1 promoter through the TEAD family of transcription factors and enhances its activity. Critically, TAZ-induced IRS1 upregulation contributes to the proliferation of TAZ-overexpressing MCF10A in 3-dimensional (3D) Matrigel culture. Therefore, we offer compelling evidence that TAZ regulates signaling through the insulin pathway in breast cancer cells. These findings highlight an additional mechanism by which TAZ may promote breast cancer tumourigenesis and progression by modulating cancer cell responses to exogenously produced factors.

Elevated expression of IRS-1 associates with phosphorylated Akt expression and predicts poor prognosis of breast invasive ductal carcinoma

Overexpression of insulin receptor substrate 1 (IRS-1) has been reported to promote cell growth, atypical hyperplasia, and carcinogenesis, and phosphorylated Akt (p-Akt) is certified to be involved in many types of cancers such as breast invasive ductal carcinoma (BIDC). However, the relationship between IRS-1 and Akt, as well as the role of expression of IRS-1 in BIDC, has never been reported. The purpose of this research is to investigate the association between expression of IRS-1 and p-Akt proteins and clinicopathological features of BIDC by immunohistochemistry, as well as the survival status. The results showed that the percentage of either elevated expression of IRS-1 or positive p-Akt expression in BIDC was significantly higher than that in control breast tissue from noncancer patients (P < .001 and P = .001, respectively). Overexpression of IRS-1 was evidently associated with positive expression of p-Akt (r = 0.337, P < .001). Also, positive percentage of p-Akt expression was statistically different among different molecular subtypes of BIDC (highest in luminal B BIDC, P = .009). Furthermore, significantly worse overall survival was found in BIDC patients with high expression of IRS-1 and p-Akt than in patients with low expression (P = .006 and P = .004, respectively). The multivariate Cox proportional hazard regression analysis showed that high expression of IRS-1 and positive expression of p-Akt protein were independent poor prognostic factors for patients with BIDC (P = .022 and P = .046, respectively). In conclusion, we report for the first time that overexpression of IRS-1 protein is associated with expression of p-Akt, and overexpression of IRS-1 and positive expression of p-Akt might be independent biomarkers for poor prognosis in BIDC.

MicroRNA-145 Negatively Regulates Cell Proliferation Through Targeting IRS1 in Isolated Ovarian Granulosa Cells From Patients With Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a complex, heterogeneous endocrine and metabolic disorder affecting 5% to 10% of reproductive-age women. A high rate of granulosa cell (GC) proliferation contributes to the abnormal folliculogenesis in patients with PCOS. Evidence has proved that dysregulation of microRNAs is involved in the pathogenesis of PCOS. In this study, we investigated the effect of miR-145 on cell proliferation and the underlying mechanism of miR-145 in isolated human GCs from the aspirated follicular fluid in women with PCOS. Our findings showed that miR-145 is downregulated in human GCs from PCOS. The miR-145 mimics suppress cell proliferation and promoted cell apoptosis in human GCs from PCOS. However, miR-145 inhibitor promotes cell proliferation and inhibited cell apoptosis. Moreover, using a dual-luciferase reporter assay, we confirmed that the insulin receptor substrate 1 (IRS1) gene is a direct target of miR-145. The miR-145 mimics inhibited messenger RNA and protein IRS1 expression levels, and silencing of IRS1 by small interfering RNA inhibits human GC proliferation, but IRS1 overexpression abrogates the suppressive effect of miR-145 mimics. Furthermore, miR-145 mimics can inhibit the activation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase (ERK). The IRS1 overexpression abrogates the suppressive effect of miR-145 mimics on MAPK/ERK signaling pathways. Together, miR-145 mimics suppress cell proliferation by targeting and inhibiting IRS1 expression to inhibit MAPK/ERK signaling pathways. Our study further found that high concentrations of insulin decreases the miR-145 expression, upregulates IRS1, and promotes cell proliferation. These observations showed that miR-145 is a novel and promising molecular target for improving the dysfunction of GCs in PCOS.

Insulin decreases atherosclerosis by inducing endothelin receptor B expression.

Endothelial cell (EC) insulin resistance and dysfunction, caused by diabetes, accelerates atherosclerosis. It is unknown whether specifically enhancing EC-targeted insulin action can decrease atherosclerosis in diabetes. Accordingly, overexpressing insulin receptor substrate-1 (IRS1) in the endothelia of Apoe-/- mice (Irs1/Apoe-/-) increased insulin signaling and function in the aorta. Atherosclerosis was significantly reduced in Irs1/ApoE-/- mice on diet-induced hyperinsulinemia and hyperglycemia. The mechanism of insulin's enhanced antiatherogenic actions in EC was related to remarkable induction of NO action, which increases endothelin receptor B (EDNRB) expression and intracellular [Ca2+]. Using the mice with knockin mutation of eNOS, which had Ser1176 mutated to alanine (AKI), deleting the only known mechanism for insulin to activate eNOS/NO pathway, we observed that IRS1 overexpression in the endothelia of Aki/ApoE-/- mice significantly decreased atherosclerosis. Interestingly, endothelial EDNRB expression was selectively reduced in intima of arteries from diabetic patients and rodents. However, endothelial EDNRB expression was upregulated by insulin via P13K/Akt pathway. Finally EDNRB deletion in EC of Ldlr-/- and Irs1/Ldlr-/- mice decreased NO production and accelerated atherosclerosis, compared with Ldlr-/- mice. Accelerated atherosclerosis in diabetes may be reduced by improving insulin signaling selectively via IRS1/Akt in the EC by inducing EDNRB expression and NO production.

MicroRNA-126 induces autophagy by altering cell metabolism in malignant mesothelioma.

Autophagy favors both cell survival and cancer suppression, and increasing evidence reveals that microRNAs (MIRs) regulate autophagy. Previously we reported that MIR126 is downregulated in malignant mesothelioma (MM). Therefore, we investigated the role of MIR126 in the regulation of cell metabolism and autophagy in MM models. We report that MIR126 induces autophagic flux in MM cells by downregulating insulin receptor substrate-1 (IRS1) and disrupting the IRS1 signaling pathway. This was specific to MM cells, and was not observed in non-malignant cells of mesothelial origin or in MM cells expressing MIR126-insensitive IRS1 transcript. The MIR126 effect on autophagy in MM cells was recapitulated by IRS1 silencing, and antagonized by IRS1 overexpression or antisense MIR126 treatment. The MIR126-induced loss of IRS1 suppressed glucose uptake, leading to energy deprivation and AMPK-dependent phosphorylation of ULK1. In addition, MIR126 stimulated lipid droplet accumulation in a hypoxia-inducible factor-1α (HIF1α)-dependent manner. MIR126 also reduced pyruvate dehydrogenase kinase (PDK) and acetyl-CoA-citrate lyase (ACL) expression, leading to the accumulation of cytosolic citrate and paradoxical inhibition of pyruvate dehydrogenase (PDH) activity. Simultaneous pharmacological and genetic intervention with PDK and ACL activity phenocopied the effects of MIR126. This suggests that in MM MIR126 initiates a metabolic program leading to high autophagic flux and HIF1α stabilization, incompatible with tumor progression of MM. Consistently, MIR126-expressing MM cells injected into immunocompromised mice failed to progress beyond the initial stage of tumor formation, showing that increased autophagy has a protective role in MM.

MicroRNA-126 inhibits the migration and invasion of endometrial cancer cells by targeting insulin receptor substrate 1

MicroRNAs (miRs) have been demonstrated to serve important roles in the development and progression of human cancer, primarily through the direct targeting of oncogenes or tumor suppressors. It has been previously suggested that miR-126 may be associated with endometrial cancer (EC). However, the exact role of miR-126 in the migration and invasion of EC cells has not yet been studied. The present study demonstrated that the expression of miR-126 was significantly decreased in EC tissues when compared with matched normal adjacent tissues. The current study reverse transcription-quantitative polymerase chain reaction was performed in order to examine the expression level of miR-126. Wound healing and transwell assays were used to examine cell migration and invasion. A luciferase reporter assay was used to determine the targeting relationship and western blotting assay was performed to detect the protein expression. Furthermore, the overexpression of miR-126 significantly inhibited EC SKOV3 cell migration and invasion. Molecular mechanism investigation established that insulin receptor substrate 1 (IRS1) functioned as a direct miR-126 target, and its expression was negatively regulated by miR-126 at a post-transcriptional level in the SKOV3 cells. Additionally, the overexpression of IRS1 reversed the inhibitory effect of miR-126 overexpression on SKOV3 cell migration and invasion. In conclusion, the current study demonstrated that miR-126 inhibited EC cell migration and invasion, at least partially through the direct targeting of IRS1, suggesting that miR-126 may aid the treatment of EC metastasis.

Activation of signal transduction pathways during hepatic oncogenesis

Background and aimsUnderstanding the molecular pathogenesis of hepatocellular carcinoma (HCC) is essential to identify therapeutic targets. A hepatitis B virus (HBV) related double transgenic murine model was developed. MethodsLiver specific expression of HBV X protein (HBx) and insulin receptor substrate 1 (IRS1) was achieved and transgenic mice were followed from birth to age 21 months. Liver and tumor tissue were assessed for histologic changes as well as activation of signal transduction pathways by qRT-PCR and multiplex ELISA protein assays. ResultsOverexpression of HBx and IRS1 stimulates liver cell proliferation in the double transgenic mice. Only the male mice developed HCC starting at age 15–18 months. The IN/IGF1/IRS1/MAPK/ERK and IN/IGF1/IRS1/PI3K/AKT/GSK3β cascades were activated early (6–9 months) in the liver followed by WNT/β-catenin and Notch signaling. Aspartate β-hydroxylase (ASPH) was found to link these upstream growth factor signaling pathways to downstream Notch activation in tumor tissues. ConclusionsSustained overexpression of HBx and IRS1 led to constitutive activation of a tripartite growth factor signal transduction cascade in the liver and was necessary and sufficient to promote HCC development and progression.