Ursolic Acid Inhibits Breast Cancer Metastasis by Suppressing Glycolytic Metabolism via Activating SP1/Caveolin-1 Signaling.

Breast cancer remains the most common malignancy and the leading causality of cancer-associated mortality among women worldwide. With proven efficacy, Oldenlandia diffusa has been extensively applied in breast cancer treatment in Traditional Chinese Medicine (TCM) for thousands of years. However, the bioactive compounds of Oldenlandia diffusa accounting for its anti-breast cancer activity and the underlying biological mechanisms remain to be uncovered. Herein, bioactivity-guided fractionation suggested ursolic acid as the strongest anti-breast cancer compound in Oldenlandia diffusa. Ursolic acid treatment dramatically suppressed the proliferation and promoted mitochondrial-mediated apoptosis in breast cancer cells while brought little cytotoxicities in nonmalignant mammary epithelial cells in vitro. Meanwhile, ursolic acid dramatically impaired both the glycolytic metabolism and mitochondrial respiration function of breast cancer cells. Further investigations demonstrated that ursolic acid may impair the glycolytic metabolism of breast cancer cells by activating Caveolin-1 (Cav-1) signaling, as Cav-1 knockdown could partially abrogate the suppressive effect of ursolic acid on that. Mechanistically, ursolic acid could activate SP1-mediated CAV1 transcription by promoting SP1 expression as well as its binding with CAV1 promoter region. More meaningfully, ursolic acid administration could dramatically suppress the growth and metastasis of breast cancer in both the zebrafish and mouse xenotransplantation models of breast cancer in vivo without any detectable hepatotoxicity, nephrotoxicity or hematotoxicity. This study not only provides preclinical evidence supporting the application of ursolic acid as a promising candidate drug for breast cancer treatment but also sheds novel light on Cav-1 as a druggable target for glycolytic modulation of breast cancer.

Extracellular vesicles derived from microRNA-150-5p-overexpressing mesenchymal stem cells protect rat hearts against ischemia/reperfusion.

An intriguing area of research has demonstrated the ability of extracellular vesicles (EVs) as biological vehicles for microRNAs (miRNAs) transfer. Mesenchymal stem cells (MSCs) produce large amounts of EVs. Rat models of ischemia/reperfusion (I/R) were established to explore the expression profile of thioredoxin-interacting protein (TXNIP), which was then knocked-down to investigate its effects on myocardial remodeling, followed by detection on myocardial infarction size (MIS), myocardial collagen volume fraction (CVF) and cardiomyocyte apoptosis. MSCs-derived EVs carrying miR-150-5p were cultured with neonatal cardiomyocytes under hypoxia/hypoglycemia condition for in vitro exploration and intramyocardially injected into I/R rats for in vivo exploration. I/R-induced rats presented higher TXNIP levels and lower miR-150-5p levels, along with increased cardiomyocyte apoptosis. miR-150-5p in MSCs was transferred through EVs to cardiomyocytes, leading to suppressed myocardial remodeling, as reflected by smaller MIS and CVF and suppressed cardiomyocyte apoptosis. I/R-treated rats injected with MSCs-derived EVs containing miR-150-5p showed a reduction in myocardial remodeling associated with the downregulation of TXNIP, which may be clinically applicable for treatment of I/R.

The Value of Geriatric Nutritional Risk Index in Evaluating Postoperative Complication Risk and Long-Term Prognosis in Elderly Colorectal Cancer Patients.

PURPOSE: The geriatric nutritional risk index (GNRI) has been reported as a useful tool for predicting the prognosis of many diseases; however, there is currently little research on the relationship between GNRI and outcomes in elderly colorectal cancer (CRC) patients. This study aimed to explore the value of GNRI in evaluating postoperative complication risk and long-term prognosis in elderly CRC patients. PATIENTS AND METHODS: The medical records of 230 CRC patients aged≥65 years who underwent surgery between January 2012 and December 2014 were retrospectively analyzed. Patients were divided into abnormal and normal GNRI groups by modified binary classification. Logistic regression analysis was used to evaluate the correlation between GNRI and complication risk. The Kaplan-Meier method with log-rank test was used to construct survival curves. The Cox proportional hazard model was used for univariate, multivariate and subgroup survival analyses to assess the relationship between GNRI and long-term prognosis. RESULTS: Multivariate logistic regression analysis showed that GNRI (p = 0.009, HR 2.280, 95% CI: 1.224-4.247) was an independent risk factor for postoperative complications in elderly CRC patients. Kaplan-Meier survival curves revealed that the abnormal GNRI group had significantly lower disease-free survival (DFS; p = 0.005) and overall survival (OS; p=0.007) than the normal GNRI group had, especially in TNM I stage. In multivariate survival analysis, GNRI was an independent prognostic factor for DFS (p = 0.003, HR 1.842, 95% CI: 1.229-2.760) and OS (p = 0.003, HR 1.852, 95% CI: 1.231-2.787). CONCLUSION: GNRI is a simple and effective tool for predicting the risk of postoperative complications and the long-term prognosis of postoperative elderly CRC patients and can provide a scientific basis for early nutrition interventions in elderly CRC patients.

[Effect of 27nt-miRNA on the differentiation of mesenchymal stem cells into vascular smooth muscle cells].

To investigate the effect of 27nt-miRNA on the differentiation of mesenchymal stem cells into vascular smooth muscle cells. The highly expression plasmids of 27nt-miRNA and anti-27nt-miRNA, and negative control plasmids were constructed, packaged with lentivirus and transfected into human umbilical cord mesenchymal stem cells (hUCMSCs). Collagen IV was added to induce hUCMSCs differentiation into blood vessel smooth muscle cells (VSMCs). The cell viability was measured by MTT assay. The expression of SMA, SM22α at mRNA and protein levels was determined by RT-PCR, immunocytochemical staining and Western blotting. Compared with the negative control group, the viability of the 27nt-miRNA overexpression group was decreased by 20.48% (P<0.05), and the expression of SMA mRNA and SM22α mRNA and protein was significantly increased (P<0.05); the viability of Anti-27nt-miRNA group was increased 18.07% (P<0.05), and the expression of SMA mRNA and SM22α mRNA and protein was decreased (P<0.05). In summary, 27nt-miRNA promotes mesenchymal stem cells differentiation into vascular smooth muscle cells and inhibits cells viability.

[Regulation of miR-24 on vascular endothelial cell function and its role in the development of cardiovascular disease].

microRNA-24 (miR-24) belongs to miR-23~27~24 cluster. It is highly expressed in vascular endothelial cells (VECs), and plays an important role in the regulation of VECs specific gene expression. Recent studies indicate that miR-24 is involved in the regulation of VECs function such as proliferation, apoptosis, angiopoiesis, inflammation, and differentiation. The dysregulation of miR-24 is associated with dysfunction or even damage of VECs, and contributes to the development of cardiovascular disease. In this review, we summarized the molecular mechanisms of miR-24 in regulating VECs function, and its role and importance during the development of cardiovascular disease.

Roles of miRNA-24 in regulating endothelial nitric oxide synthase expression and vascular endothelial cell proliferation.

This study is to investigate the effect of miRNA-24 on endothelial nitric oxide synthase (eNOS) expression and vascular endothelial cell proliferation. Constructed high expression miRNA-24 plasmids were introduced into human umbilical vein endothelial cells (HUVECs) by liposome. Cell numbers were counted by a Hemocytometer, and cell proliferation was detected by MTT assay. The expression levels of eNOS and specificity protein 1 (Sp1) at mRNA and protein levels were, respectively, examined by real-time PCR, immunohistochemistry, and Western blotting. Compared with the control group, endothelial cell proliferation in miRNA-24 group significantly decreased by 64.24 % (6.53 ± 0.11 vs 18.26 ± 0.45, P < 0.01). The expression of eNOS at mRNA and protein levels in miRNA-24 group decreased by 64.21 % (0.34 ± 0.01 vs 0.95 ± 0.02, P < 0.05) and 82.86 % (0.072 ± 0.06 vs 0.42 ± 0.06, P < 0.05), respectively. Meanwhile, the expression of Sp1 at mRNA and protein levels in miRNA-24 group decreased by 64.64 % (0.35 ± 0.01 vs 0.99 ± 0.03, P < 0.05) and 60.34 % (0.23 ± 0.05 vs 0.58 ± 0.07, P < 0.05), respectively. In anti-miRNA-24 group, endothelial cell proliferation decreased by 33.46 % compared with the control group (12.15 ± 0.21 vs 18.26 ± 0.45, P < 0.01), while it increased by 46.25 % compared with the miRNA-24 group (12.15 ± 0.21 vs 6.53 ± 0.11, P < 0.01). The expression of eNOS at mRNA and protein levels in anti-miRNA-24 group decreased by 44.21 % (0.53 ± 0.04 vs 0.95 ± 0.02, P < 0.05) and by 30.95 %(0.29 ± 0.05 vs 0.42 ± 0.06, P < 0.05) compared with the control group, while it increased by 35.84 % (0.53 ± 0.04 vs 0.34 ± 0.01, P < 0.05) and by 75.17 % (0.29 ± 0.05 vs 0.072 ± 0.06, P < 0.05) compared with miRNA-24 group. The expression of Sp1 at mRNA and protein levels in ant-miRNA-24 group decreased by 36.36 % (0.63 ± 0.04 vs 0.99 ± 0.03, P < 0.05) and by 22.41 % (0.45 ± 0.06 vs 0.58 ± 0.07, P < 0.05) compared with the control group, while it increased by 44.44 % (0.63 ± 0.04 vs 0.35 ± 0.01, P < 0.05) and by 48.88 % (0.45 ± 0.06 vs 0.23 ± 0.05, P < 0.05) compared with miRNA-24 group. HUVECs proliferation and eNOS expression are significantly inhibited by miRNA-24. Sp1, which is regulated by miRNA-24, might act as one of the important factors involved in eNOS gene expression.

Suppression of endothelial nitric oxide synthase expression and endothelial cell proliferation by an intronic 27-ntmiRNA and it's a novel link to AP-1.

This study aims to investigate the role of activator protein 1 (AP-1) in the effects of 27nt-miRNA on expression of endothelial nitric oxide synthase (eNOS) gene and proliferation of endothelial cells. Cell proliferation was analyzed by cell number counting, colony formation assay and MTT assay. Cell migration and invasion was detected by transwell assay and invasion assay. Expression of eNOS and AP-1 was measured by real-time RT-PCR (mRNA level) and Western blotting (protein level). Luciferase reporter assay was performed to detect the binding of 27nt-miRNA to AP-1. Overexpression of 27nt-miRNA significantly inhibited endothelial cells proliferation, invasion and migration in vitro. And, eNOS and AP-1 expression at mRNA and protein levels were down-regulated by overexpression of 27nt-miRNA. Interestingly, overexpression of AP-1 protein partially restored eNOS expression and endothelial cell proliferation. Furthermore, the luciferase reporter assay demonstrated that AP-1 was a direct target of 27nt-miRNA. These data demonstrate that overexpression of 27nt-miRNA inhibits endothelial cell proliferation, invasion, migration, eNOS expression and AP-1 expression. Moreover, AP-1, a direct target of 27nt-miRNA, reverses the inhibitory effects of 27nt-miRNA. Thus, the effects of 27nt-miRNA might be acted through targeting AP-1.

Activation of miR-21 by STAT3 induces proliferation and suppresses apoptosis in nasopharyngeal carcinoma by targeting PTEN gene.

The present study is to investigate the role of microRNA-21 (miR-21) in nasopharyngeal carcinoma (NPC) and the mechanisms of regulation of PTEN by miR-21. Fifty-four tissue samples were collected from 42 patients with NPC and 12 healthy controls. Human NPC cell lines CNE-1, CNE-2, TWO3 and C666-1 were used for cell assays. To investigate the expression of miR-21, RT-PCR was employed. RT-PCR, Western blotting, and immunohistochemistry were used to measure the expression of STAT3 mRNA and STAT3 protein. To test the effect of miR-21 on the cell growth and apoptosis of NPC cells in vitro, transfection of CNE1 and CNE2 cell lines and flow cytometry were performed. TUNEL assay was used to detect DNA fragmentation. To validate whether miR-21 directly recognizes the 3'-UTRs of PTEN mRNA, luciferase reporter assay was employed. miR-21 expression was increased in NPC tissues compared with control and the same result was found in NPC cell lines. Notably, increased expression of miR-21 was directly related to advanced clinical stage and lymph node metastasis. STAT3, a transcription factor activated by IL-6, directly activated miR-21 in transformed NPC cell lines. Furthermore, miR-21 markedly inhibited PTEN tumor suppressor, leading to increased AKT activity. Both in vitro and in vivo assays revealed that miR-21 enhanced NPC cell proliferation and suppressed apoptosis. miR-21, activated by STAT3, induced proliferation and suppressed apoptosis in NPC by targeting PTEN-AKT pathway.

MicroRNA-21 inhibits platelet-derived growth factor-induced human aortic vascular smooth muscle cell proliferation and migration through targeting activator protein-1.

OBJECTIVES: This study is to investigate whether microRNA (miR)-21 inhibits platelet-derived growth factor-induced human aortic vascular smooth muscle cell (VSMC) proliferation and migration through targeting activator protein-1 (AP-1). METHODS: VSMCs were transfected with the miR-21 or miR-21 inhibitor. Cell proliferation was determined using methyl thiazolyl tetrazolium assay. Cell migration was detected by transwell assay. Luciferase reporter assay was used to study the interaction between miR-21 and AP-1. The levels of mRNA were determined using quantitative real-time polymerase chain reaction, while protein expression was measured using Western blotting assay. RESULTS: Low expression of miR-21 significantly inhibited VSMC proliferation, invasion and migration. The mRNA levels and protein expression of α-SMA and AP-1 were down-regulated by low expression of miR-21. In addition, luciferase reporter assay demonstrated that AP-1 might be a direct target gene of miR-21 in VSMC initiation and development. Moreover, up-regulation of AP-1 was critical for miR-21-mediated inhibitory effects on platelet-derived growth factor-induced cell proliferation and migration in human VSMCs. CONCLUSIONS: In summary, miR-21 is a key molecule in regulating human VSMC proliferation and migration by targeting AP-1, suggesting that specific modulation of miR-21 in human VSMCs may become an attractive approach for the treatment of proliferative vascular diseases.

miR-21 inhibitor suppresses proliferation and migration of nasopharyngeal carcinoma cells through down-regulation of BCL2 expression.

This study is to investigate the expression of miR-21 in nasopharyngeal carcinoma (NPC) cells, and the effect of miR-21 in the biological behavior and expression of B-cell lymphoma 2 (BCL2) in NPC cells. Paired NPC and adjacent non-tumor tissues were obtained from 53 patients who underwent primary surgical resection of NPC tissues. Luciferase reporter assay was performed to test whether BCL2 is a direct target of miR-21. Methylthiazolyl blue tetrazolium assay and colony assay were used to evaluate the effect of miR-21 on NPC cell proliferation. Transwell and wound-healing assays were carried out to test the effect of low expression of miR-21 on cancer cell migration and invasion. QRT-PCR and Western blotting were used to measure the levels of mRNA and protein expression, respectively. Tumor tissues showed a positive correlation between the levels of miR-21 and BCL2 protein expression. Cells transfected with miR-21 inhibitor healed slower compared the control (P < 0.05). In addition, cell migration was notably inhibited by the down-regulation of miR-21 in vitro (P < 0.05). The reduction in miR-21 expression showed a remarkable effect on the biological behavior of NPC cell clone formation (P < 0.05). Low expression of miR-21 by transfection with miRNA expression plasmid led to a decrease in BCL2 expression, which was accompanied by reduced migration and proliferation of the cancer cells. Our results demonstrated that miR-21 inhibitor down-regulated BCL2 expression level, suggesting that BCL2 might be a target gene for the initiation and development of NPC cells.

The effect of nitidine chloride on the proliferation and apoptosis of nasopharyngeal carcinoma cells.

PURPOSE: This study was conducted to investigate the effect of nitidine chloride (NC) on the proliferation and apoptosis of nasopharyngeal carcinoma cell line CNE1, CNE2, TWO3, and C666-1, and to explore its antitumor mechanism. METHODS: NC was dissolved in IMDM medium and cultured with nasopharyngeal carcinoma cell line CNE1, CNE2, TWO3 and C666-1. Cell morphology, cell proliferation, cell apoptosis, p53 mRNA and p53 protein levels were assessed. RESULTS: After incubation with NC for 24 h, typical apoptotic morphology was observed. NC inhibited the proliferation and induced apoptosis of all 4 cell lines in a time-dose dependent manner. p53 mRNA and p53 protein levels were significantly increased. CONCLUSIONS: NC inhibited proliferation and induced apoptosis of nasopharyngeal carcinoma cells with upregulation of p53 gene.

Effect of trichosanthin on apoptosis and telomerase activity of nasopharyngeal carcinomas in nude mice.

PURPOSE: To evaluate the effect of trichosanthin (TCS) on telomerase activity and apoptosis in nasopharyngeal carcinoma cells implanted into nude mice. METHODS: Nude mice implanted with CNE1 and CNE2 nasopharyngeal carcinoma cell lines were randomly divided into 3 groups, including TCS group, control group (treatment with saline only), and cyclophosphamide (CTX) group. The weight of mice and the tumor volume were measured. The ultra-microstructural changes were observed by transmission electron microscope. Expression of Bcl-2 and Bax were investigated by immunohistochemistry. Apoptosis of CNE1 and CNE2 cells was determined by the TUNEL method, and telomerase activity by TRAP-ELISA. RESULTS: Compared with the normal control group (saline group), TCS significantly inhibited the growth of both CNE1 and CNE2 tumor cells. CTX also inhibited the growth of both CNE1 and CNE2 tumor cells but this inhibition was slightly slower compared with the TCS. CONCLUSION: TCS suppresses the growth of CNE1 and CNE2 cells lines in vivo. Its anticancer effects were associated with induction of apoptosis and, at least partially, with suppression of telomerase activity.

An intronic miRNA regulates expression of the human endothelial nitric oxide synthase gene and proliferation of endothelial cells by a mechanism related to the transcription factor SP-1.

OBJECTIVE: This study was to investigate the molecular mechanisms underlying the 27nt-miRNA-mediated regulation of expression of the endothelial nitric oxide synthase (eNOS) gene. METHODS: Cell lines overexpressing 27nt-miRNA or its mutant were established by transfecting the miRNA expression vector into the endothelial cells. eNOS mRNA and protein expression were examined by RT-PCR and Western Blotting, respectively. Luciferase activity reporter system was used to study the target of 27nt-miRNA. RESULTS: The results showed that overexpression of 27nt-miRNA significantly inhibited eNOS mRNA level and protein expression, and reduced the eNOS transcriptional efficiency. Such inhibitory effects of 27nt-miRNA were attenuated by the sequence mutations in 27nt-miRNA. Interestingly, the transcription factor SP-1 expression was reduced by 27nt-miRNA. Meanwhile, overxpression of SP-1 protein partially restored eNOS expression, and rescued the 27nt-miRNA-mediated reduction of endothelial cell proliferation. Moreover, certain sites in the SP-1 mRNA were found to be the direct target of 27nt-miRNA by a luciferase reporter system. CONCLUSIONS: These results demonstrate that the 27nt-miRNA suppresses eNOS gene expression and SP-1 expression in vascular endothelial cells. The 27nt-miRNA directly target to SP-1 mRNA, thereby contributing to proliferation of endothelial cells.

Role of microRNA-21 in regulating 3T3-L1 adipocyte differentiation and adiponectin expression.

MicroRNAs are endogenous small RNAs with a high degree of conservation, participating in a variety of vital activities. In present study, to explore the effect of microRNAs on 3T3-L1 adipocyte differentiation and adiponectin expression, the adipo-related microRNAs were screened and identified by micorRNA microarray. The highly expression plasmid of microRNA-21 with obvious expression up-regulation (miR-21) and its anti-sense (miR-21 inhibitor) were constructed and transfected into 3T3-L1 preadipocytes. The effect of miR-21 on 3T3-L1 adipocyte differentiation was observed, and the protein and mRNA expression level of adiponectin and AP-1 were analyzed. Results showed that, the expression profiles of microRNAs significantly changed during 3T3-L1 adipocyte differentiation. The expression of miR-21 was obviously up-regulated. miR-21 could significantly promote adipocyte differentiation, increase adiponectin mRNA and protein expression, while decrease AP-1 protein level. Meanwhil, miR-21 inhibitor blocked the effects of miR-21 mentioned above. The overexpression of AP-1 could absolutely reverse the stimulatory effect of miR-21 on adiponectin. miR-21 plays an important role in regulating adipocyte differentiation and adiponectin expression by inhibiting AP-1 expression.

miR-129-2 suppresses proliferation and migration of esophageal carcinoma cells through downregulation of SOX4 expression.

We report the emerging role of microRNA (miRNA) deregulation associated with activation of an oncogene SOX4 (a member of the SRY-related HMG-box) in esophageal carcinoma. Paired esophageal cancer and adjacent non-tumor tissues were obtained from 42 patients who underwent primary surgical resection for esophageal cancer. Experiments such as real-time PCR, western blot analysis, luciferase-reporter assay, cell proliferation and colony formation assays, in vitro migration and invasion assays, and a wound-healing assay were performed to determine the effects of miR-129-2. We found that SOX4 expression was elevated (P<0.005) in esophageal tumors (n=42) when compared with its expression in the controls (n=42). Compared with the normal esophageal tissues, the expression of miR-129-2 was downregulated in 27 of 31 primary esophageal tumors, while the expression of SOX4 was upregulated (P<0.001). Restoration of miR-129-2 by transfection with an miRNA expression plasmid led to a decrease in SOX4 expression, which was accompanied by reduced migration and proliferation of the cancer cells. These results suggest that aberrant expression of SOX4 is associated with repression of miR-129-2, and restoration of miR-129-2 suppresses the migration and proliferation of esophageal cancer cells. Our results demonstrated that the deregulation of miR-129-2 leads to aberrant SOX4 expression, presenting a new paradigm in which the restoration of miRNA suppresses its oncogenic target in esophageal cancer.

Intronic microRNA suppresses endothelial nitric oxide synthase expression and endothelial cell proliferation via inhibition of STAT3 signaling.

Intronic microRNA (miRNAs) suppressed the expression of endothelial nitric oxide synthase (eNOS) gene in endothelial cells (ECs). This study was to investigate the role of signal transducer and activator of transcription 3 (STAT3) in the regulation of eNOS expression and vascular EC proliferation by the intronic 27-nucleotide (nt) miRNA derived from the 27-base pair repeats in intron 4 of eNOS gene. A detectable level of the 27-nt miRNA was present in the control ECs. Overexpression of the 27-nt miRNA dramatically suppressed the expression of eNOS and STAT3 at both transcription and translation levels in ECs in association with significant inhibition of EC proliferation. Mutation of the 27-nt miRNA at the 3'-terminal region resulted in substantial reduction of the inhibitory effect of miRNA on eNOS and STAT3 expression, and EC proliferation. Overexpression of active STAT3 significantly reversed the inhibitory effect of the 27-nt miRNA on eNOS expression and EC proliferation. In summary, we demonstrated that the 27-nt intronic miRNA functioned as a negative regulator for the expression of its host gene eNOS and cell proliferation in ECs. The sequence in 3'-terminal region played a key role in the function of the 27-nt miRNA. The regulatory effect of the intronic miRNA on eNOS gene expression was associated with miRNA polymorphisms, and mediated through inhibition of STAT3 signaling in ECs.

MicroRNA-375 promotes 3T3-L1 adipocyte differentiation through modulation of extracellular signal-regulated kinase signalling.

1. Adipocyte hypertrophy and hyperplasia are important processes in the development of obesity. To understand obesity and its associated diseases, it is important to elucidate the molecular mechanisms governing adipogenesis. MicroRNA-375 has been shown to inhibit differentiation of neurites, and participate in the regulation of insulin secretion and blood homeostasis. However, it is unknown whether miR-375 plays a role in adipocyte differentiation. 2. To investigate the role of miR-375 in adipocyte differentiation, we compared the miR-375 expression level between 3T3-L1 pre-adipocytes and adipocytes using miRNA microarray and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis. Furthermore, we evaluated the effects of overexpression or inhibition of miR-375 on 3T3-L1 adipocyte differentiation. 3. In the present study, we found that miR-375 expression was increased after induction of adipogenic differentiation. Overexpression of miR-375 enhanced 3T3-L1 adipocyte differentiation, as evidenced by its ability to increase mRNA levels of both CCAAT/enhancer binding protein-α (C/EBPα) and peroxisome proliferator-activated receptor-γ (PPARγ2), and induction of adipocyte fatty acid-binding protein (aP2) and triglyceride (TG) accumulation. Furthermore, we found overexpression of miR-375 suppressed phosphorylation levels of extracellular signal-regulated kinases 1/2 (ERK1/2). In contrast, anti-miR-375 increased ERK1/2 phosphorylation levels and inhibited mRNA expression of C/EBPα, PPARγ2 and aP2 in 3T3-L1 adipocyte, accompanied by decreased adipocyte differentiation. 4. Taken together, these data suggest that miR-375 promotes 3T3-L1 adipocyte differentiation, possibly through modulating the ERK-PPARγ2-aP2 pathway.

CHANGES IN microRNA (miR) profile and effects of miR-320 in insulin-resistant 3T3-L1 adipocytes.

1. MicroRNAs (miRNAs) play essential roles in many biological processes. It is known that aberrant miRNA expression contributes to some pathological conditions. However, it is not known whether miRNAs play any role in the development of insulin resistance in adipocytes, a key pathophysiological link between obesity and diabetes. 2. To investigate the function of miRNAs in the development of insulin resistance, using miRNA microarray analysis we compared miRNA expression profiles between normal insulinsensitive 3T3-L1 adipocytes and 3T3-L1 adipocytes rendered insulin resistant following treatment with high glucose (25mmol/L) and high insulin (1 mol/L). Furthermore, adipocytes were transfected with specific antisense oligonucleotides against miRNA-320 (anti-miR-320 oligo) and the effects on the development of insulin resistance were evaluated. 3. We identified 50 upregulated and 29 downregulated miRNAs in insulin-resistant (IR) adipocytes, including a 50-fold increase in miRNA-320 (miR-320) expression. Using bioinformatic techniques, the p85 subunit of phosphatidylinositol 3-kinase (PI3-K) was found to be a potential target of miR-320. In experiments with anti-miR-320 oligo, insulin sensitivity was increased in IR adipocytes, as evidenced by increases in p85 expression, phosphorylation of Akt and the protein expression of the glucose transporter GLUT-4, as well as insulin-stimulated glucose uptake. These beneficial effects of anti-miR-320 oligo were observed only in IR adipocytes and not in normal adipocytes. 4. In conclusion, the miRNA profile changes in IR adipocytes compared with normal 3T3-L1 adipocytes. Anti-miR-320 oligo was found to regulate insulin resistance in adipocytes by improving insulin­PI3-K signalling pathways. The findings provide information regarding a potentially new therapeutic strategy to control insulin resistance.