Combination of Metformin, Sodium Oxamate and Doxorubicin Induces Apoptosis and Autophagy in Colorectal Cancer Cells via Downregulation HIF-1α.

Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide in both sexes. Current therapies include surgery, chemotherapy, and targeted therapy; however, prolonged exposure to chemical agents induces toxicity in patients and drug resistance. So, we implemented a therapeutic strategy based on the combination of doxorubicin, metformin, and sodium oxamate called triple therapy (Tt). We found that Tt significantly reduced proliferation by inhibiting the mTOR/AKT pathway and promoted apoptosis and autophagy in CRC derived cells compared with doxorubicin. Several autophagy genes were assessed by western blot; ULK1, ATG4, and LC3 II were overexpressed by Tt. Interestingly, ULK1 was the only one autophagy-related protein gradually overexpressed during Tt administration. Thus, we assumed that there was a post-transcriptional mechanism mediating by microRNAs that regulate UKL1 expression during autophagy activation. Through bioinformatics approaches, we ascertained that ULK1 could be targeted by mir-26a, which is overexpressed in advanced stages of CRC. In vitro experiments revealed that overexpression of mir-26a decreased significantly ULK1, mRNA, and protein expression. Contrariwise, the Tt recovered ULK1 expression by mir-26a decrease. Due to triple therapy repressed mir-26a expression, we hypothesized this drug combination could be involved in mir-26a transcription regulation. Consequently, we analyzed the mir-26a promoter sequence and found two HIF-1α transcription factor recognition sites. We developed two different HIF-1α stabilization models. Both showed mir-26a overexpression and ULK1 reduction in hypoxic conditions. Immunoprecipitation experiments were performed and HIF-1α enrichment was observed in mir-26a promoter. Surprisingly, Tt diminished HIF-1α detection and restored ULK1 mRNA expression. These results reveal an important regulation mechanism controlled by the signaling that activates HIF-1α and that in turn regulates mir-26a transcription.

Knockdown of miR-26a in zebrafish leads to impairment of the anti-inflammatory function of TnP in the control of neutrophilia

Our recent data show the valuable potential of TnP for the development of a new and safe anti-inflammatory drug due to its ability to control the traffic and activation of leukocytes in response to inflammation. Although there is considerable knowledge surrounding the cellular mechanisms of TnP, less is known about the mechanistic molecular role of TnP underlying its immunomodulatory functions. Here, we conducted investigations to identify whether miRNAs could be one of the molecular bases of the therapeutic effect of TnP. Using a zebrafish model of neutrophilic inflammation with a combination of genetic gain- and loss-of-function approaches, we showed that TnP treatment was followed by up-regulation of only four known miRNAs, and mature dre-miR-26a-1, herein referred just as miR-26a was the first most highly expressed. The knockdown of miR-26a ubiquitously resulted in a significant reduction of miR-26a in embryos, accompanied by impaired TnP immunomodulatory function observed by the loss of the control of the removal of neutrophils in response to inflammation, while the overexpression increased the inhibition of neutrophilic inflammation promoted by TnP. The striking importance of miR-26a was confirmed when rescue strategies were used (morpholino and mimic combination). Our results identified miR-26a as an essential molecular regulator of the therapeutic action of TnP, and suggest that miR-26a or its targets could be used as promising therapeutic candidates for enhancing the resolution of inflammation.

Plasma microRNA expression levels in HIV-1-positive patients receiving antiretroviral therapy.

MicroRNAs (miRNAs/miRs) may serve as therapeutic agents or targets in diseases in which the expression of proteins plays an important role. The aim of the present study was to compare the expression levels of specific miRNAs, as well as their correlation with markers of response to antiretroviral (ARV) therapy, in patients with human immunodeficiency virus type 1 (HIV-1) infection with and without resistance to highly active antiretroviral therapy (HAART). METHODS: miRNA assays were performed on plasma samples obtained from 20 HIV-1-positive patients. A total of ten patients were divided into two groups: HAART-responsive and HAART-resistant (n=5 per group). Commercial arrays were subsequently used to identify 84 miRNAs. A total of three differentially expressed miRNAs were selected and analyzed by quantitative PCR (qPCR). Five other patients were subsequently added to each group for a new relative expression analysis. The absolute expression level of the two miRNAs was obtained and compared using the Student's t test. Receiver operating characteristic (ROC) curves were used to identify patients with antiretroviral therapy (ART) resistance. RESULTS: The array analysis revealed that miR-15b-5p, miR-16-5p, miR-20a-5p, miR-26a-5p, miR-126-3p and miR-150-5p were down-regulated in patients with HAART-resistance comparing with HAART-responsive. The expression levels of miR-16-5p, miR-26a-5p and miR-150-5p were confirmed using qPCR. The area under the ROC curve was 1.0 for the three miRNAs. CONCLUSIONS: The lower expression levels of miR-16-5p and miR-26a-5p in patients with HAART-resistance suggested that these may serve as potential biomarkers for the identification of HAART-responsive patients.

miR-26a-5p protects against myocardial ischemia/reperfusion injury by regulating the PTEN/PI3K/AKT signaling pathway

Reperfusion strategies in acute myocardial infarction (AMI) can cause a series of additional clinical damage, defined as myocardial ischemia/reperfusion (I/R) injury, and thus there is a need for effective therapeutic methods to attenuate I/R injury. miR-26a-5p has been proven to be an essential regulator for biological processes in different cell types. Nevertheless, the role of miR-26a-5p in myocardial I/R injury has not yet been reported. We established an I/R injury model in vitro and in vivo. In vitro, we used cardiomyocytes to simulate I/R injury using hypoxia/reoxygenation (H/R) assay. In vivo, we used C57BL/6 mice to construct I/R injury model. The infarct area was examined by TTC staining. The level of miR-26a-5p and PTEN was determined by bioinformatics methods, qRT-PCR, and western blot. In addition, the viability and apoptosis of cardiomyocytes were separately detected by MTT and flow cytometry. The targeting relationship between miR-26a-5p and PTEN was analyzed by the TargetScan website and luciferase reporter assay. I/R and H/R treatment induced myocardial tissue injury and cardiomyocyte apoptosis, respectively. The results showed that miR-26a-5p was down-regulated in myocardial I/R injury. PTEN was found to be a direct target of miR-26a-5p. Furthermore, miR-26a-5p effectively improved viability and inhibited apoptosis in cardiomyocytes upon I/R injury by inhibiting PTEN expression to activate the PI3K/AKT signaling pathway. miR-26a-5p could protect cardiomyocytes against I/R injury by regulating the PTEN/PI3K/AKT pathway, which offers a potential approach for myocardial I/R injury treatment.

Cell migration and proliferation are regulated by miR-26a in colorectal cancer via the PTEN-AKT axis.

BACKGROUND: Invasion and metastasis are determinant events in the prognosis of Colorectal cancer (CRC), a common neoplasm worldwide. An important factor for metastasis is the acquired capacity of the cell to proliferate and invade adjacent tissues. In this paper, we explored the role of micro-RNA-26a in the regulation of proliferation and migration in CRC-derived cells through the negative regulation of PTEN, a key negative regulator of the AKT pathway. METHODS: Expression levels of PTEN and mir-26a were surveyed in normal and CRC-derived cell lines; paraffin embedded human tissues, TCGA CRC expression data and a Balb/c mice orthotopic induced CRC model. CRC was induced by an initial intraperitoneal dose of the colonic carcinogen Azoxymethane followed by inflammatory promoter Dextran Sulfate Sodium Salt. Luciferase assays provide information about miR-26a-PTEN 3'UTR interaction. Proliferation and migration by real time cell analysis and wound-healing functional analyses were performed to assess the participation of mir-26a on important hallmarks of CRC and its regulation on the PTEN gene. RESULTS: We observed a negative correlation between PTEN and mir-26a expression in cell lines, human tissues, TCGA data, and tissues derived from the CRC mouse model. Moreover, we showed that negative regulation of PTEN exerted by miR-26a affected AKT phosphorylation levels directly. Functional assays showed that mir-26a directly down-regulates PTEN, and that mir-26a over-expressing cells had higher proliferation and migration rates. CONCLUSIONS: All this data proposes an important role of mir-26a as an oncomir in the progression and invasion of CRC. Our data suggested that mir-26a could be used as a biomarker of tumor development in CRC patients, however more studies must be conducted to establish its clinical role.

miR-26a-5p suppresses tumor metastasis by regulating EMT and is associated with prognosis in HCC.

OBJECTIVE: To determine the role of miR-26a-5p in tumor invasion and metastasis in hepatocellular carcinoma (HCC). METHODS: We evaluated miR-26a-5p expression in HCC tissues by quantitative PCR and then analyzed its clinical significance using a Cox regression model. Transwell and nude mouse models were used to examine tumor metastasis in vitro and in vivo, respectively. The relationship between miR-26a-5p and epithelial-mesenchymal transition was also investigated by q-PCR and western blot. RESULTS: Strong downregulation of miR-26a-5p was observed in tumor tissues compared to paired adjacent normal tissues. Moreover, patients with low miR-26a-5p expression had a significantly poorer prognosis than those with high expression. The multivariate analysis indicated that miR-26a-5p expression was an independent prognostic indicator. The experimental transwell model and athymic mouse model revealed that miR-26a-5p depressed tumor metastasis in vitro and in vivo, respectively. In addition, the decreased miR-26a-5p level observed in HCC was associated with reduced E-cadherin expression and upregulation of vimentin, which affects the molecular mechanism of EMT. CONCLUSION: Downregulation of miR-26a-5p promotes tumor metastasis by targeting EMT and influences the prognosis of HCC patients. Therefore, miR-26a-5p has potential as a new biomarker and therapeutic target.

IL-6 upregulation contributes to the reduction of miR-26a expression in hepatocellular carcinoma cells

A recent study showed that miR-26a is downregulated in hepatocellular carcinoma tissues and that this downregulation is an independent predictor of survival. Interestingly, the same study also reported that miR-26a downregulation causes a concomitant elevation of IL-6 expression. Because miR-26a expression was found to be transcriptionally downregulated by oncogene c-Myc in various cancers, and the expression of c-Myc was increased by IL-6 stimulation, we hypothesized that IL-6 contributes to reduction of miR-26a in hepatocellular carcinoma. Serum IL-6 was measured by ELISA and miR-26a was detected by qRT-PCR. The data of 30 patients with hepatocellular carcinoma who had undergone surgical tumor resection revealed that serum IL-6 could be considered to be a predictor of survival up to 5 years for hepatocellular carcinoma patients (log-rank test, P < 0.05). We observed that the serum IL-6 concentration was inversely correlated with miR-26a expression in cancerous tissues (Pearson correlation test, r = -0.651, P < 0.01). Furthermore, by in vitro experiments with HepG2 cells, we showed that IL-6 stimulation can lead to miR-26a suppression via c-Myc activation, whereas in normal hepatocyte LO2 cells incubation with IL-6 had no significant effect on miR-26a expression. Taken together, these results indicate that miR-26a reduction in hepatocellular carcinoma might be due to IL-6 upregulation.