Oncogenic microRNAs characterization in clear cell renal cell carcinoma

A key challenge for the improvement of clear cell renal cell carcinoma (ccRCC) management could derive from a deeper characterization of the biology of these neoplasms that could greatly improve the diagnosis, prognosis and treatment choice. The aim of this study was to identify specific miRNAs that are deregulated in tumor vs. normal kidney tissues and that could impact on the biology of ccRCC. To this end we selected four miRNAs (miR-21-5p, miR-210-3p, miR-185-5p and miR-221-3p) and their expression has been evaluated in a retrospective cohort of formalin-fixed paraffin-embedded (FFPE) tissues from 20 ccRCC patients who underwent surgical nephrectomy resection. miR-21-5p and miR-210-3p resulted the most significantly up-regulated miRNAs in this patient cohort, highlighting these onco-miRNAs as possible relevant players involved in ccRCC tumorigenesis. Thus, this study reports the identification of specific oncogenic miRNAs that are altered in ccRCC tissues and suggests that they might be useful biomarkers in ccRCC management

Argonaute 2 drives miR-145-5p-dependent gene expression program in breast cancer cells

To perform their regulatory functions, microRNAs (miRNAs) must assemble with any of the four mammalian Argonaute (Ago) family of proteins, Ago1–4, into an effector complex known as the RNA-induced silencing complex (RISC). While the mature miRNA guides the RISC complex to its target mRNA, the Ago protein represses mRNA translation. The specific roles of the various Ago members in mediating miRNAs activity, however, haven’t been clearly established. In this study, we investigated the contribution of Ago2, the only human Ago protein endowed with nuclease activity, to the function of tumor-suppressor miR-145-5p in breast cancer (BC). We show that miR-145-5p and Ago2 protein are concomitantly downregulated in BC tissues and that restoration of miR-145-5p expression in BC cells leads to Ago2 protein induction through the loosening of Ago2 mRNA translational repression. Functionally, miR-145-5p exerts its inhibitory activity on cell migration only in presence of Ago2, while, upon Ago2 depletion, we observed increased miR-145/Ago1 complex and enhanced cell motility. Profiling by microarray of miR-145-5p target mRNAs, in BC cells depleted or not of Ago2, revealed that miR-145-5p drives Ago2-dependent and -independent activities. Our results highlight that the Ago2 protein in cancer cells strictly dictates miR-145-5p tumor suppressor activity

Contribution of miR-145-5p/Ago2 complex to the regulation of epithelial-mesenchymal transition

The epithelial-mesenchymal transition (EMT) is essential for cell fate determination during development but it is involved in pathological processes like cancer as well, being one of the first steps in the mechanisms leading to metastasis. miR-145-5p is one of the most widely recognized tumor-suppressor miRNAs, able to regulate cell migration and EMT through the contribution of the RISC complex in which Argonaute (Ago) proteins are required for target recognition and gene silencing [1]. Ago2 is an important member of the Ago family and its overexpression correlates with a transformed phenotype in breast cancer cells [2]. With the aim to unravel miR-145-5p/Ago2 contribution to the suppression of cancer progression in epithelial tumors, here we show that: i) miR-145-5p and Ago2 are down-regulated in breast tumor vs normal tissues; ii) the restored expression of miR-145-5p in breast cancer cell lines results in the reduction of tumor phenotype; iii) Ago2 expression is positively and specifically regulated by miR-145-5p; iv) miR-145-5p-dependent Ago2 induction is necessary for the inhibition of cell migration; v) when Ago2 is depleted, the formation of an alternative miR-145-5p/Ago1 active complex redirects miR-145-5p tumor suppressor function and correlates with a more invasive phenotype in breast cancer cells. These results open to the identification of miR-145-5p/Ago2-dependent molecular networks involved in the maintenance and progression of cancer phenotype

Retinoic acid sensitizes acute myeloid leukemia cells to ER stress

Acute myeloid leukemia (AML) is caused by the blockade of hematopoietic myeloid precursors at different stages of differentiation. A subtype of AML, acute promyelocytic leukemia (APL), is a paradigm of differentiation therapy since retinoic acid (RA) is able to induce leukemic blast terminal differentiation leading to cure rates exceeding 80% when administered in combination with chemotherapy. Although APL patients refractory to RA or who relapsed are very effectively treated with arsenic trioxide (ATO) in combination with RA, the elevated costs limit its use in developing countries and in first line therapy so that RA plus chemotherapy currently remain the standard of care (1, 2). Most importantly non-APL acute myeloid leukemia do not respond to RA indicating the need for novel strategies to sensitize AML cells to RA. Here we show that RA-triggered differentiation of APL cells induces endoplasmic reticulum (ER) stress slightly activating the unfolded protein response (UPR). This is sufficient to render leukemic cell lines and human primary blasts very sensitive to doses of ER stress inducing drugs, like tunicamycin (Tm), that are not toxic for the same cells in the absence of RA or for most cell types. Furthermore we observed that low doses of Tm, even in the absence of RA, are sufficient to strongly increase ATO toxicity. Indeed both RA-sensitive and RA-resistant APL cell lines resulted sensitive to Tm-ATO combined treatment at low doses of ATO that are ineffective in the absence of ER stress. The use of inhibitors targeting specific UPR branches indicate that the Protein Kinase RNA-like Endoplasmic Reticulum kinase (PERK) pathway protects differentiating APL cells from ER stress rendering it an interesting therapeutic molecular target. Finally, we extended our observations in a non-APL model, assessing that RA sensitize the non-APL cell line HL60 to ER stress. Altogether our data indicate ER stress as a possible target for designing novel combination therapeutic strategies in AML. Contribution of AIRC (StG 4841) and FILAS-RU-2014-1020 to FF was greatly appreciated

Argonaute 2 as novel molecular determinant for myeloid differentiation

microRNAs (miRNAs) are emerging as crucial factors for the establishment of complex regulatory circuitries involved in the regulation of hematopoietic cell fate determination. These small non-coding RNAs to exert their functional activity are assembled in RNA-induced silencing complexes (RISCs), where a member of Argonaute (Ago) family of proteins plays a central role in miRNA-mRNA target interaction and gene silencing. In human cells the miRNAs-Ago complex can also localize in the nucleus where Ago proteins can associate with promoter gene sequences to impact heterochromatin genomic structure and transcriptional silencing (Janowski BA et al., 2006; Meister G., 2013). By using human myeloid cell lines and acute myeloid leukemia (AML) primary blasts we highlight Ago2 as a new player in myeloid cell fate determination. We observed that: i) Ago2 protein levels are strongly increased during 1,25-dihydroxyvitamin D3 (D3)-induced monocyte differentiation, whereas are down-regulated during Retinoic Acid (RA)-induced granulocyte differentiation; ii) Ago2 depletion by shRNA or small chemical compounds disrupting both miRNA-Ago2 complex interaction and Ago2 chromatin localization, results in a strong improvement of the RA-dependent myeloid differentiation. These results are bringing out that the down-regulation of Ago2 expression/functional activity is required during RA-dependent myeloid differentiation and may represent a molecular determinant for the improvement of RA-treatment response in leukemic myeloid progenitors cells

Surface plasmon resonance: a useful strategy for the identification of small molecule argonaute 2 protein binders

Surface plasmon resonance (SPR) is one of the most important techniques for the detection and the characterization of molecular interactions. SPR technology is a label-free approach for monitoring biomolecular interactions in real time. The binding of analytes to molecules immobilized on a thin metal film (ligand) determines a change in the refractive index and, therefore in the angle of extinction of light, is reflected when polarized light hits the film, monitored in real time as a change in the position of the dip in reflected intensity. Since SPR detects mass, the technique is label-free. Here, we describe the use of SPR techniques to study the interaction between Argonaute 2 and small molecular compounds selected by means of high-throughput docking screening

Small molecules targeting the miRNA-binding domain of argonaute 2: from computer-aided molecular design to RNA immunoprecipitation

The development of small-molecule-based target therapy design for human disease and cancer is object of growing attention. Recently, specific microRNA (miRNA) mimicking compounds able to bind the miRNA-binding domain of Argonaute 2 protein (AGO2) to inhibit miRNA loading and its functional activity were described. Computer-aided molecular design techniques and RNA immunoprecipitation represent suitable approaches to identify and experimentally determine if a compound is able to impair the loading of miRNAs on AGO2 protein. Here, we describe these two methodologies that we recently used to select a specific compound able to interfere with the AGO2 functional activity and able to improve the retinoic acid-dependent myeloid differentiation of leukemic cell

What is the impact of vitamin D supplementation on glycemic control in people with type-2 diabetes: a systematic review and meta-analysis of randomized controlled trails

Abstract Background There is conflicting evidence on the effect of vitamin D on glycemic control. Therefore, in the current meta-analyses, we aimed to assess the effect of vitamin D supplementation on the glycemic control of type 2 diabetes (T2D) patients. Methods We conducted a comprehensive search in electronic databases including; PubMed/Medline, Web of Science, Scopus, Embase, Cochrane Central Register of Controlled Trials (CENTRAL), and NIH’s Clinical Trials Registry, from the inception of each database up to January first, 2021. Results A total of 46 randomized controlled trials (RCTs) consisting of 2164 intervention subjects and 2149 placebo controls were included in this meta-analysis. Pooled analyses for HbA1c showed a significant change between the intervention and placebo group, the weighted mean difference (WMD)(95% confidence interval(CI)) was -0.20%(-0.29, -0.11) with P < 0.001. Analyses for assessing changes in FPG found a significant reduction in the intervention group after vitamin D supplementation, the WMD (95%CI) was -5.02 mg/dl (-6.75,-3.28) with P < 0.001. The result of pooled analyses for HOMA-IR revealed a significant change between the intervention and control group, the WMD (95%CI) was -0.42(-0.76, -0.07) with P = 0.019. The subgroup analyses showed the most efficacy in a higher dose and short intervention period and in subjects with deficient vitamin D status. Conclusion Vitamin D supplementation might be beneficial for the reduction of FPG, HbA1c, and HOMA-IR in type 2 diabetes patients with deficient vitamin D status. This effect was especially prominent when vitamin D was given in large doses and for a short period of time albeit with substantial heterogeneity between studies and a probability of publication bias

Identification of post-transcriptional regulatory networks during myeloblast-to-monocyte differentiation transition

Treatment of leukemia cells with 1, 25-dihydroxyvitamin D3 may overcome their differentiation block and lead to the transition from myeloblasts to monocytes. To identify microRNA-mRNA networks relevant for myeloid differentiation, we profiled the expression of mRNAs and microRNAs associated to the low- and high-density ribosomal fractions in leukemic cells and in their differentiated monocytic counterpart. Intersection between mRNAs shifted across the fractions after treatment with putative target genes of modulated microRNAs showed a series of molecular networks relevant for the monocyte cell fate determination, as for example the post-transcriptional regulation of the Polo-like kinase 1 (PLK1) by miR-22–3p and let-7e-5p