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Hyperglycemia and fibroblast migration in 3-D extracellular matrices, matrix composition and remodeling - potential roles of microRNAs 31, 196a e 29b


Deficient wound healing in diabetic patients is one of the most common complications of hyperglycemia (HG) and is associated with microangiopathy, neuropathy and fibroblasts dysfunction. Some studies using fibroblasts from diabetic patients or fibroblasts cultured under conditions that simulate HG showed changes in cell proliferation and ECM protein synthesis. Few studies, however, have addressed the effects of HG on the migratory behavior of these cells, on the physical properties of the ECM and on its remodeling, which are essential for proper tissue healing. Previous studies from this group showed that fibroblasts derived from dermis of diabetic rats or exposed in vitro to a high concentration of glucose migrate less on two-dimensional substrates (2-D) consisting of fibronectin or collagen I. Under these conditions the cells showed reduced speed and directionality, increased number of simultaneous non-productive cellular protrusions (unstable) and reduced maturation of cell adhesions to the ECM. These effects were reversed in the presence of the antioxidant N-acetylcysteine (NAC). The effects of HG on migration of fibroblasts in three-dimensional environments (3-D), which better simulate in vivo conditions, as well as its effects on the synthesis of ECM, which in turn affects cell migration, are not known. Recently, microRNAs (miRs) have been demonstrated as important modulators of cell migration (miR-31, for example) and ECM synthesis (miR-196a and miR-29b, for example). MicroRNAs are post-transcriptional regulators of gene expression by promoting the degradation of target mRNAs, or blocking their translation. We have previously demonstrated that the expression of miR-31 increased by 3 times in fibroblasts from diabetic animals compared to fibroblasts from control animals. In NIH-3T3 fibroblasts, HG increased expression of miR-31 by 50% after 3 days of exposure. In these cells, treatment with the antioxidant NAC prevented the increase of miR-31 by keeping it at normal levels and avoiding the increase in the number of non productive protrusions. Expression of exogenous miR-31 in NIH-3T3 increased the number of simultaneous unstable cellular protrusions and reduced cell directionality, without effects on cellular speed (Gomes, 2015). A hypothesis of this study is that high glucose regulates the expression of miR-31 by generating reactive oxygen species. This miR, in turn, negatively regulates the expression of proteins important for cell migration. Other potentially relevant miRs that regulate the ECM, such as miR-196a and miR-29b, altered during the final phase of ECM remodeling in skin wound healing in diabetics, may also be less expressed in fibroblasts, changing the proportion collagen III/collagen I and fibronectin. Thus, this study aims to: 1) In NIH-3T3 fibroblasts, evaluate the expression of miR-31 (miR-31-5p) targets (predicted by bioinformatics) that may be responsible for the phenotype observed in these cells maintained under high glucose: RhoA, Radixin, Rock1, Rock2, integrins ±v and ±5. To validate these targets by luciferase assays and, in cells exposed to high glucose, inhibit the expression of miR-31-5p using antagomir and evaluate a potential reversal (even partial) of the migratory phenotype and 2) using fibroblasts derived from human dermis, evaluate the effects of HG on cell migration in three-dimensional environments (3-D) and on ECM production and remodeling. The expression of ECM and adhesion molecules will be studied by RT PCR Array (84 genes). Three-dimensional cultures will be performed inin cell-derived ECM (CDM) and collagen I / fibronectin gels. In CDM, changes in biochemical composition and organization promoted by HG will also be evaluated. Proteoglycans and glicosaminoglycans (including hyaluronan) will be analyzed.The potential regulation of ECM by microRNAs miR-29b and miR-196a will be evaluated in cultures, both in cells and in extracellular millieu (secreted microvesicles). (AU)

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(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
MONTEIRO, MARIANA M.; LIMA, CILENE R.; GOMES, CIBELE C.; CRUZ, MARIO C.; HORLIANA, ANNA C. R. T.; SANTOS, MARINILCE F.. Lowered Expression of MicroRNAs 221 and 222 Mediate Apoptosis Induced by High Glucose in Human Periodontal Ligament Cells. Cell Biochemistry and Biophysics, v. 78, n. 3, SI, . (15/19645-7, 12/24508-0, 12/03990-9, 11/18936-7)
LIMA, CILENE REBOUCAS; GOMES, CIBELE CRASTEQUINI; SANTOS, MARINILCE FAGUNDES. Role of microRNAs in endocrine cancer metastasis. Molecular and Cellular Endocrinology, v. 456, n. C, SI, p. 62-75, . (15/19645-7, 11/18936-7)

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