High glucose impairs human periodontal ligament cells migration through lowered microRNAs 221 and 222.

OBJECTIVE: To investigate the effects of miR-221 and miR-222 and high glucose on human periodontal ligament (PL) cells morphology, cytoskeleton, adhesion, and migration. BACKGROUND: Chronic hyperglycemia is common in uncontrolled diabetes mellitus (DM) and plays a central role in long-term DM complications, such as impaired periodontal healing. We have previously shown that high glucose increases apoptosis of human PL cells by inhibiting miR-221 and miR-222 and consequently augmenting their target caspase-3. However, other effects of miR-221/222 downregulation on PL cells are still unknown. METHODS: Cells from young humans' premolar teeth were cultured for 7 days under 5 or 30 mM glucose. Directional and spontaneous migration on fibronectin were studied using transwell and time-lapse assays, respectively. F-actin staining was employed to study cell morphology and the actin cytoskeleton. MiR-221 and miR-222 were inhibited using antagomiRs, and their expressions were evaluated by real-time RT-PCR. RESULTS: High glucose inhibited PL cells early adhesion, spreading, and migration on fibronectin. Cells exposed to high glucose showed reduced polarization, velocity, and directionality. They formed several simultaneous unstable and short-lived protrusions, suggesting impairment of adhesion maturation. MiR-221 and miR-222 inhibition also reduced migration, decreasing cell directionality but not significantly cell velocity. After miR-221 and miR-222 downregulation cells showed morphological resemblance with cells exposed to high glucose. CONCLUSION: High glucose impairs human PL cells migration potentially through a mechanism involving reduction of microRNA-221 and microRNA-222 expression. These effects may contribute to the impairment of periodontal healing, especially after surgery and during guided regeneration therapies.

Age-related increase of let-7 family microRNA in rat retina and vitreous.

Vitreous alterations occur from early stages and continue through the normal aging, with gradual lamellae formation and the appearance of liquefied spaces, which eventually leads to complications, such as retinal tear, retinal detachment, and intravitreal hemorrhage. The aim of the present study was to investigate the expression of let-7 miRNA family in the vitreous and retina in newborn (1-3- day-old), young adult (2-month-old), and aging (12-month-old) rats, as well as their role as regulators of vitreous components. MicroRNAs are small, non-coding RNAs that post-transcriptionally regulate gene expression. Our results showed detection of all investigated let-7 isoforms (let-7a, let-7b, let-7c, let-7d, let-7e, let-7f and let-7i) in the retina and vitreous. Although most let-7 members were significantly upregulated in the vitreous during development, only let-7b, let-7c, and let-7e followed this same expression pattern in the retina. Let-7b and -7c increased in aging vitreous as well, and were expressed in vitro by Müller glial cells and their extracellular vesicles. Moreover, let-7 targeted hyaluronan synthase 2 (Has2) mRNA, a synthesizing enzyme of hyaluronan. These observations indicate that let-7 function is important during retina and vitreous development, and that isoforms of let-7 increased with aging, potentially modulating hyaluronan content.

Anti-HER2 monoclonal antibody based-radioimmunoconjugates: Assessment of the chelating agent influence.

In the present work, the radioimmunoconjugates 111In-DTPA-trastuzumab and 177Lu-DOTA-trastuzumab were evaluated regarding the influence of the chelating agents on the physical-chemical parameters and human epidermal growth factor receptor 2 (HER2) tumor cell binding. Data showed that both chelating agents, at predetermined molar ratios (antibody:chelator - 1:10 and 1:20), did not influence the immunoconjugates integrity, the radiolabeling process and the radiolabeled antibodies stability. However, differences were observed in the lipophilic feature between DOTA and DTPA radioimmunoconjugates and in the specific binding to SK-BR-3 tumor cells (HER2 positive). Therefore, this study showed the importance of assessing the influence of chelating agents and their molar ratios in the development process of radioimmunoconjugates.

Role of microRNAs in endocrine cancer metastasis.

The deregulation of transcription and processing of microRNAs (miRNAs), as well as their function, has been involved in the pathogenesis of several human diseases, including cancer. Despite advances in therapeutic approaches, cancer still represents one of the major health problems worldwide. Cancer metastasis is an aggravating factor in tumor progression, related to increased treatment complexity and a worse prognosis. After more than one decade of extensive studies of miRNAs, the fundamental role of these molecules in cancer progression and metastasis is beginning to be elucidated. Recent evidences have demonstrated a significant role of miRNAs on the metastatic cascade, acting either as pro-metastatic or anti-metastatic. They are involved in distinct steps of metastasis including epithelial-to-mesenchymal transition, migration/invasion, anoikis survival, and distant organ colonization. Studies on the roles of miRNAs in cancer have focused mainly on two fronts: the establishment of a miRNA signature for different tumors, which may aid in early diagnosis using these miRNAs as markers, and functional studies of specific miRNAs, determining their targets, function and regulation. Functional miRNA studies on endocrine cancers are still scarce and represent an important area of research, since some tumors, although not frequent, present a high mortality rate. Among the endocrine tumors, thyroid cancer is the most common and best studied. Several miRNAs show lowered expression in endocrine cancers (i.e. miR-200s, miR-126, miR-7, miR-29a, miR-30a, miR-137, miR-206, miR-101, miR-613, miR-539, miR-205, miR-9, miR-195), while others are commonly overexpressed (i.e. miR-21, miR-183, miR-31, miR-let7b, miR-584, miR-146b, miR-221, miR-222, miR-25, miR-595). Additionally, some miRNAs were found in serum exosomes (miR-151, miR-145, miR-31), potentially serving as diagnostic tools. In this review, we summarize studies concerning the discovery and functions of miRNAs and their regulatory roles in endocrine cancer metastasis, which may contribute for the finding of novel therapeutic targets. The review focus on miRNAs with at least some identified targets, with established functions and, if possible, upstream regulation.

MiRNA-146b-5p upregulates migration and invasion of different Papillary Thyroid Carcinoma cells.

BACKGROUND: Tumor invasiveness is directly related to the ability of tumor cells to migrate and invade surrounding tissues, usually degrading extracellular matrix. Despite significant progress in the knowledge about migration and invasion, there is much more to elucidate about their regulatory mechanisms, especially in cancer cells. MicroRNAs (miRs) were recently described as important regulators of migration. Differential expression of miRs in cancer is frequently associated with progression, invasion and metastasis. In papillary thyroid carcinoma (PTC), miR-146b-5p is highly expressed and positively correlated to the degree of malignancy. METHODS: This study aimed to investigate the role of miR-146b-5p on the migratory and invasive behaviors of thyroid cells, using a non tumor rat thyroid follicular cell line (PCCl3) transfected with the miR-146b-5p genomic region, and two PTC cell lines (TPC-1 and BCPAP, bearing distinct oncogenic backgrounds), which express high levels of miR-146b-5p, after miR-146b inhibition by antagomiR and miR-146b overexpression by mimics-miR. Migration and invasion were studied by time-lapse and transwell assays (with and without Matrigel®). Gelatin degradation assays were also employed, as well as F-actin staining. RESULTS: Migration and invasion of PCCl3 were increased 2-3x after miR-146b-5p overexpression (10X) and large lamellipodia were evident in those cells. After miR-146b-5p inhibition, TPC-1 and BCPAP migration and invasion were significantly reduced, with cells showing several simultaneous processes and low polarity. Gelatin degradation was inhibited in TPC-1 cells after inhibition of miR-146b-5p, but was unaffected in BCPAP cells, which did not degrade gelatin. The inhibition of miR-146b-5p in PCCl3 also inhibited migration and invasion, and additional (exogenous) overexpression of this miR in TPC-1 and BCPAP cells increased migration and invasion, without effects on cell morphology or gelatin degradation. The overexpression of SMAD4 in BCPAP cells, a validated target of miR-146b-5p and key protein in the TGF-ß signaling pathway, inhibited migration similarly to the effects observed with the antagomiR 146b-5p. CONCLUSIONS: miR-146b-5p positively regulates migration and invasion of thyroid normal and tumor follicular cells (independently from their original mutation, either BRAF or RET/PTC), through a mechanism that involves the actin cytoskeleton but not an increased capacity of matrix degradation.

Changes in glycosaminoglycans and proteoglycans of normal breast and fibroadenoma during the menstrual cycle.

BACKGROUND: Fibroadenoma is the most common breast tumor in young women, and its growth and metabolism may be under hormonal control. In the present paper we described the proteoglycan (PG) composition and synthesis rate of normal breast and fibroadenoma during the menstrual cycle. METHODS: Samples of fibroadenoma and adjacent normal breast tissue were obtained at surgery. PGs were characterized by agarose gel electrophoresis and enzymatic degradation with glycosaminoglycan (GAG) lyases, and immunolocalized by confocal microscopy. To assess the synthesis rate, PGs were metabolic labeled by 35S-sulfate. RESULTS: The concentration of PGs in normal breast was higher during the secretory phase. Fibroadenoma contained and synthesized more PGs than their paired controls, but the PG concentrations varied less with the menstrual cycle and, in contrast to normal tissue, peaked in the proliferative phase. The main mammary GAGs are heparan sulfate (HS, 71%-74%) and dermatan sulfate (DS, 26%-29%). The concentrations of both increased in fibroadenoma, but DS increased more, becoming 35%-37% of total. The DS chains contained more ß-d-glucuronic acid (IdoUA/GlcUA ratios were >10 in normal breast and 2-7 in fibroadenoma). The 35S-sulfate incorporation rate revealed that the in vitro synthesis rate of DS was higher than HS. Decorin was present in both tissues, while versican was found only in fibroadenoma. CONCLUSIONS: In normal breast, the PG concentration varied with the menstrual cycle. It was increased in fibroadenoma, especially DS. GENERAL SIGNIFICANCE: PGs are increased in fibroadenoma, but their concentrations may be less sensitive to hormonal control.