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This study aimed to study the roleof microRNA (miR)‐181b and its target TIMP3 in the development of diabetic nephropathy (DMN) via inhibiting the apoptosis of mesangial cells. Real‐time polymerase chain reaction (RT‐PCR) was adopted to compare the miR‐181b expression between subjects with diabetic nephropathy (DN) and normal control. In addition, luciferase assays were utilized to explore the regulatory relationship between TIMP3 and miR‐181b. Real‐time PCR and densitometry analysis were conducted to measure the levels of TIMP3 mRNA/protein in DMN or in cells treated by miR‐181b inhibitors, miR‐181b mimics, and TIMP3 siRNA. And the 3‐(4,5‐dimethythiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay was adopted to study the effect of miR‐181b on cell survival and apoptosis. miR‐181b expression was much higher in the DN group, and the results of computational analysis identified TIMP3 as a miR‐181b target. The luciferase activity of cells transfected with wild‐type TIMP3 and mutant2 TIMP3 was significantly reduced, whereas the luciferase activity of cells transfected with mutant1 TIMP3 was evidently higher. Furthermore, a negative regulatory relationship was established between TIMP3 and miR‐181b expression with a correlation efficient of −0.5351. The levels of TIMP3 mRNA/protein expression were apparently increased in the DN group. In addition, the treatment of cells with miR‐181b mimics and TIMP3 siRNA remarkably lowered the levels of TIMP3 mRNA/protein, whereas the transfection of cells with miR‐181b inhibitors notably elevated the expression of TIMP3 mRNA/protein. miR‐181b promoted the survival of cells and inhibited their apoptosis. The miR‐181b expression was related to the development of DMN and could be used as a prognosis biomarker of DMN in the patients with DM. [ABSTRACT FROM AUTHOR]

Abstract: This study aimed to investigate the relationship between the expression of microRNA (miR)‐181b, protein inhibitor of activated STAT3 (PIAS3) and STAT3, and to examine the function of the miR‐181b/PIAS3/STAT3 axis on the Warburg effect and xenograft tumour growth of colon cancer. Moreover, a positive feedback loop between miR‐181b and STAT3 that regulated the Warburg effect in colon cancer was explored. A luciferase reporter assay was used to identify whether PIAS3 was a direct target of miR‐181b. The gain‐of‐function and loss‐of‐function experiments were performed on HCT 116 cells to investigate the effect of miR‐181b/PIAS3/STAT3 on the Warburg effect and xenograft tumour growth of colon cancer, as determined by commercial kits and xenograft experiments. The relationship between the expression of miR‐181b, PIAS3 and STAT3 in HCT 116 and HT‐29 cells was determined using RT‐qPCR and Western blot. We found miR‐181b was a direct regulator of PIAS3. miR‐181b promoted the Warburg effect and the growth of colon cancer xenografts; however, these effects could be reversed by PIAS3. miR‐181b expression interacted with STAT3 phosphorylation in a positive feedback loop in colon cancer cells via regulating PIAS3 expression. In conclusion, this study for the first time demonstrated that miR‐181b contributed to the Warburg effect and xenograft tumour growth of colon cancer by targeting PIAS3. Moreover, a positive feedback loop between miR‐181b and STAT3 that regulated the Warburg effect in colon cancer was also demonstrated. This study suggested miR‐181b/PIAS3/STAT3 axis as a novel target for colon cancer treatment. [ABSTRACT FROM AUTHOR]

Background The identification of large-artery stiffness as a major, independent risk factor for cardiovascular disease-associated morbidity and death has focused attention on identifying therapeutic strategies to combat this disorder. Genetic manipulations that delete or inactivate the translin/trax microRNA-degrading enzyme confer protection against aortic stiffness induced by chronic ingestion of high-salt water (4%NaCl in drinking water for 3 weeks) or associated with aging. Therefore, there is heightened interest in identifying interventions capable of inhibiting translin/trax RNase activity, as these may have therapeutic efficacy in large-artery stiffness. Methods and Results Activation of neuronal adenosine A 2A receptors (A 2A Rs) triggers dissociation of trax from its C-terminus. As A 2A Rs are expressed by vascular smooth muscle cells (VSMCs), we investigated whether stimulation of A 2A R on vascular smooth muscle cells promotes the association of translin with trax and, thereby increases translin/trax complex activity. We found that treatment of A7r5 cells with the A 2A R agonist CGS21680 leads to increased association of trax with translin. Furthermore, this treatment decreases levels of pre-microRNA-181b, a target of translin/trax, and those of its downstream product, mature microRNA-181b. To check whether A 2A R activation might contribute to high-salt water-induced aortic stiffening, we assessed the impact of daily treatment with the selective A 2A R antagonist SCH58261 in this paradigm. We found that this treatment blocked aortic stiffening induced by high-salt water. Further, we confirmed that the age-associated decline in aortic pre-microRNA-181b/microRNA-181b levels observed in mice also occurs in humans. Conclusions These findings suggest that further studies are warranted to evaluate whether blockade of A 2A Rs may have therapeutic potential in treating large-artery stiffness.

Our study aims to explore the role of microRNA-181b (miR-181b) and TLR in the regulation of cell proliferation of human epidermal keratinocytes (HEKs) in psoriasis. Twenty-eight patients diagnosed with psoriasis vulgaris were selected as a case group with their lesional and non-lesional skin tissues collected. A control group consisted of 20 patients who underwent plastic surgery with their healthy skin tissues collected. Real-time quantitative fluorescence polymerase chain reaction (RT-qPCR), in situ hybridization and immunohistochemistry were used to detect the expressions of miR-181b and TLR4 in HEKs of healthy skin, psoriatic lesional skin and non-lesional skin respectively. The 3' untranslated region (3'UTR) of TLR4 combined with miR-181b was verified by a dual-luciferase reporter assay. Western blotting and bromodeoxyuridine were applied for corresponding detection of TLR4 expression and cell mitosis. The expression of miR-181b in HEKs of psoriatic lesional skin was less than healthy skin and psoriatic non-lesional skin. In psoriatic lesional and non-lesional skin, TLR4-positive cell rates and the number of positive cells per square millimetre were higher than healthy skin. The dual-luciferase reporter assay verified that miR-181b targets TLR4. HEKs transfected with miR-181b mimics had decreased expression of TLR4, along with the decrease of mitotic indexes and Brdu labelling indexes. However, HEKs transfected with miR-181b inhibitors showed increased TLR4 expression, mitotic indexes and Brdu labelling indexes. HEKs transfected with both miR-181b inhibitors and siTLR4 had decreased mitotic indexes and Brdu labelling indexes. These results indicate that miR-181b can negatively regulate the proliferation of HEKs in psoriasis by targeting TLR4., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)