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.
Keywords: TIMP3; apoptosis; diabetic nephropathy; mesangial cells; microRNA-181b.
© 2019 Wiley Periodicals, Inc.