Mir 125a 5p

Expression of IL-16 gene and its regulator microRNA miR-125a-5p was examined in representative specimens (8 premenopausal ovaries, 6 early menopausal ovaries, and 4 late menopausal ovaries) by quantitative real-time polymerase chain reaction (qRT-PCR). Total RNA was extracted from all specimens using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's recommendation. RNA was then measured at an optical density (OD) of 260 nm and an OD of 260/280 nm absorbance ratio ≥1.7 was used to evaluate the purity, as previously reported [30 (link)].
The expression of IL-16 messenger RNA (mRNA) and miR-125a-5p in normal ovaries and fimbriae was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The human specific IL-16 primer (QT00075138) designed by QuantiTech and miR-125a-5p designed by Applied Biosystems (Foster City, CA) were used for qRT-PCR analyses. β-Actin was used as housekeeping gene in qRT-PCR experiments. Gene expression amplification was determined using the method of the differences (δ) in cycle threshold (ΔCt) for IL-16 mRNA and miR-125a-5p according to the manufacturer's recommendation. Subtracting ΔCt from each group from the average ΔCt determined the ΔΔCt. 2^–ΔΔCt was used to calculate the fold change in the differences in IL-16 mRNA and miR-125a-5p expression levels.

miRNA qRTPCR analysis was performed using Taqman assays (Applied Biosystems) (Cat #4429795): miR-145 (Assay #2278), miR-125a-5p (Assay #2198). The endogenous control was U6 snRNA (Assay #1973). Analysis of mRNA expression for Ret and DAT used (Cat #4331182, Rn00562224_m1) and (Cat #4331182, Rn01463098_m1) respectively. GAPDH (Cat #4331182, Rn01775763_m1) was selected as an endogenous control [56 (link)]. Analyses were performed with a final volume of 10 µL of miRNA cDNA, or 20 µL of mRNA cDNA and Universal PCR Mastermix (#4369016) in a Bio-Rad CFX Connect Real-time system cycler (Bio-Rad, CA, USA). Each sample was run in triplicate. Expression was normalized (∆Ct) using the appropriate endogenous control; small nuclear RNA U6 for the miRNA, and GAPDH for the mRNA analyses. A one-tailed T-test (comparison of means) was used to test for significance, in line with the differential expression observed for the array data.

The H295R human adrenocortical cell line (a gift from Professor William Rainey, Medical College of Georgia, USA) [24 (link)] and HeLa cells (European Collection of Animal Cell Cultures, Wiltshire, UK) were maintained as previously described [8 (link)] and used between passage numbers 15 and 25. Cells were transfected using siPORT NeoFX Transfection Agent (Applied Biosystems, Warrington, UK) according to the manufacturer's instructions; H295R cells were seeded to a final density of 4.8 × 10⁵ cells/well in 6-well plates and HeLa cells to 8 × 10⁴ cells/well in 24-well plates. Pre-miR™ or Anti-miR™ molecules (miR-125a-5p: product code 12561; miR-125b-5p miR-134-3p 10341; miR-495-3p: 11526; and miR-320a-3p: 11621, Applied Biosystems) were transfected to a final concentration of 50 nM and prevalidated siRNA molecules (Dicer 1A: product code s23755; Dicer 1B: s23756, Applied Biosystems) to a final concentration of 30 nM. Reporter constructs were cotransfected with pEZX construct (500 ng) and either a Pre-miR or an Anti-miR.