Ticular tissue fixative (Servicebio, Wuhan, China) for 24 h then transferred
Ticular tissue fixative (Servicebio, Wuhan, China) for 24 h and then transferred to 70 ethanol for storage. Soon after embedding of tissues in paraffin, 5-m thick sections had been obtained. Tissue morphology was observed applying hematoxylin and eosin (HE) staining in line with the manufacturer’s guidelines (Solarbio, Beijing, China).TUNEL assayParaffin-embedded testicular tissue sections had been used for the TUNEL assay to establish apoptotic cells in tissues. TUNEL-positive cells were detected utilizing a DNA Fragmentation Detection Kit (Merck Millipore, Billerica, MA, USA), as outlined by the advisable protocol.Cell culture, transfection, and reagentsR2C cells purchased in the China Infrastructure of Cell Line Sources (Beijing, China) have been transfected with miRNA mimics for gain-of-function experiments, and miRNA inhibitors (GenePharma, Shanghai, China) for loss-of-function experiments. Cell transfection was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s guidelines. miR504 mimic (sense:5-AGACCCUGGUCUGCA CUCUGUC-3, antisense: 5-CAGAGUGCAGACCAG GGUCUUU-3), mi504 inhibitor (5-GACAGAGUG CAGACCAGGGUCU-3), miR935 mimic (sense:5-CCA GUUACCGCUUCCGCUACCGC-3, antisense: 5-GGU AGCGGAAGCGGUAACUGGUU-3), mi935 inhibitor (5-GCGGUAGCGGAAGCGGUAACUGG-3), mimicNC (sense:5-UUCUCCGAACGUGUCACGUTT-3, antisense: 5-ACGUGACACGUUCGGAGAATT-3) and inhibitor NC(5-CAGUACUUUUGUGUAGUACAA-3) have been transfected at a final concentration of 50 nM for 24 h. Cell culture was maintained in DMEM (GIBCO, Grand Island, NY, USA) supplemented with ten FBS (GIBCO,) inside a humidified air incubator with 5 CO2 at 37 . Leydig cells were exposed to regular (5 mM) or moderately high (15 mM) or higher (30 mM) glucose concentrations for 48 h in accordance with the preceding study (Karpova et al. 2020).Realtime quantitative PCR (RTqPCR)extracted from blood using a QIAamp RNA Blood Mini Kit (QIAGEN, Duesseldorf, P2Y2 Receptor Agonist site Germany). Total RNA from tissues and cells was extracted working with a TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Tokyo, Japan) following the manufacturer’s guidelines. For the quantification of miRNA by qPCR, reverse transcription and Topo II Inhibitor Accession RT-qPCR had been performed applying the Mir-X miRNA RT-qPCR TB GreenKit (TaKaRa) and normalized to U6. The complete sequence of mature miRNA was employed as miRNA specific, 5 primer (miR-504, 5-AGACCCUGG UCUGCACUCUGUC-3′ miR-935, 5-CCAGUUACC GCUUCCGCUACCGC-3; miR-484, 5-UCAGGCUCA GUCCCCUCCCGAU-3; miR-301a-5p, 5-GCUCUG ACUUUAUUGCACUAC-3; U6, 5-CGTTCACGAATT TGCGTGTCAT-3). The 3 primer utilized within the qPCR was the mRQ three primer supplied together with the kit. Reverse transcription of mRNA was performed utilizing the PrimeScriptTM RT Master Mix (TaKaRa), whilst RT-qPCR was performed making use of the One particular Step TB GreenPrimeScriptTM RT-qPCR Kit II (TaKaRa) and normalized to -actin. The primers utilised were as follows: MEK5 forward primer 5-TCGTGCCATGGAGAACCA-3, reverse primer 5-CGCGCCACTATTTGGAATCT-3; MEF2C forward primer 5-ACCACCACCCCATCGAGATA-3, reverse primer 5-GGAGTGGAATTCGTTCCGGT-3; -actin forward primer 5-ATGGATGACGATATCGCTGC-3, reverse primer 5-CTTCTGACCCATACCCACCA-3. The 2Cq approach was employed to compare the relative levels of expression of miRNA and mRNA (Livak and Schmittgen 2001).Western blot analysisBlood samples were obtained from individuals with diabetes and healthful donors at Shenzhen University Common Hospital. This project was approved by the ethics committee of the Shenzhen University. Total RNA wasWestern blot analysis was performed accordin.