Rnai mate

Normal culture medium was used, and cells were exposed to ambient air until transfection. The TREM2 siRNA mixtures (TREM2-siRNA1, 5′-GAGGGUGUCAUGUACUUAUTT-3′; TREM2-siRNA2,5′-CCUCUAGAUGACCAAGAUTT-3′;TREM2-siRNA3,5′-GGAAUCAAGAGACCUCCUUTT-3′) or control siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′) was transfected into primary microglia cells for 36h using siRNA or RNAi-Mate (a mixture of control siRNA and RNAi-Mate, GenePharma, Shanghai, China) using the protocol provided by the manufacturer. Following transfection, the cells were exposed to OGDR as described above.

Human cervical cancer cell line HeLa obtained from American Type Culture Collection were grown in DMEM (Hyclone, USA) at 37 °C with 5% CO₂ in air containing 10% fetal bovine serum (FBS), 2 mM glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin. For silencing the expression level of HOTAIR, 20 nM of siRNA targeted against HOTAIR (siHOTAIR-I or siHOTAIR-II) or a negative control siRNA (siNC) were transfected into HeLa cells using RNAi-mate (GenePharma, Shanghai, China). The RNA expression of HOTAIR was measured at 48 h after transfection by quantitative real-time PCR (qRT-PCR) as described below. The siRNA sequences were listed in Additional file 1: Table S1–1.
For MKL1 gene silencing, HeLa cells were transfected with 20 nM of siRNA targeted against MKL1 (siMKL1-I, siMKL1-II or siMKL1-III) and a negative control siRNA (siNC). Transfection with 20 nM siMKL1 or siNC was performed using RNAi-mate (GenePharma, China). Cells were harvested at 48 h after transfection and MKL1 gene knockdown was assessed by Western blotting. All the siRNAs were purchased from GenePharma Co. Ltd. The siRNA sequences were listed in Additional file 1: Table S1–1.

For miR-26a overexpression, miR-26a agomir (GenePharma, China) was added to the complexes at final concentrations of 50 nM. For miR-26a knockdown, the miR-26a antagomir (GenePharma, China) was added to the complexes at final concentrations of 100 nM. Knockdown of MAPK6 expression was performed using MAPK6 siRNA-smart pool (Dharmacon^TM), with all negative siRNAs (GenePharma, China) as a control. Overexpression of MAPK6 was performed by inserting full-length rat MAPK6 cDNA into the pcDNA3.3 expression vector. Twelve hours after they were seeded into the 6-well plates, cells were transfected using either RNAi-MATE (GenePharma, China) or PolyJet™ DNA in vitro Transfection Reagent (SignaGen Laboratories, China) according to the manufacture’s protocol. Transfection medium was replaced by regular cell culture medium after 6 h of transfection.