Trizol reagent

Total RNA was isolated from cells using the Trizol reagent (ELK Biotechnology, Wuhan, China). Next, RNA samples were reversed transcribed into cDNA using an EntiLink™ 1st-Strand cDNA Synthesis Kit (ELK Biotechnology). Then, RT-qPCR was carried out using the EnTurbo™ SYBR Green PCR SuperMix (ELK Biotechnology) on the StepOne™ Real-Time PCR System (Thermo Fisher Scientific). The 2^−ΔΔCT method was used for data analysis. The U6 gene worked as an internal control. The primers were as follows: U6, forward, 5′-CTCGCTTCGGCAGCACAT-3′, reverse, 5′-AACGCTTCACGAATTTGCGT-3′; hsa-miR-181b-5p, forward, 5′-TCGGTGGGTCTCAACTGAATT-3′, reverse, 5′-CTCAACTGGTGTCGTGGAGTC-3′. PDHX, forward, 5′-AAGATTACCGAC TCCAGACCAA-3′, reverse: 5′-TGTCCAGGAGTTGATACTGCTG-3′. Actin, forward, 5′-GTCCACCGCAAATGCTTCTA-3′, reverse: 5′-TGCTGTCACCTTCACCGTTC-3′.

Total RNA was isolated from tissues using the Trizol reagent (ELK Biotechnology, Wuhan, China). Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to qualify the concentration of RNAs. Affymetrix Expression Console Software was used for microarray analysis. DESeq2 was used for differentially expressed gene analysis (15 (link)). DEGs were identified with p ≤ 0.05 and AveExpr ≥ 3.

Total RNA was isolated using the TRIzol reagent (ELK Biotechnology, Wuhan, China) according to the manufacturer’s protocol. cDNA was synthesized using the EntiLink™ 1st Strand cDNA Synthesis Kit. All real-time polymerase chain reactions (PCRs) were performed using EnTurbo™ SYBR Green PCR SuperMix Kit on a StepOne™ Real-Time PCR system (Life Technologies, New York, NY, USA). GAPDH was used as an internal control for normalization of the relative expression levels. Gene expression levels were calculated using the 2^–ΔΔCT method relative to that of the internal control. Primers are listed in Table 3.

Total RNA was extracted from brain tissue using TRIzol reagent (ELK Biotechnology, China) according to the manufacturer’s instructions. RNA concentrations were equalized and converted to cDNA using the EntiLink™ 1st Strand cDNA Synthesis Kit (ELK Biotechnology, China). Gene expression was measured using a StepOne™ Real-Time PCR system. The sequences of primers used in these experiments were listed in the Supplementary material.

Total RNA was extracted from HUVECs using a TRIzol^® reagent (ELK Biotechnology, Co., Ltd.) according to the manufacturer's instructions. Subsequently, total RNA was reverse transcribed into complementary DNA (cDNA) using the EntiLink™ 1st Strand cDNA Synthesis Kit (ELK Biotechnology, Co., Ltd.). qPCR was performed using the EnTurbo™ SYBR Green PCR SuperMix kit (ELK Biotechnology, Co., Ltd.) on the StepOne™ Real-Time PCR system (Thermo Fisher Scientific, Inc.). The thermocycling conditions were used as follows: 3 min at 95°C, followed by 40 cycles of 10 s at 95°C, 30 s at 58°C, and 30 s at 72°C. The primer sequences used were as follows: For β-actin, forward, 5′-GTCCACCGCAAATGCTTCTA-3′, and reverse, 5′-TGCTGTCACCTTCACCGTTC-3′; for LINC00452, forward, 5′-GTCCACTGTGAAGCTCGACG-3′, and reverse, 5′-GAGCACCACTCTGTCCACTCAG-3′; and for IGF1R, forward, 5′-GCATCATCATAACCTGGCACC-3′, and reverse, 5′-GTAAACGGCGTACTGAGTCCAG-3′. The RT-qPCR results were calculated using the 2^−ΔΔCq method (25 (link)), and β-actin was used as the internal control for normalization.