β actin

Total RNA from breast cancer cells was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A transcriptional First-Strand cDNA Synthesis kit (Takara Bio, Inc.) was used for the reverse transcription, and qPCR was performed using SYBR Green PCR Master Mix (Takara Bio, Inc.). The thermocycling conditions of the PCR were as follows: 15 sec at 95°C, and 60 sec at 60°C for 45 cycles. Primers for eIF5A2, β-actin, miR-33a-5p and U6 were obtained from GeneCopoeia, Inc. U6 was used as the internal control for miR-33a-5p. β-actin was used as the internal control for eIF5A2. Data were analyzed using the 2^−ΔΔCq method (17 (link)). The primer sequences were as follows: miR-33a-5p: 5′-GTGCATTGTAGTTGCATTGCA-3′; eIF5A2: Forward, 5′-TATGCAGTGCTCGGCCTTG-3′; reverse, 5′-TTGGAACATCCATGTTGTGAGTAGA-3′; β-actin: Forward, 5′-TTCCAGCCTTCCTTCCTG-3′; reverse, 5′-CTTTGCGGATGTCCACGT-3′.

A total RNA extraction kit (Tiangen Biotech, Beijing, China) was used to extract total RNA from BV2 cells pretreated with VD11 for 12 hours. The RNA purity and concentration were measured using an ultra-microspectrophotometer (Thermo Fisher Scientific). The cDNA was reverse transcribed using a SureScript kit (GeneCopoeia, Guangzhou, China) in a total volume of 20 μL at 25°C for 5 minutes, 42°C for 15 minutes, and 85°C for 5 minutes, and then held at 4°C. A BlazeTaq kit was used for polymerase chain reaction (PCR) amplification (GeneCopoeia, Guangzhou, China). β-Actin (5′-TCA TCA CTA TTG GCA ACG AGC-3′, 5′-AAC AGT CCG CCT AGA AGC AC-3′), NGF (5′-TCT ATA CTG GCC GCA GTG AG-3′, 5′-GGA CAT TGC TAT CTG TGT ACG G-3′), and BDNF (5′-CTC CTG GGT TCC TGA GCA TC-3′, 5′-TTC ACT CCC TGA GTC ACA GC-3′) primers were purchased from GeneCopoeia. A Life Technologies thermocycler (Thermo, Waltham, MA, USA) was used to perform the qPCR reaction. The 2^–ΔΔCt method was used to calculate the mRNA expression of NGF and BDNF, which was normalized to β-Actin (Yin et al., 2021).