RT-qPCR and western blotting

RNAs were reverse transcribed into cDNA using miScript Reverse Transcription Kit (Qiagen, Dusseldorf, Germany), and RT-qPCR was performed with the diluted cDNA, special primers and Fast SYBR™ Green Master Mix (Applied Biosystems). Primers for circ_0054537, PSME4, miR-640, NPTX2, U6, and β-actin were designed using Primer3 software and synthesized by GENEWIZ (Beijing, China). The primer sequences are presented in Table 2. The melting curve was drawn to ensure primer specificity, and RNA expression levels were determined by cycle threshold (CT) method [²¹]. The relative RNA expression was normalized to U6 or β-actin.

Total proteins were isolated from tissues and cells in RIPA reagent (Sigma-Aldrich), and 20 μg aliquot of that were subjected to western blotting assay to evaluate expression of special proteins related to apoptosis (Bcl2-associated X protein [Bax] [²²]), autophagy (LC3 [²³]) and metastasis (matrix metalloproteinase 9 [MMP-9] [²⁴]). The primary antibodies including anti-LC3 (4600-1-AP, 1:2,500; Proteintech, Wuhan, China), anti-NPTX2 (10,889-1-AP, 1:1,000; Proteintech), anti-MMP9 (ab228402; 1:5,000; Abcam, Shanghai, China), anti-Bax (ab216494; 1:500; Abcam), and anti-β-actin (20,536-1-AP, 1:5,000; Proteintech) were incubated overnight at 4°C, and secondary antibody anti-Rabbit IgG (SA00001-2, 1:10,000, Proteintech) was incubated for 2 h at 25°C. Eventually, proteins were detected using ECL Western Blotting Substrate (Pierce, Rockford, IL, USA). Protein expression was determined by gray density of blots analyzed on Image J software (NIH, Bethesda, MD, USA), and relative protein expression was normalized to β-actin.