Validation of Hub Genes Expression and Quantitative Real-Time PCR Analysis

To verify the expression levels of biomarker genes in patients, we collected the blood samples from patients in Zhongnan Hospital (Hubei, China). They were divided into four groups: fracture healing without T2DM(n=3), fracture healing with T2DM(n=3), non-union without T2DM(n=3), non-union with T2DM(n=3). We used red blood cell lysis solution (Solarbio, China) to lyse red blood cells. Cell precipitates were collected by centrifugation at 450 rpm for 10 min, which were further lysed with TRIzol Reagent (TIANGEN, China). RNA was extracted and purified using chloroform, isopropanol, and ethanol solutions. We used Nanodrop 2000 to detect RNA concentration and adjusted the final RNA concentration to 1000 ng/µL. Next, the RevertAid First Strand cDNA Synthesis Kit (Thermo, German) was utilized to reverse transcribe RNA into cDNA. Finally, we performed qRT-PCR analysis of cDNA by FastStart Universal SYBR Green Master (Roche, Switzerland). ANXA3 gene primers: 5’-ACCGCGCTTTGGATTAGTGT-3’(forward), 5’-CAGCATCCACTGATGGGCTA-3’(reverse). GAPDH gene primers: 5’-GAGAAGGCTGGGGCTCATTT-3’ (forward), 5’-TAAGCAGTTGGTGGTGCAGG-3’ (reverse).