Suitable reference gene selection for gene expression studies in knee osteoarthritis synovium using quantitative PCR analysis

Osteoarthritis (OA) is the leading cause of musculoskeletal disability in the elderly. Insights into the biological features of OA are obtained by characterization of the molecular features by gene expression profiling using reverse transcription-quantitative PCR (RT-qPCR). However, it has recently become evident that the use of suitable reference genes is required for appropriate normalization of this technique. Here total RNA was isolated from the synovium of 18 men and 20 women who underwent total knee arthroplasty for knee OA (KOA). We validated the expression stability of 7 candidate housekeeping genes (ACTB, B2M, GAPDH, HPRT1, RPL13A, SDHA, and YWHAZ) in the synovium of KOA with 3 commonly used algorithms (geNorm, NormFinder, and BestKeeper). Additionally, we evaluated expression profiles of the steroid hormone receptor (AR, ESR1, ESR2, GR, MR, and PR) and proinflammatory cytokines (IL1B and IL6) genes in the synovium and their correlations with the risk factors of KOA, using the most and least stable housekeeping genes for comparison. Results showed that HPRT1 was the most stable gene, whereas B2M was the least stable. RT-qPCR analysis revealed sexually dimorphic expression of AR, IL1B, and IL6; intercorrelations between steroid hormone receptor expression levels and female-specific correlations of IL1B expression with ESR1 and PR expression, IL6 expression with ESR1 and GR expression, and body mass index with AR and PR expression; and the choice of the least stable reference gene altered several correlations and statistical significances. In conclusion, HPRT1 was identified as the suitable reference gene for normalization in the OA synovium.

骨关节炎（Osteoarthritis, OA）是导致老年人肌肉骨骼残疾的首要病因。通过逆转录定量聚合酶链反应（reverse transcription-quantitative PCR, RT-qPCR）开展基因表达谱分析以表征OA的分子特征，可帮助研究者深入了解其生物学特性。不过近期研究证实，采用该技术时需选用合适的内参基因以实现准确的归一化校正。本研究从18名男性及20名因膝关节骨关节炎（knee OA, KOA）接受全膝关节置换术患者的滑膜组织中提取总RNA。我们采用geNorm、NormFinder、BestKeeper这3种常用算法，对KOA滑膜中的7个候选持家基因（ACTB、B2M、GAPDH、HPRT1、RPL13A、SDHA及YWHAZ）的表达稳定性进行了验证。此外，本研究分别选用稳定性最优与最差的持家基因作为参照，分析了滑膜组织中类固醇激素受体基因（AR、ESR1、ESR2、GR、MR及PR）与促炎细胞因子基因（IL1B及IL6）的表达谱，并评估其与KOA危险因素的相关性。研究结果显示，HPRT1是稳定性最高的基因，而B2M的稳定性最低。RT-qPCR分析表明，AR、IL1B及IL6的表达存在性别二态性；类固醇激素受体表达水平与多项指标存在关联：IL1B表达与ESR1、PR表达呈女性特异性相关，IL6表达与ESR1、GR表达呈相关，体质量指数与AR、PR表达呈相关；且选用稳定性最差的内参基因会改变部分相关性及统计学显著性。综上，本研究确定HPRT1可作为OA滑膜研究中用于归一化校正的适宜内参基因。