Department of Genetics and Molecular Biology, Xi'an Jiaotong University College of Medicine, Xi'an, Shaanxi, People's Republic of China.
Rheumatology (Oxford). 2011 Dec;50(12):2176-86. doi: 10.1093/rheumatology/ker230. Epub 2011 Oct 4.
Primary OA and Kashin-Beck disease (KBD) show similar pathological changes in articular cartilage. The objective was to screen differentially expressed genes between OA and normal cartilage, confirm the candidate gene expression among OA, KBD and normal cartilage, and then clarify its role in vitro.
Differentially expressed genes in OA cartilage were screened by suppression subtractive hybridization (SSH) and verified by real-time quantitative PCR (Q-PCR) analysis. Heparan sulphate 6-O-sulphotransferase 2 (HS6ST2) expression was identified by Q-PCR and immunohistochemistry. After suppressing HS6ST2 by RNA interference in C28/I2 human chondrocyte line, the effects were analysed through determining the cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), the aggrecan contents by toluidine blue staining and the mRNA expression levels of SRY-type high mobility group box 9 (SOX9), AGC1, MMP3, a disintegrin and metalloproteinase domain with thrombospondin motifs 4 (ADAMTS4) and ADAMTS5 by Q-PCR.
HS6ST2 in the reverse subtraction library was identified as a down-regulated gene in OA and KBD at both mRNA and protein levels. The percentage of safranion O staining area was correlated positively with the percentage of HS6ST2-positive chondrocytes in OA and KBD cartilage. After HS6ST2-specific short interfering RNA (siRNA) transfection to C28/I2 cells, the cell viability was inhibited significantly, and the mRNA expression levels of SOX9 and AGC1 were reduced markedly, while MMP3 expression was increased significantly. CONCLUSION; HS6ST2 down-regulation was identified in both OA and KBD cartilage. The findings first suggest that HS6ST2 may participate in the pathogenesis of OA and KBD by influencing aggrecan metabolism.
原发性骨关节炎（OA）和大骨节病（KBD）的关节软骨表现出相似的病理变化。本研究旨在筛选 OA 与正常软骨之间差异表达的基因，验证 OA、KBD 和正常软骨中候选基因的表达，并在体外阐明其作用。
通过抑制性消减杂交（SSH）筛选 OA 软骨中的差异表达基因，并通过实时定量 PCR（Q-PCR）分析进行验证。通过 Q-PCR 和免疫组织化学检测硫酸乙酰肝素 6-O-磺基转移酶 2（HS6ST2）的表达。在 C28/I2 人软骨细胞系中通过 RNA 干扰抑制 HS6ST2 后，通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）测定细胞活力、甲苯胺蓝染色测定聚集蛋白含量、Q-PCR 测定 SRY 型高迁移率族框 9（SOX9）、AGC1、基质金属蛋白酶 3（MMP3）、解整合素和金属蛋白酶域 4（ADAMTS4）和 ADAMTS5 的 mRNA 表达水平，分析其作用。
在 OA 和 KBD 软骨中，HS6ST2 在 RNA 和蛋白质水平均下调。OA 和 KBD 软骨中 safranin O 染色面积与 HS6ST2 阳性软骨细胞的百分比呈正相关。用 HS6ST2 特异性短发夹 RNA（siRNA）转染 C28/I2 细胞后，细胞活力明显受到抑制，SOX9 和 AGC1 的 mRNA 表达水平明显降低，而 MMP3 的表达明显增加。
OA 和 KBD 软骨中均存在 HS6ST2 下调。研究结果首次表明，HS6ST2 可能通过影响聚集蛋白代谢参与 OA 和 KBD 的发病机制。
