Down-regulated HS6ST2 in osteoarthritis and Kashin-Beck disease inhibits cell viability and influences expression of the genes relevant to aggrecan metabolism of human chondrocytes
Down-regulated HS6ST2 in osteoarthritis and Kashin-Beck disease inhibits cell viability and influences expression of the genes relevant to aggrecan metabolism of human chondrocytes
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DOI：
10.1093/rheumatology/ker230
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发表时间：
2011-12-01
期刊：
影响因子：
5.5
通讯作者：
Lu, Shemin
中科院分区：
文献类型：
--
作者：
Wang, Wei;Zhong, Bo;Lu, Shemin
方法。采用抑制减法杂交（suppression subtractive hybridization， SSH）筛选OA软骨中差异表达基因，并用实时定量PCR （real-time quantitative PCR, Q-PCR）进行验证。采用Q-PCR和免疫组化检测硫酸肝素6- o -硫转移酶2 （HS6ST2）的表达。采用RNA干扰法抑制C28/I2人软骨细胞系HS6ST2后，采用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑（MTT）法测定细胞活力，甲苯胺蓝染色法测定聚集蛋白含量，q - pcr法测定sry型高迁移率组框9 （SOX9）、AGC1、MMP3、溶栓素和金属蛋白酶结构域4 （ADAMTS4）和ADAMTS5 mRNA表达量。反向减文库中的HS6ST2在OA和KBD中mRNA和蛋白水平均被下调。safranion O染色面积百分比与OA和KBD软骨中hs6st2阳性软骨细胞百分比呈正相关。转染hs6st2特异性短干扰RNA （siRNA）后，C28/I2细胞活力明显受到抑制，SOX9和AGC1 mRNA表达水平明显降低，MMP3表达水平明显升高。在OA和KBD软骨中均发现HS6ST2下调。研究结果首次提示HS6ST2可能通过影响聚集蛋白代谢参与OA和KBD的发病机制。
Methods. 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.Results. 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.