Effective reduction of the interleukin-1β transcript in osteoarthritis-prone guinea pig chondrocytes via short hairpin RNA mediated RNA interference influences gene expression of mediators implicated in disease pathogenesis.
Effective reduction of the interleukin-1β transcript in osteoarthritis-prone guinea pig chondrocytes via short hairpin RNA mediated RNA interference influences gene expression of mediators implicated in disease pathogenesis.
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DOI：
10.1016/j.joca.2011.09.004
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发表时间：
2011-12
影响因子：
7
通讯作者：
Bertone, A. L.
中科院分区：
文献类型：
--
作者：
Santangelo, K. S.;Bertone, A. L.
关键词：
确定一种基于病毒载体的短发夹RNA （shRNA）能够减少骨关节炎（OA）易发软骨细胞中白细胞介素-1β (IL-1β)的转录，并检测几种关键疾病介质表达模式的相应变化。筛选来自2月龄Hartley豚鼠的培养软骨细胞，以质粒为基础递送u6驱动的shRNA序列，以减少IL-1β转录物。鉴定出一种成功的质粒/shRNA敲低组合，并用于构建腺相关病毒血清型5 （AAV5）载体进行进一步评估。使用相对实时逆转录聚合酶链反应（RTPCR）定量IL-1β和其他9个基因在该靶向敲低载体转导后的体外转录变化。为了验证体外研究结果，将AAV5载体注射到一只膝盖，同时将等量的生理盐水载体（3只动物）或非靶向对照载体（3只动物）注射到另一只膝盖。使用比较CT（2−ΔΔCT）方法计算相对于对照组的折叠差异和随后的百分比基因表达水平。与模拟转导对照组和非靶向载体对照组相比，靶向敲低载体在体外均实现了IL-1β表达的统计学显著降低。使用这种靶向敲低载体，合成代谢转化生长因子-β (TGF-β)的转录水平显著增加。与模拟转导和非靶向载体对照组相比，这种靶向AAV5载体的转导也显著降低了关键炎症因子[肿瘤坏死因子-α (TNF-α)、IL-2、IL-8和IL-12]和分解代谢因子[基质金属蛋白酶（MMP）13、MMP2、干扰素-γ (IFN-γ)和诱导型氧化亚氮合成酶（iNOS）]的转录水平。在体内应用这种靶向敲低载体，相对于载体或非靶向载体对照暴露的软骨，IL-1β转录物分别减少50% （P= 0.0045）或90% （P= 0.0001）。通过RNA干扰（RNAi）技术成功地减少了IL-1β转录物。重要的是，这种改变显著影响了OA发病机制中几个主要参与者在疾病修饰方向上的转录水平。有必要研究靶向敲低AAV5载体对体内IL-1β转录物的减少所影响的其他基因表达变化。
To ascertain a viral vector-based short hairpin RNA (shRNA) capable of reducing the interleukin-1β (IL-1β) transcript in osteoarthritis (OA)-prone chondrocytes and detect corresponding changes in the expression patterns of several critical disease mediators. Cultured chondrocytes from 2-month-old Hartley guinea pigs were screened for reduction of the IL-1β transcript following plasmid-based delivery of U6-driven shRNA sequences. A successful plasmid/shRNA knockdown combination was identified and used to construct an adeno-associated virus serotype 5 (AAV5) vector for further evaluation. Relative real-time reverse transcription polymerase chain reaction (RTPCR) was used to quantify in vitro transcript changes of IL-1β and an additional nine genes following transduction with this targeting knockdown vector. To validate in vitro findings, this AAV5 vector was injected into one knee, while either an equivalent volume of saline vehicle (three animals) or non-targeting control vector (three animals) were injected into opposite knees. Fold differences and subsequent percent gene expression levels relative to control groups were calculated using the comparative CT (2−ΔΔCT) method. Statistically significant decreases in IL-1β expression were achieved by the targeting knockdown vector relative to both the mock-transduced control and non-targeting vector control groups in vitro. Transcript levels of anabolic transforming growth factor-β (TGF-β) were significantly increased by use of this targeting knockdown vector. Transduction with this targeting AAV5 vector also significantly decreased the transcript levels of key inflammatory cytokines [tumor necrosis factor-α (TNF-α), IL-2, IL-8, and IL-12] and catabolic agents [matrix metalloproteinase (MMP)13, MMP2, interferon-γ (IFN-γ), and inducible nitrous oxide synthase (iNOS)] relative to both mock-transduced and non-targeting vector control groups. In vivo application of this targeting knockdown vector resulted in a >50% reduction (P= 0.0045) or >90% (P= 0.0001) of the IL-1β transcript relative to vehicle-only or non-targeting vector control exposed cartilage, respectively. Successful reduction of the IL-1β transcript was achieved via RNA interference (RNAi) techniques. Importantly, this alteration significantly influenced the transcript levels of several major players involved in OA pathogenesis in the direction of disease modification. Investigations to characterize additional gene expression changes influenced by targeting knockdown AAV5 vector-based diminution of the IL-1β transcript in vivo are warranted.
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