RNA结合蛋白PUM2在骨关节炎软骨细胞凋亡中的作用机制研究

The role of RNA binding protein PUM2 in the apoptosis of osteoarthritis chondrocytes

目的 研究PUM2在骨关节炎中的表达情况及其参与调控软骨细胞凋亡的作用机制。方法 软骨细胞系C28/I2利用IL-1β诱导模拟软骨细胞损伤,构建沉默PUM2(转染lenti-si-PUM2)和过表达PUM2(转染lenti-PUM2)C28/I2细胞系,real-time PCR检测C28/I2细胞PUM2和FOXO3的mRNA表达,Western blot检测C28/I2细胞PUM2和FOXO3的蛋白表达;MTT检测C28/I2细胞的活力,流式细胞术检测C28/I2细胞的凋亡,RNA pull-down检测PUM2和FOXO3的相互作用。结果 过表达PUM2促进软骨细胞的凋亡,降低细胞活性;IL-1β处理显著促进PUM2的表达,而抑制PUM2的表达则逆转了IL-1β引起的细胞凋亡增加,增加了细胞活性;PUM2能够与FOXO3 3′-UTR结合,负调控FOXO3的表达;IL-1β处理条件下,干扰FOXO3衰减Lenti-si-PUM2的促细胞活性作用,逆转Lenti-si-PUM2引起的细胞凋亡抑制作用,过表达FOXO3可缓解PUM2引起的细胞凋亡。结论 PUM2负调控FOXO3的表达,介导由IL-1β处理引起的软骨细胞活性降低及凋亡增加。

Objective To study the expression of PUM2 in osteoarthritis and its mechanism of regulating chondrocyte apoptosis.Methods Chondrocyte cell line C28/I2 was treated by IL-1βto mimic chondrocyte injury,and the silencing PUM2(transfected lenti-si-PUM2)and overexpressing PUM2(transfected lenti-PUM2)C28/I2 cell lines were constructed.The mRNA expression of PUM2 and FOXO3 in C28/I2 cells was detected by real-time PCR,and the protein expression of PUM2 and FOXO3 was detected by Western blot.MTT was used to detect cell viability of C28/I2,and cell apoptosis was detected by flow cytometry.RNA pull-down was performed to determine the interaction between PUM2 and FOXO3.Results Overexpression of PUM2 promoted cell apoptosis of chondrocytes,and inhibited cell viability.IL-1βtreatment promoted PUM2 expression,while PUM2 interference reversed cell apoptosis and viability that induced by IL-1β.PUM2 could combine with FOXO33′-UTR,and negatively regulate FOXO3 expression.Cells treated by IL-1βand futher knockdown of FOXO3 could diminish the role of Lenti-si-PUM2 on cell viability and apoptosis,while overexpression of FOXO3 could weaken cell apoptosis that induced by PUM2.Conclusion PUM2 negatively regulated the expression of FOXO3 and mediated the decrease in chondrocytes viability and the increase in apoptosis induced by IL-1βtreatment.