长链非编码RNAKCNK15-AS1靶向微RNA-140-5p对骨关节炎软骨细胞增殖、凋亡及炎症因子表达的影响

Effect of the long noncoding RNA KCNK15-AS1 targeting miR-140-5p on proliferation,apoptosis,and inflammatory factors in osteoarthritic chondrocytes

目的:探讨长链非编码RNA(lncRNA)KCNK15-AS1是否通过靶向微RNA-140-5p(miR-140-5p)影响骨关节炎(OA)软骨细胞增殖、凋亡,炎症因子表达及细胞外基质(ECM)合成。方法:实时荧光定量PCR(qRT-PCR)检测KCNK15-AS1和miR-140-5p在OA软骨组织中的表达。在OA软骨细胞中转染si-KCNK15-AS1,噻唑蓝(MTT)和流式细胞术分别检测细胞增殖与凋亡,蛋白质印迹法(WB)检测细胞周期蛋白D1(Cyclin D1)、p21、Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤/白血病-2(Bcl-2)、白细胞介素-6(IL-6)、IL-8、肿瘤坏死因子-α(TNF-α)、蛋白聚糖(Aggrecan)、Ⅱ型胶原(Collagen typeⅡ)、基质金属蛋白酶-3(MMP-3)、MMP-9、MMP-13的表达。双荧光素酶活性检测分析KCNK15-AS1与miR-140-5p的靶向关系。si-KCNK15-AS1和anti-miR-140-5p共转染,观察干扰miR-140-5p表达对抑制KCNK15-AS1表达诱导的OA软骨细胞增殖、凋亡,炎症因子表达及ECM合成的影响。结果:与正常软骨组织比较,OA软骨组织中KCNK15-AS1表达量明显增加(P<0.05),miR-140-5p表达量显著减少(P<0.05)。抑制KCNK15-AS1表达明显提高OA软骨细胞24 h、48 h、72 h的吸光度(OD)值和CyclinD1、Bcl-2蛋白表达量、Aggrecan、Collagen typeⅡ水平(P<0.05),显著降低细胞凋亡率、p21、Bax蛋白水平、IL-6、TNF-α、IL-8、MMP-3、MMP-9、MMP-13水平(P<0.05)。KCNK15-AS1靶向miR-140-5p抑制miR-140-5p的表达。干扰miR-140-5p表达逆转了抑制KCNK15-AS1表达对OA软骨细胞增殖、ECM合成的促进作用,对细胞凋亡、炎症因子表达的抑制作用。结论:LncRNA KCNK15-AS1通过靶向调控miR-140-5p表达影响OA软骨细胞增殖、凋亡、炎症因子表达及ECM合成。

Objective:To investigate whether the long noncoding RNA(lncRNA)KCNK15-AS1 affects proliferation,apoptosis,expression of inflammatory factor and extracellular matrix(ECM)synthesis in osteoarthritic(OA)chondrocytes by targeting miR-140-5p.Methods:quantitative real-time PCR(qRT‒PCR)was used to detect the expression levels of KCNK15-AS1 and miR-140-5p in OA cartilage tissue.Si-KCNK15-AS1 was transfected into OA chondrocytes.Cell proliferation and apoptosis were assessed using the MTT assay and flow cytometry,respectively.Western blotting was employed to evaluate the expression of cyclin D1,p21,Bax,Bcl-2,interleukin-6(IL-6),IL-8,tumor necrosis factor-alpha(TNF-α),aggrecan,collagen type Ⅱ,matrix metalloproteinase-3(MMP-3),MMP-9,and MMP-13.A dual-luciferase reporter assay was utilized to analyze the relationship between KCNK15-AS1 and miR-140-5p.Co-transfection of si-KCNK15-AS1 and anti-miR-140-5p was performed to observe the impact of interfering with miR-140-5p expression on the inhibition of KCNK15-AS1-induced proliferation,apoptosis,expression of inflammatory factors,and ECM synthesis in OA chondrocytes.Results:Compared to the normal cartilage tissue,the expression level of KCNK15-AS1 was significantly increased(P<0.05),while the expression level of miR-140-5p was markedly decreased(P<0.05)in OA cartilage tissue.KCNK15-AS1 inhibition significantly increased the optical density(OD)of OA chondrocytes at 24 h,48 h,and 72 h,as well as the protein expression levels of Cyclin D1,Bcl-2,Aggrecan and Collagen typeⅡ(P<0.05).It also significantly decreased the percentage of apoptotic cells;the protein levels of p21 and Bax;and the levels of IL-6,TNF-α,IL-8,MMP-3,MMP-9,and MMP-13(P<0.05).KCNK15-AS1 targeted miR-140-5p to inhibit its expression.Interference with miR-140-5p expression reversed the promoting effect of inhibiting KCNK15-AS1 expression on OA chondrocyte proliferation and ECM synthesis and the inhibitory effect on cell apoptosis and the expression of inflammatory factors.Conclusions:LncRNA KCNK15-AS1 affects the proliferation,apoptosis,expression of inflammatory factors and ECM synthesis in OA chondrocytes by targeting and regulating miR-140-5p expression.