lncRNA-H19通过靶向miR-106a-5p在骨关节炎软骨基质降解和钙化中的调控作用

Regulation of lncRNA-H19 in cartilage matrix degradation and calcification in osteoarthritis by targeting miR-106a-5p

目的:探讨长链非编码RNA H19通过靶向microRNA(miR)-106a-5p对骨关节炎(OA)软骨基质降解和钙化的调控作用。方法:收集临床骨关节炎软骨组织和健康软骨组织,采用实时荧光定量PCR检测软骨组织中lncRNA-H19、miR-106a-5p mRNA表达水平;将人软骨细胞系(HC-A)分为H19干扰组(si-H19)、阴性转染组(si-Control)、miR-106a-5p mimic组(miR-106a-5p)、mimic阴性对照组(miR-106a-5p-NC)、miR-106a-5p抑制剂组(inhibitor)、阴性抑制剂对照组(inhibitor-NC)组、inhibitor+si-H19和inhibitor-NC+si-H19组,检测细胞中碱性磷酸酶(ALP)、骨钙素(OCN)和骨涎蛋白(BSP)mRNA表达水平;Western blot检测软骨细胞基质金属蛋白酶(MMP)-1、MMP-13和II型α1胶原纤维(COL2A1)的水平;CCK-8检测细胞增殖率;流式细胞术检测细胞凋亡率。结果:OA软骨组织中lncRNA-H19 mRNA表达上调(P<0.05),miR-106a-5p mRNA表达下调(P<0.05);沉默lncRNA-H19可明显抑制软骨细胞凋亡,促进软骨细胞增殖(P<0.05),并下调MMP-1和MMP-13的表达水平(P<0.05),上调COL2A1的表达(P<0.05)。沉默lncRNA-H19可抑制软骨细胞ALP、OCN、BSP mRNA水平和ALP活性(P<0.05)。沉默miR-106a-5p逆转了沉默lncRNA-H19对软骨细胞基质降解和钙化标志物的抑制作用(P<0.05)。结论:lncRNA-H19可通过抑制miR-106a-5p表达,促进细胞外软骨基质的降解和钙化,从而参与OA的进展。

Objective:To investigate the regulatory effects of long-stranded non-coding RNA H19(lncRNA-H19)on cartilage matrix degradation and calcification in osteoarthritis(OA)by targeting microRNA(miR-106a-5p).Methods:Collect clinical osteoarthritis cartilage tissue and healthy cartilage tissue,the mRNA expression levels of lncRNA-H19 and miR-106a-5p in cartilage tissues were detected by real-time quantitative PCR.The cells were divided into H19 interference group(si-H19),negative transfection group(si-Control),miR-106a-5p mimic group(miR-106a-5p),mimic-negative control group(miR-106a-5p-NC),miR-106a-5p inhibitor group(inhibitor),negative inhibitor control group(inhibitor-NC),inhibitor+si-H19 and inhibitor-NC+si-H19 group.Test cells,alkaline phosphatase(ALP),osteocalcin(OCN)and bone sialoprotein(BSP)mRNA expression level.Western blot was used to detect the levels of matrix metalloproteinase(MMP-1),MMP-13 and type IIα1 collagen fibers(COL2A1).CCK-8 was used to detect cell proliferation rate,and flow cytometry was used to detect cell apoptosis rate.Results:The expression of lncRNA-H19 mRNA was up-regulated in OA cartilage tissue(P<0.05),down-regulation of miR-106a-5p mRNA expression(P<0.05).Silent lncRNA-H19 could significantly inhibit the apoptosis of chondrocytes,promote the proliferation of chondrocytes(P<0.05),and down-regulate the expression levels of MMP-1 and MMP-13(P<0.05).The expression of COL2A1 was up-regulated(P<0.05).Silenced lncRNA-H19 inhibited the mRNA levels of ALP,OCN and BSP and the activity of ALP in chondrocytes(P<0.05).Silencing miR-106a-5p significantly reversed the inhibitory effects of H19 on chondrocyte matrix degradation and calcification markers(P<0.05).Conclusion:LncRNA-H19 may promote the degradation and calcification of extracellular cartilage matrix by inhibiting the expression of miR-106a-5p,thus participating in the progression of OA.