lncRNA-H19/miR-140-5p 在骨关节炎软骨基质降解和钙化中的调控作用。
Regulation of lncRNA-H19/miR-140-5p in cartilage matrix degradation and calcification in osteoarthritis.
机构信息
Department of Nephrology Endocrinology and Rheumatology, The 960th Hospital of the Chinese People's Liberation Army, Zibo 255300, China.
出版信息
Ann Palliat Med. 2020 Jul;9(4):1896-1904. doi: 10.21037/apm-20-929. Epub 2020 Jun 16.
BACKGROUND
To study the effect of long non-coding RNA H19/microRNA (miR)-140-5p regulatory axis on the degradation and calcification of osteoarthritis (OA) cartilage matrix.
METHODS
Twenty cases of clinical OA cartilage tissue and healthy cartilage tissue were collected. Construction of small interfering RNA vector (si-H19) and negative control (si-Control), miR-140-5p mimic and negative control mimic-NC, miR-140-5p inhibitor, and negative control inhibitor-NC transfected human chondrocytes HC-A cells. Chondrocytes were categorized into classes of si-H19, si-Control, mimic, mimic-NC, inhibitor, NC inhibitor, inhibitor + si-H19, NC inhibitor + si-H19. The expression levels of H19, miR-140-5p, alkaline phosphatase (ALP), osteocalcin (OCN), and bone sialoprotein (BSP) in cartilage tissue and chondrocytes were detected using real-time fluorescent quantitative PCR (RT-qPCR). In each group of chondrocytes, Western blotting was used to detect the levels of matrix metalloproteinase (MMP)- 1, MMP-13, and type II α1 collagen fibers (COL2A1). CCK-8 was used to detect the cell proliferation rate, and flow cytometry was used to detect the cell apoptosis rate.
RESULTS
H19 was up-regulated in OA cartilage tissue (P<0.05); Silencing H19 not only prevented chondrocyte apoptosis but also facilitated chondrocyte proliferation (both P<0.05), down-regulated MMP1 and MMP-13 expression levels (both P<0.05), and up-regulated COL2A1 expression rates (P<0.05). Also, silenced H19 inhibited the mRNA level of ALP, OCN, and BSP and ALP activity of chondrocytes (all P<0.05). There was a negative correlation between miR-140-5p and H19 in OA cartilage tissue and chondrocytes (r=0.98, P<0.001). Silenced miR-140-5p significantly reversed the inhibitory effects of silenced H19 on matrix degradation and calcification markers in chondrocytes (all P<0.05).
CONCLUSIONS
The present study showed that the H19/ miR-140-5p regulatory axis could affect the degradation and calcification of the cartilage matrix in OA cells.
背景
研究长链非编码 RNA H19/微小 RNA (miR)-140-5p 调控轴对骨关节炎 (OA) 软骨基质降解和钙化的影响。
方法
收集 20 例临床 OA 软骨组织和健康软骨组织。构建小干扰 RNA 载体 (si-H19) 和阴性对照 (si-Control)、miR-140-5p 模拟物和阴性对照模拟物-NC、miR-140-5p 抑制剂和阴性对照抑制剂-NC，转染人软骨细胞 HC-A 细胞。将软骨细胞分为 si-H19、si-Control、mimic、mimic-NC、inhibitor、NC-inhibitor、inhibitor+si-H19、NC-inhibitor+si-H19 组。采用实时荧光定量 PCR (RT-qPCR)检测软骨组织和软骨细胞中 H19、miR-140-5p、碱性磷酸酶 (ALP)、骨钙素 (OCN) 和骨涎蛋白 (BSP) 的表达水平。用 Western blot 检测各组软骨细胞中基质金属蛋白酶 (MMP)-1、MMP-13 和 II 型α1 胶原纤维 (COL2A1) 的水平。CCK-8 法检测细胞增殖率，流式细胞术检测细胞凋亡率。
结果
OA 软骨组织中 H19 表达上调 (P<0.05)；沉默 H19 不仅能阻止软骨细胞凋亡，还能促进软骨细胞增殖 (均 P<0.05)，下调 MMP1 和 MMP-13 的表达水平 (均 P<0.05)，上调 COL2A1 的表达率 (P<0.05)。此外，沉默 H19 抑制软骨细胞中 ALP、OCN 和 BSP 的 mRNA 水平和 ALP 活性 (均 P<0.05)。OA 软骨组织和软骨细胞中 miR-140-5p 与 H19 呈负相关 (r=0.98，P<0.001)。沉默 miR-140-5p 显著逆转了沉默 H19 对软骨细胞基质降解和钙化标志物的抑制作用 (均 P<0.05)。
结论
本研究表明，H19/miR-140-5p 调控轴可能影响 OA 细胞中软骨基质的降解和钙化。
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