Effects of LncRNA NEF on differentiation and expression of related proteins of osteoblasts and osteoclasts
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摘要:目的
探讨长链非编码RNA NEF(LncRNA NEF)在骨质疏松症大鼠中的表达及其对成骨细胞和破骨细胞分化的影响。
方法通过鼠尾悬吊法制备骨质疏松大鼠为实验组(n=6), 另设置正常大鼠为对照组(n=6), 采用双能X射线骨密度仪与微计算机断层扫描技术测定2组大鼠股骨骨量及结构; 采用实时荧光定量聚合酶链反应(qRT-PCR)法检测2组大鼠股骨组织中LncRNA NEF、碱性磷酸酶(ALP)、抗酒石酸酸性磷酸酶(TRAP)、组织蛋白酶K(CTSK)的水平变化。体外培养及诱导分化大鼠前成骨细胞系MC3T3-E1细胞、巨噬细胞系RAW264.7细胞。ALP活性检测试剂盒测定成骨细胞ALP活性，茜素红染色、TRAP染色分别观测成骨分化、破骨分化效果。探讨沉默LncRNA NEF表达对MC3T3-E1细胞ALP、LncRNA NEF表达、成骨效果的影响，以及对RAW264.7细胞CTSK mRNA及其蛋白表达和破骨效果的影响。
结果实验组大鼠股骨骨密度、骨体积分数(BV/TV)、骨小梁数量(Tb.N)、骨小梁厚度(Tb.Th)低于对照组，骨小梁间距(Tb.Sp)高于对照组，差异有统计学意义(P < 0.05)。实验组大鼠股骨组织中LncRNA NEF、ALP水平低于对照组, TRAP、CTSK水平高于对照组，差异有统计学意义(P < 0.05)。Pearson相关分析显示，在骨质疏松症大鼠中LncRNA NEF与ALP表达呈正相关(r=0.532, P < 0.05), 与TRAP、CTSK表达呈负相关(r=-0.624、-0.573，P < 0.05)。MC3T3-E1细胞成骨分化过程中，与第0天相比，第15天LncRNA NEF水平、ALP活性上调，差异有统计学意义(P < 0.05); RAW264.7细胞破骨分化过程中，与第0天相比，第6天LncRNA NEF水平下调, CTSK mRNA及其蛋白水平上调，差异有统计学意义(P < 0.05)。敲低LncRNA NEF表达可显著抑制MC3T3-E1细胞成骨分化，促进RAW264.7细胞破骨分化。
结论LncRNA NEF在成骨分化过程中异常低表达，在破骨分化过程中异常高表达, LncRNA NEF沉默可能通过阻滞成骨细胞分化及促进破骨细胞分化而诱发骨质疏松。
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关键词:
- 长链非编码RNA NEF /
- 骨质疏松症 /
- 成骨细胞 /
- 破骨细胞 /
- 分化
Abstract:ObjectiveTo investigate the expression of long non-coding RNA NEF (LncRNA NEF) in osteoporosis rats and its effect on the differentiation of osteoblasts and osteoclasts.
MethodsOsteoporotic rats were prepared as the experimental group (n=6) by the tail suspension method, and another normal control group (n=6) was set up.The bone mass and structure of rat femur in two groups were measured by dual energy X-ray absorptiometry and microcomputer tomography; the quantitative real-time polymerase chain reaction (qRT-PCR) method was used to detect the changes of LncRNA NEF, ALP, tartrate-resistant acid phosphatase (TRAP), CTSK levels in the femoral tissues of the two groups of rats.The MC3T3-E1 cells of the rat pre-osteoblast cell line and RAW264.7 cells of the macrophage cell line were cultured and induced differentiation in vitro. The ALP activity detection kit was used to determine the ALP activity of osteoblasts. Alizarin red staining and TRAP staining were used to observe the effects of osteogenic differentiation and osteoclast differentiation, respectively. The effects of silencing LncRNA NEF expression on ALP and LncRNA NEF expression and osteogenic effects in MC3T3-E1 cells, as well as CTSK mRNA and its protein expression and osteoclast effect in RAW264.7 cells were explored.
ResultsThe bone mineral density, bone volume fraction (BV/TV), number of trabeculae(Tb.N) and thickness of trabeculae (Tb.Th) in the experimental group were significantly lower, and the trabecular spacing (Tb.Sp) was significantly higher than that in the control group (P < 0.05).The levels of LncRNA NEF and ALP in the femur of rats in the experimental group were significantly, while the levels of TRAP and CTSK were significantly higher than those in the control group (P < 0.05). Pearson correlation analysis showed that LncRNA NEF was positively correlated with ALP expression (r=0.532, P < 0.05), and negatively correlated with TRAP and CTSK expression in osteoporosis rats (r=-0.624, -0.573, P < 0.05). During osteogenic differentiation of MC3T3-E1 cells, the level of LncRNA NEF and ALP activity were significantly up-regulated on day 15 compared with day 0 (P < 0.05); during osteoclast differentiation of RAW264.7 cells, the level of LncRNA NEF was significantly down-regulated, and the CTSK mRNA and its protein levels were significantly up-regulated on day 6 compared with day 0 (P < 0.05). Knockdown of the expression of LncRNA NEF could significantly inhibit the osteogenic differentiation of MC3T3-E1 cells, and promote the osteoclast differentiation of RAW264.7 cells.
ConclusionLncRNA NEF expression is abnormally low during osteogenic differentiation and abnormally high during osteoclast differentiation.LncRNA NEF silencing may induce osteoporosis by blocking osteoblast differentiation and promoting osteoclast differentiation.
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Keywords:
- long non-coding RNA NEF /
- osteoporosis /
- osteogenesis /
- osteoclasts /
- differentiation
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表 1 引物序列
基因 正向引物5′—3′ 反向引物5′—3′ LncRNA NEF TGGAGTTGGGGGTTTCTGTA TTCAGTGCACAGGTTCAAGC ALP CTGATGTGGAATACGAACTGGA AGTGGGAATGCTTGTGTCTGG TRAP CGCCAAGCAAATCGGCAAAG CGGTCACTGAACACGTCCTCGA CTSK GTCAACGGATTTGGTCTGTATT AGTCTTCTGGGTGGCAGTGAT β-actin GCAGAAGTGCGAAGAGGAGG GCTTGATGGAGTTGTCGGTGTA 
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表 2 大鼠股骨结构参数比较(x±s)
组别 股骨骨密度/(g/cm2) (BV/TV)/% Tb.N/(1/mm) Tb.Th/mm Tb.Sp/mm 对照组(n=6) 0.18±0.03 0.52±0.08 3.94±0.35 0.21±0.03 0.28±0.06 实验组(n=6) 0.14±0.02* 0.27±0.07* 2.01±0.31* 0.05±0.01* 0.56±0.11* BV/TV: 骨体积分数; Tb.N: 骨小梁数量; Tb.Th: 骨小梁厚度; Tb.Sp: 骨小梁间距。与对照组比较, *P < 0.05。
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表 3 LncRNA NEF、ALP、TRAP、CTSK在大鼠股骨组织中的表达情况(x±s)
组别 LncRNA NEF/β-actin ALP/β-actin TRAP/β-actin CTSK/β-actin 对照组(n=6) 1.01±0.15 1.00±0.12 1.05±0.14 1.02±0.12 实验组(n=6) 0.50±0.07* 0.62±0.08* 1.54±0.17* 1.63±0.18* ALP: 碱性磷酸酶; CTSK: 组织蛋白酶K; TRAP: 抗酒石酸酸性磷酸酶。与对照组比较, *P < 0.05。
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表 4 骨质疏松症大鼠LncRNA NEF与ALP、TRAP、CTSK相关性分析
指标 LncRNA NEF r P ALP 0.532 < 0.001 TRAP -0.624 < 0.001 CTSK -0.573 < 0.001
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表 5 成骨分化过程中ALP活性与LncRNA NEF水平表达变化(x±s)(n=6)
时点 ALP活性/(U/mg) LncRNA NEF/β-actin 第0天 6.66±0.99 0.99±0.15 第15天 15.78±2.37* 1.53±0.23* 与第0天比较, *P < 0.05。
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表 6 成骨分化过程中CTSK mRNA及其蛋白表达与LncRNA NEF水平表达变化(x±s)(n=6)
时点 CTSK mRNA CTSK/β-actin LncRNA NEF/β-actin 第0天 1.00±0.15 0.79±0.12 1.02±0.16 第6天 1.45±0.22* 1.16±0.17* 0.61±0.09* 与第0天比较, *P < 0.05。
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表 7 下调LncRNA NEF表达对成骨诱导ALP活性及LncRNA NEF的影响(x±s)
组别 ALP活性/(U/mg) LncRNA NEF/β-actin NC-shRNA组(n=6) 11.97±1.78 1.01±0.15 shRNA-LncRNA NEF组(n=6) 8.95±1.34* 0.47±0.07* 与NC-shRNA组比较, *P < 0.05。
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表 8 下调LncRNA NEF表达对破骨诱导CTSK mRNA与蛋白及LncRNA NEF影响(x±s)
组别 CTSK mRNA CTSK/β-actin LncRNA NEF/β-actin NC-shRNA组(n=6) 1.02±0.16 0.95±0.12 1.03±0.15 shRNA-LncRNA NEF组(n=6) 1.69±0.25* 1.45±0.22* 0.37±0.06* 与NC-shRNA组比较, *P < 0.05。
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[1] OVERGAARD K, HANSEN M A, JENSEN S B, et al. Effect of salcatonin given intranasally on bone mass and fracture rates in established osteoporosis: a dose-response study[J]. BMJ, 1992, 305(6853): 556-561.
[2] YAO R H, NISHⅡ K, KITO T, et al. A novel device to prevent osteoporosis by promoting bone metabolism using a newly developed double-loading stimulation with vibration and shaking[J]. Okajimas Folia Anat Jpn, 2019, 96(1): 13-21. doi: 10.2535/ofaj.96.13
[3] 王如梦, 彭山萍, 徐宏. 长非编码RNA对骨质疏松症中成骨和破骨细胞分化及功能的调节作用[J]. 南昌大学学报: 医学版, 2021, 61(2): 89-93. doi: 10.3969/j.issn.1006-0448.2021.02.009 [4] LIU C, CAO Z, BAI Y, et al. LncRNA AK077216 promotes RANKL-induced osteoclastogenesis and bone resorption via NFATc1 by inhibition of NIP45[J]. J Cell Physiol, 2019, 234(2): 1606-1617. doi: 10.1002/jcp.27031
[5] CHEN X, YANG L, GE D W, et al. Long non-coding RNA XIST promotes osteoporosis through inhibiting bone marrow mesenchymal stem cell differentiation[J]. Exp Ther Med, 2019, 17(1): 803-811.
[6] MA X Y, GUO Z X, GAO W S, et al. LncRNA-NEF is downregulated in postmenopausal osteoporosis and is related to course of treatment and recurrence[J]. J Int Med Res, 2019, 47(7): 3299-3306. doi: 10.1177/0300060519847854
[7] 王鹏, 夏晓枫, 左斌, 等. 槲皮素对大鼠废用性骨质疏松的预防作用及ERK1/2-MAPK信号通路的影响[J]. 解放军医药杂志, 2020, 32(11): 6-10. doi: 10.3969/j.issn.2095-140X.2020.11.002 [8] 章坤, 时哲敏, 任怡, 等. 长链非编码RNA Kcnqlot1促进成骨细胞分化和抑制破骨细胞分化[J]. 南方医科大学学报, 2021, 41(1): 31-38. https://www.cnki.com.cn/Article/CJFDTOTAL-DYJD202101004.htm [9] ZHENG H Y, FENG H T, ZHANG W Z, et al. Targeting autophagy by natural product Ursolic acid for prevention and treatment of osteoporosis[J]. Toxicol Appl Pharmacol, 2020, 409: 115271.
[10] SHAH G M, GONG H S, CHAE Y J, et al. Evaluation and management of osteoporosis and sarcopenia in patients with distal radius fractures[J]. Clin Orthop Surg, 2020, 12(1): 9-21.
[11] 朱治同, 李泽平, 董立明. 培哚普利对糖皮质激素诱导的MSCs凋亡及成骨分化的影响[J]. 中国骨质疏松杂志, 2022, 28(2): 230-236. https://www.cnki.com.cn/Article/CJFDTOTAL-ZGZS202202013.htm [12] 乔建新, 刘明, 刘熙鹏. miR-29a对胶质母细胞瘤细胞增殖、迁移、侵袭及PTEN/AKT/GSK-3β通路的影响[J]. 现代医学, 2021, 49(8): 884-892. https://www.cnki.com.cn/Article/CJFDTOTAL-TDYX202108011.htm [13] 刘琳. DXA影像评价与骨密度测定联合诊断原发性骨质疏松症的临床探讨[J]. 影像研究与医学应用, 2019, 3(17): 50-51. https://www.cnki.com.cn/Article/CJFDTOTAL-YXYY201917023.htm [14] KIM E N, KIM Y G, LEE J H, et al. 6, 7, 4'-Trihydroxyflavone inhibits osteoclast formation and bone resorption in vitro and in vivo[J]. Phytother Res, 2019, 33(11): 2948-2959.
[15] ABDI E, LATIFI-NAVID S, ABDI F, et al. Emerging circulating MiRNAs and LncRNAs in upper gastrointestinal cancers[J]. Expert Rev Mol Diagn, 2020, 20(11): 1121-1138.
[16] ZENG J, WANG C, DING Z H, et al. Identification and functional prediction of lncRNAs during cassava post-harvest physiological deterioration[J]. Agron J, 2020, 112(6): 4914-4925.
[17] SONG X, LIU Z Y, YU Z Y. LncRNA NEF is downregulated in triple negative breast cancer and correlated with poor prognosis[J]. Acta Biochim Biophys Sin: Shanghai, 2019, 51(4): 386-392.
[18] YANG Q L, YU H Y, YIN Q S, et al. lncRNA-NEF is downregulated in osteosarcoma and inhibits cancer cell migration and invasion by downregulating miRNA-21[J]. Oncol Lett, 2019, 17(6): 5403-5408.
[19] XIE J Q, LIU Y, DU X L, et al. TGF-β1 promotes the invasion and migration of papillary thyroid carcinoma cells by inhibiting the expression of lncRNA-NEF[J]. Oncol Lett, 2019, 17(3): 3125-3132.
[20] CUI X Y, FANG N, CUI Y, et al. Long non-coding RNA NEF inhibits proliferation and promotes apoptosis of laryngeal squamous cell carcinoma cells by inhibiting Wnt/β-catenin signaling[J]. Oncol Lett, 2019, 17(6): 4928-4934.
[21] VAN HOOF V O, LEPOUTRE L G, HOYLAERTS M F, et al. Improved agarose electrophoretic method for separating alkaline phosphatase isoenzymes in serum[J]. Clin Chem, 1988, 34(9): 1857-1862.
[22] LI R, ZHOU R, WANG H, et al. Gut microbiota-stimulated cathepsin K secretion mediates TLR4-dependent M2 macrophage polarization and promotes tumor metastasis in colorectal cancer[J]. Cell Death Differ, 2019, 26(11): 2447-2463.
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