LncRNA MALAT1通过miR-34c/SATB2轴对脂肪间充质干细胞成骨分化的促进作用

doi:10.13481/j.1671-587x.20200511

摘要/Abstract

摘要： 目的：探讨长非编码RNA肺癌转移相关转录本1（LncRNA MALAT1）通过微小RNA（miR）-34c/特异AT序列结合蛋白2（SATB2）轴对脂肪来源的间充质干细胞（ADSCs）成骨分化的影响，并阐明其作用机制。方法：人ADSCs转染Lenti-NC、Lenti-MALAT1、sh-NC、sh-MALAT1、miR-NC和miR-34c，RT-PCR法检测ADSCs中LncRNA MALAT1、miR-34c和SATB2 mRNA表达水平；miRcode和TargetScan 7.1网站预测并通过荧光素酶报告基因实验验证miR-34c与LncRNA MALAT1、miR-34c与SATB2之间的靶向结合作用；Western blotting法检测ADSCs中成骨标志物Runt相关转录因子2（Runx2）、骨桥蛋白（OPN）和骨钙蛋白（OCN）表达水平；碱性磷酸酶（ALP）染色和茜素红S（ARS）染色检测ADSCs中ALP和ARS水平及钙盐沉积。结果：与第0天比较，ADSCs成骨诱导第3、7、14和21天后，细胞中LncRNA MALAT1、SATB2、Runx2、OPN和OCN蛋白表达水平明显升高（P<0.05），miR-34c表达水平明显降低（P<0.05）。与Lenti-NC组和sh-NC组比较，Lenti-MALAT1组ADSCs中LncRNA MALAT1表达水平，Runx2、OPN和OCN蛋白表达水平，ALP和ARS水平明显升高（P<0.01），sh-MALAT1组上述指标明显降低（P<0.01）。与miR-NC+Lenti-NC组比较，miR-34c+Lenti-NC组ADSCs中Runx2、OPN和OCN蛋白表达水平明显降低（P<0.01），ALP和ARS水平明显降低（P<0.01），miR-34c+Lenti-MALAT1组ADSCs中上述各项指标比较差异无统计学意义（P>0.05）。与Lenti-NC+miR-NC组比较，Lenti-SATB2+miR-NC组ADSCs中Runx2、OPN和OCN蛋白表达水平及ALP和ARS水平明显升高（P<0.01），Lenti-SATB2+miR-34c组上述各项指标差异无统计学意义（P>0.05）。结论：LncRNA MALAT1通过miRNA-34c/SATB2轴促进ADSCs的成骨分化。

关键词: 长链非编码RNA, 肺癌转移相关转录本1, 脂肪间充质干细胞, 成骨分化, 微小RNA-34c, 特异AT序列结合蛋白2

Abstract: Objective: To explore the effect of LncRNA MALAT1 on the osteogenic differentiation of adipose-derived mesenchymal stem cells(ADSCs) through the microRNA-34c/SATB2 axis, and to clarify its mechanism. Methods: The human ADSCs were transfected with Lenti-NC, Lenti-MALAT1, sh-NC, sh-MALAT1, miR-NC and miR-34c. RT-PCR method was used to detect the expression levels of LncRNA MALAT1, miR-34c and SATB2 mRNA in the ADSCs. MiRcode and TargetScan 7.1 website were used to predicte and the targeted binding effect between miR-34c and LncRNA MALAT1, miR-34c and SATB2 were verified through luciferase reporter gene experiment; Western blotting method was used to detect the expression levels of osteogenic markers Runx2, OPN and OCN proteins in the ADSCs; ALP staining and Alizarin red S(ARS) staining were used to detect the levels of ALP and ARS and calcium salt deposition in the ADSCs. Results: Compared with day 0, the expression levels of LncRNA MALAT1, SATB2, Runx2, OPN, and OCN proteins in the cells were significantly increased on the 3rd, 7th, 14th, and 21st days after osteogenic induction of ADSCs (P<0.05); the expression levels were significantly reduced (P<0.05). Compared with Lenti-NC group or sh-NC group, the expression level of LncRNA MALAT1,the expression levels of Runx2, OPN and OCN proteins, and the levels of ALP and ARS in the ADSCs in Lenti-MALAT1 group were significantly increased (P<0.01), and the indexes mentioned obove in sh-MALAT1 group were significantly decreased (P<0.01). Compared with miR-NC+Lenti-NC group, the expression levels of Runx2, OPN and OCN proteins in the ADSCs in miR-34c+Lenti-NC group were significantly reduced (P<0.01), and the levels of ALP and ARS were decreased(P<0.01),but the indexes mentioned obove in miR-34c+Lenti-MALAT1 group had no statistically significant differences (P>0.05). Compared with Lenti-NC+miR-NC group, the expression levels of Runx2, OPN and OCN proteins in the ADSCs in Lenti-SATB2+miR-NC group were significantly increased (P<0.01), and the levels of ALP and ARS were significantly increased (P<0.01);but the indexes mentioned above in Lenti-SATB2+miR-34c group had no statistically significant differences(P>0.05). Conclusion: LncRNA MALAT1 promotes the osteogenic differentiation of ADSCs through the miRNA-34c/SATB2 axis.

Key words: long non-coding RNA, metastasis-associated lung adenocarcinoma transcript 1, adipose-derived mesenchymal stem cells, osteogenic differentiation, microRNA-34c, special AT-rich sequence binding protein 2

中图分类号:

Q254

郭玮玮, 秦悦, 杨海波, 米占虎. LncRNA MALAT1通过miR-34c/SATB2轴对脂肪间充质干细胞成骨分化的促进作用[J]. 吉林大学学报(医学版), 2020, 46(05): 963-971.

GUO Weiwei, QIN Yue, YANG Haibo, MI Zhanhu. Promotion effect of LncRNA MALAT1 on osteogenic differentiation of adipose-derived mesenchymal stem cells through miR-34c/SATB2 axis[J]. Journal of Jilin University(Medicine Edition), 2020, 46(05): 963-971.

参考文献

[1] MADHURAKKAT PERIKAMANA S K, LEE J, AHMAD T, et al. Harnessing biochemical and structural cues for tenogenic differentiation of adipose derived stem cells (ADSCs) and development of an in vitro tissue interface mimicking tendon-bone insertion graft[J]. Biomaterials, 2018, 165:79-93.
[2] FERNANDES M, VALENTE S G, SABONGI R G, et al. Bone marrow-derived mesenchymal stem cells versus adipose-derived mesenchymal stem cells for peripheral nerve regeneration[J]. Neural Regen Res, 2018, 13(1):100-104.
[3] 宋孟晓,王燕,刘进忠.miR-222-5p在人根尖乳头干细胞成骨/成牙本质向分化中的作用[J].山东大学学报(医学版),2020,58(3):87-93,112.
[4] PENG S P, CAO L H, HE S W, et al. An overview of long noncoding RNAs involved in bone regeneration from mesenchymal stem cells[J]. Stem Cells Int, 2018, 2018:8273648.
[5] GAO X R, GE J, LI W Y, et al. LncRNA KCNQ1OT1 promotes osteogenic differentiation to relieve osteolysis via Wnt/β-catenin activation[J]. Cell Biosci, 2018, 8:19.
[6] HUANG G X, KANG Y, HUANG Z Y, et al. Identification and characterization of long non-coding RNAs in osteogenic differentiation of human adipose-derived stem cells[J]. Cell Physiol Biochem, 2017, 42(3):1037-1050.
[7] XIAO X X, ZHOU T W, GUO S C, et al. LncRNA MALAT1 sponges miR-204 to promote osteoblast differentiation of human aortic valve interstitial cells through up-regulating Smad4[J]. Int J Cardiol, 2017, 243:404-412.
[8] NARAYANAN A, SRINAATH N, ROHINI M, et al. Regulation of Runx2 by MicroRNAs in osteoblast differentiation[J]. Life Sci, 2019, 232:116676.
[9] BAE Y J, YANG T, ZENG H C, et al. miRNA-34c regulates Notch signaling during bone development[J]. Hum Mol Genet, 2012, 21(13):2991-3000.
[10] YU L J, XU Y S, QU H, et al. Decrease of MiR-31 induced by TNF-α inhibitor activates SATB2/RUNX2 pathway and promotes osteogenic differentiation in ethanol-induced osteonecrosis[J]. J Cell Physiol, 2019, 234(4):4314-4326.
[11] 王洋,宋卓悦,连晓磊,等.脂肪和滑膜来源间充质干细胞成软骨分化能力的比较[J].郑州大学学报(医学版),2019,54(3):394-398.
[12] NADERI N, COMBELLACK E J, GRIFFIN M, et al. The regenerative role of adipose-derived stem cells (ADSC) in plastic and reconstructive surgery[J]. Int Wound J, 2017, 14(1):112-124.
[13] AGHEBATI-MALEKI L, DOLATI S, ZANDI R, et al. Prospect of mesenchymal stem cells in therapy of osteoporosis:A review[J]. J Cell Physiol, 2019, 234(6):8570-8578.
[14] RANSOHOFF J D, WEI Y N, KHAVARI P A. The functions and unique features of long intergenic non-coding RNA[J]. Nat Rev Mol Cell Biol, 2018, 19(3):143-157.
[15] HUYNH N P, ANDERSON B A, GUILAK F, et al. Emerging roles for long noncoding RNAs in skeletal biology and disease[J]. Connect Tissue Res, 2017, 58(1):116-141.
[16] HUANG X Z, HUANG J, LI W Z, et al. LncRNA-MALAT1 promotes osteogenic differentiation through regulating ATF4 by sponging miR-214:Implication of steroid-induced avascular necrosis of the femoral head[J]. Steroids, 2020, 154:108533.
[17] YOSHIOKA H, YOSHIKO Y. The roles of long non-protein-coding RNAs in osteo-adipogenic lineage commitment[J]. Int J Mol Sci, 2017, 18(6):E1236.
[18] JIN C Y, ZHENG Y F, HUANG Y P, et al. Long non-coding RNA MIAT knockdown promotes osteogenic differentiation of human adipose-derived stem cells[J]. Cell Biol Int, 2017, 41(1):33-41.
[19] FAN F Y, DENG R, LAI S H, et al. Inhibition of microRNA-221-5p induces osteogenic differentiation by directly targeting smad3 in myeloma bone disease mesenchymal stem cells[J]. Oncol Lett, 2019, 18(6):6536-6544.
[20] LAN C G, LONG L Z, XIE K G, et al. MiRNA-429 suppresses osteogenic differentiation of human adipose-derived mesenchymal stem cells under oxidative stress via targeting SCD-1[J]. Exp Ther Med, 2020, 19(1):696-702.
[21] GAO Y, XIAO F, WANG C L, et al. Long noncoding RNA MALAT1 promotes osterix expression to regulate osteogenic differentiation by targeting miRNA-143 in human bone marrow-derived mesenchymal stem cells[J]. J Cell Biochem, 2018, 119(8):6986-6996.
[22] YU C, LI L F, XIE F, et al. LncRNA TUG1 sponges miR-204-5p to promote osteoblast differentiation through upregulating Runx2 in aortic valve calcification[J]. Cardiovasc Res, 2018, 114(1):168-179.
[23] WANG Q J, LI Y X, ZHANG Y, et al. LncRNA MEG3 inhibited osteogenic differentiation of bone marrow mesenchymal stem cells from postmenopausal osteoporosis by targeting miR-133a-3p[J]. Biomed Pharmacother, 2017, 89:1178-1186.
[24] ZARATE Y A, STEINRATHS M, MATTHEWS A, et al. Bone health and SATB2-associated syndrome[J]. Clin Genet, 2018, 93(3):588-594.
[25] GONG Y M, XU F, ZHANG L, et al. MicroRNA expression signature for Satb2-induced osteogenic differentiation in bone marrow stromal cells[J]. Mol Cell Biochem, 2014, 387(1/2):227-239.
[26] 王亮,钟武,陈睦虎.miR-34a对人脂肪间充质干细胞成骨向分化的调控[J].西安交通大学学报(医学版),2018,39(5):675-679,708.
[27] HAO J B, ZHANG L, CONG G T, et al. MicroRNA-34b/c inhibits aldosterone-induced vascular smooth muscle cell calcification via a SATB2/Runx2 pathway[J]. Cell Tissue Res, 2016, 366(3):733-746.
[28] TANG J F, ZHANG Z, JIN X Y, et al. miR-383 negatively regulates osteoblastic differentiation of bone marrow mesenchymal stem cells in rats by targeting Satb2[J]. Bone, 2018, 114:137-143.