Email Alert | RSS

北京大学学报（医学版） ›› 2022, Vol. 54 ›› Issue (2): 320-326. doi: 10.19723/j.issn.1671-167X.2022.02.020

• 论著 • 上一篇    下一篇

敲减长链非编码RNA MIR4697HG抑制骨髓间充质干细胞成脂向分化

帅婷1,刘娟2,郭艳艳1,金婵媛1,△()   

  1. 1.北京大学口腔医学院∙口腔医院第二门诊部,国家口腔医学中心,国家口腔疾病临床医学研究中心,口腔生物材料和数字诊疗装备国家工程研究中心,口腔数字医学北京市重点实验室,国家卫生健康委员会口腔医学计算机应用工程技术研究中心,国家药品监督管理局口腔生物材料重点实验室,北京 100081
    2.首都医科大学附属北京友谊医院口腔科,北京 100050
  • 收稿日期:2020-03-30 出版日期:2022-04-18 发布日期:2022-04-13
  • 通讯作者: 金婵媛 E-mail:jinchanyuanjcy@bjmu.edu.cn
  • 基金资助:
    国家自然科学基金(81800942);中国博士后基金(2018M631442)

Knockdown of long non-coding RNA MIR4697 host gene inhibits adipogenic differentiation in bone marrow mesenchymal stem cells

SHUAI Ting1,LIU Juan2,GUO Yan-yan1,JIN Chan-yuan1,△()   

  1. 1. Second Clinical Division, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices & Beijing Key Laboratory of Digital Stomatology & NHC Research Center of Engineering and Technology for Computerized Dentistry & NMPA Key Laboratory for Dental Materials, Beijing 100081, China
    2. Department of Stomatology, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China
  • Received:2020-03-30 Online:2022-04-18 Published:2022-04-13
  • Contact: Chan-yuan JIN E-mail:jinchanyuanjcy@bjmu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(81800942);Chinese Postdoctoral Science Foundation(2018M631442)

RICH HTML

   

PDF (PC)

摘要：

目的: 初步探究长链非编码RNA (long non-coding RNA,lncRNA) MIR4697宿主基因(MIR4697 host gene,MIR4697HG)对骨髓间充质干细胞 (bone marrow stem cells,BMSCs)的成脂向分化调控作用。方法: 将BMSCs进行成脂诱导,在不同的时间点(0、1、2、3、5、7、10 d)收集RNA,通过实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)技术检测成脂分化调控相关的过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor gamma,PPARγ)、CCAAT增强子结合蛋白α(CCAAT/enhanced binding protein α,CEBP/α)、脂联素(adiponectin,ADIPQ)编码基因的mRNA以及lncRNA MIR4697HG的表达水平。为了防止脱靶效应,本研究构建了两条不同序列的MIR4697HG shRNA (shMIR4697HG-1, shMIR4697HG-1)并通过慢病毒感染BMSCs,建立MIR4697HG稳定敲减的BMSCs细胞系。采用油红O染色、蛋白质印迹实验和qRT-PCR等方法检测敲减MIR4697HG对BMSCs成脂分化能力的影响。结果: 体外诱导BMSCs成脂向分化时,成脂标志基因PPARγ、CEBP/α和ADIPQ表达量均显著升高,在此过程中lncRNA MIR4697HG表达也明显增加(P<0.01)。慢病毒感染BMSCs 72 h后,荧光显微镜下可以观察到90%以上细胞成功表达绿色荧光蛋白,qRT-PCR结果显示MIR4697HG敲减效率高于60%;BMSCs敲减MIR4697HG后,在BMSCs成脂诱导7 d时,成脂基因PPARγ、CEBP/α和ADIPQ的转录物(mRNA)水平显著下降 (P<0.01),同时PPARγ和CEBP/α的蛋白质水平也显著降低 (P<0.01)。敲减MIR4697HG的BMSCs成脂向分化能力减弱。结论: lncRNA MIR4697HG对BMSCs成脂向分化有调控作用,敲减MIR4697HG可抑制BMSCs的成脂向分化,提示lncRNA MIR4697HG可能成为治疗骨质疏松症等成脂肪异常疾病的潜在靶点。

关键词: 骨髓间充质干细胞, 长链非编码RNA, MIR4697HG, 成脂向分化

Abstract:

Objective: To preliminarily investigate the role of long non-coding RNA (lncRNA) MIR4697 host gene (MIR4697HG) in regulating the adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods: For adipogenic differentiation, BMSCs were induced in adipogenic media for 10 days. The mRNA expression levels of lncRNA MIR4697HG and adipogenic marker genes including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhanced binding protein α (CEBP/α) and adiponectin (ADIPQ) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) at different time points (0, 1, 2, 3, 5, 7, 10 days). The MIR4697HG stable knockdown-BMSC cell line was generated by infection of MIR4697HG shRNA-containing lentiviruses. To avoid off-target effect, two target sequences (shMIR4697HG-1, shMIR4697HG-2) were designed. And then cells were induced to differentiate in adipogenic medium. Oil red O staining, Western blot and qRT-PCR were used to detect the effect of MIR4697HG knockdown on adipogenic differentiation of BMSCs. Results: The mRNA expression level of MIR4697HG was significantly increased during adipogenic differentiation (P<0.01), and adipogenic differentiation of BMSCs was evidenced by upregulated mRNA levels of specific adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ. Observed by fluorescence microscopy, more than 90% transfected target cells expressed green fluorescent protein successfully after shMIR4697HG-1 group, shMIR4697HG-2 group and shNC group transfection for 72 h. And the transfection efficiency of MIR4697HG examined by qRT-PCR was above 60%. Then the BMSCs were treated with adipogenic media for 7 days and showed that the mRNA expression levels of adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ were significantly decreased in the MIR4697HG knockdown group (P<0.01), while the expression levels of PPARγ and CEBP/α proteins were decreased remarkably as well (P<0.01). Consistently, MIR4697HG knockdown BMSCs formed less lipid droplets compared with the control BMSCs, which further demonstrated that MIR4697HG knockdown inhibited adipogenic differentiation of BMSCs. Conclusion: lncRNA MIR4697HG played a crucial role in regulating the adipogenic differentiation of BMSCs, and MIR4697HG knockdown significantly inhibited the adipogenic differentiation of BMSCs. These data may suggest that lncRNA MIR4697HG could serve as a therapeutic potential target for the aberrant adipogenic differentiation-associated disorders including osteoporosis.

Key words: Bone marrow mesenchymal stem cells, Long non-coding RNA, MIR4697HG, Adipogenic differentiation

中图分类号: 

  • R34

表1

qRT-PCR引物"

lncRNA name Forward primer sequences(5' to 3') Reverse primer sequences(5' to 3')
MIR4697HG GAAGTGTGTGTGCAGGCTTG GGAAAAGGCTCTGTCGTGGA
GAPDH GGTCACCAGGGCTGCTTTTA GGATCTCGCTCCTGGAAGATG
PPARγ GAGGAGCCTAAGGTAAGGAG GTCATTTCGTTAAAGGCTGA
CEBP/α CGCAAGAGCCGAGATAAAGC CACGGCTCAGCTGTTCCA
ADIPQ CTTGCAAGAACCGGCTCAGATCCTCCC GAGCTGTTCTACTGCTATTAGCTCTGC

表2

shRNA 靶序列"

shRNA shRNA sequences(5' to 3')
shNC CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAG
shMIR4697HG-1 GTGCCCTCTGCACACATATATCTCGAGATATATGTGTGCAGAGGGCAC
shMIR4697HG-2 TGCTGTACCAAAGCCTTATATCTCGAGATATAAGGCTTTGGTACAGCA

图1

BMSCs成脂诱导后MIR4697HG和成脂相关基因的表达水平"

图2

慢病毒感染效果和敲减效率检测"

图3

敲减MIR4697HG抑制BMSCs成脂分化的检测结果"

[1] Pino AM, Rosen CJ, Rodríguez JP. In osteoporosis, differentiation of mesenchymal stem cells (MSCs) improves bone marrow adipogenesis[J]. Biol Res, 2012, 45(3):279-287.
doi: 10.4067/S0716-97602012000300009
[2] Taylor DH, Chu TJ, Spektor R, et al. Long non-coding RNA regulation of reproduction and development[J]. Mol Reprod Dev, 2015, 82(12):932-956.
doi: 10.1002/mrd.22581 pmid: 26517592
[3] Sun M, Kraus WL. From discovery to function: the expanding roles of long non-coding RNAs in physiology and disease[J]. Endocr Rev, 2015, 36(1):25-64.
doi: 10.1210/er.2014-1034
[4] Luan A, Paik KJ, Li J, et al. RNA sequencing for identification of differentially expressed noncoding transcripts during adipogenic differentiation of adipose-derived stromal cells[J]. Plast Reconstr Surg, 2015, 136(4):752-763.
doi: 10.1097/PRS.0000000000001582
[5] Liu Y, Wang Y, He X, et al. LncRNA TINCR/miR-31-5p/C/EBP-α feedback loop modulates the adipogenic differentiation process in human adipose tissue-derived mesenchymal stem cells[J]. Stem Cell Res, 2018, 32:35-42.
doi: 10.1016/j.scr.2018.08.016
[6] Mao YH, Shen G, Su ZP, et al. RAD21 inhibited transcription of tumor suppressor MIR4697HG and led to glioma tumorigenesis[J]. Biomed Pharmacother, 2020, 123:109759.
doi: 10.1016/j.biopha.2019.109759
[7] Zhang LQ, Yang SQ, Wang Y, et al. Long noncoding RNA MIR4697HG promotes cell growth and metastasis in human ovarian cancer[J]. Anal Cell Pathol (Amst), 2017, 2017(4):1-9.
[8] Park SB, Kim J, Jeong JH, et al. Prevalence and incidence of osteoporosis and osteoporotic vertebral fracture in Korea: nationwide epidemiological study focusing on differences in socioecono-mic status[J]. Spine, 2016, 41(4):328.
doi: 10.1097/BRS.0000000000001291
[9] Montagner RLM, Martinez CR, Canterle DOL, et al. Factors related with osteoporosis treatment in postmenopausal women[J]. Medicine, 2018, 97(28):e11524.
doi: 10.1097/MD.0000000000011524
[10] Zhou X, Liu Z, Huang B, et al. Orcinol glucoside facilitates the shift of MSC fate to osteoblast and prevents adipogenesis via Wnt/beta-catenin signaling pathway[J]. Drug Des Devel Ther, 2019, 13:2703-2713.
doi: 10.2147/DDDT
[11] Crockett JC, Mellis DJ, Scott DI, et al. New knowledge on critical osteoclast formation and activation pathways from study of rare genetic diseases of osteoclasts: focus on the RANK/RANKL axis[J]. Osteoporos Int, 2011, 22(1):1-20.
doi: 10.1007/s00198-010-1272-8 pmid: 20458572
[12] Lorenzo J. The many ways of osteoclast activation[J]. J Clin Invest, 2017, 127(7):2530-2532.
doi: 10.1172/JCI94606 pmid: 28530641
[13] Wang QL, Li HF, Wang DP, et al. Effect of GGCX on the differentiation function of osteoporosis bone marrow mesenchymal stem cells through regulating TGFbeta/smad signaling pathway[J]. Eur Rev Med Pharmacol Sci, 2019, 23(17):7224-7231.
[14] Aimaiti A, Wahafu T, Keremu A, et al. Strontium ameliorates glucocorticoid inhibition of osteogenesis via the ERK signaling pathway[J]. Biol Trace Elem Res, 2020, 197(2):591-598.
doi: 10.1007/s12011-019-02009-6
[15] Chen X, Zhi X, Yin Z, et al. 18β-glycyrrhetinic acid inhibits osteoclastogenesis in vivo and in vitro by blocking RANKL-mediated RANK-TRAF6 interactions and NF-κB and MAPK signaling pathways[J]. Front Pharmacol, 2018, 9:647.
doi: 10.3389/fphar.2018.00647
[16] Chen X, He F, Zhong DY, et al. Acoustic-frequency vibratory stimulation regulates the balance between osteogenesis and adipogenesis of human bone marrow-derived mesenchymal stem cells[J]. Biomed Res Int, 2015, 2015:540731.
[17] James AW. Review of signaling pathways governing MSC osteoge-nic and adipogenic differentiation[J]. Scientifica, 2013, 2013:684736.
[18] Shang GW, Wang YD, Xu Y, et al. Long non-coding RNA TCONS_00041960 enhances osteogenesis and inhibits adipogenesis of rat bone marrow mesenchymal stem cell by targeting miR-204-5p and miR-125a-3p[J]. J Cell Physiol, 2018, 233(8):6041-6051.
doi: 10.1002/jcp.v233.8
[19] Wang YJ, Liu WT, Liu YD, et al. Long noncoding RNA H19 mediates LCoR to impact the osteogenic and adipogenic differentiation of mBMSCs in mice through sponging miR-188[J]. J Cell Physiol, 2018, 233(9):7435-7446.
doi: 10.1002/jcp.v233.9
[20] Yang L, Li Y, Gong R, et al. The long non-coding RNA-ORLNC1 regulates bone mass by directing mesenchymal stem cell fate[J]. Mol Ther, 2019, 27(2):394-410.
doi: S1525-0016(18)30586-0 pmid: 30638773
[21] Yuan HR, Xu XW, Feng X, et al. A novel long noncoding RNA PGC1β-OT1 regulates adipocyte and osteoblast differentiation through antagonizing miR-148a-3p[J]. Cell Death Differ, 2019, 26(10):2029-2045.
doi: 10.1038/s41418-019-0296-7
[1] 何丽杰,张春艳,王静. NEAT1、miR-27a-3p在阿尔茨海默病患者血清和脑脊液中的表达关系[J]. 北京大学学报（医学版）, 2024, 56(2): 207-212.
[2] 尤鹏越,刘玉华,王新知,王思雯,唐琳. 脱细胞猪心包膜生物相容性及成骨性能的体内外评价[J]. 北京大学学报（医学版）, 2021, 53(4): 776-784.
[3] 谢静,赵玉鸣,饶南荃,汪晓彤,方滕姣子,李晓霞,翟越,李静芝,葛立宏,王媛媛. 3种口腔颌面部来源的间充质干细胞成血管内皮分化潜能的比较研究[J]. 北京大学学报（医学版）, 2019, 51(5): 900-906.
[4] 杨飞龙,洪锴,赵国江,刘承,宋一萌,马潞林. 基于长链非编码RNA的生物信息学分析构建膀胱癌预后模型并确定预后生物标志物[J]. 北京大学学报（医学版）, 2019, 51(4): 615-622.
[5] 刘霞,李英妮,孙晓麟,彭清林,卢昕,王国春. 去整合素金属蛋白酶对成骨分化的影响[J]. 北京大学学报（医学版）, 2018, 50(6): 962-967.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
ISSN 1671-167X
CN 11-4691/R
主管单位：中华人民共和国教育部
主办单位：北京大学
地址： 北京市海淀区学院路38号 北京大学医学部 《北京大学学报（医学版）》
邮编：100191
电话：010-82801551
Email： xbbjb2@bjmu.edu.cn

《北京大学学报（医学版）》
微信公众号
版权所有 © 2018 《北京大学学报（医学版）》编辑部