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Journal of Peking University (Health Sciences) ›› 2022, Vol. 54 ›› Issue (2): 320-326. doi: 10.19723/j.issn.1671-167X.2022.02.020

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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)

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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

CLC Number: 

  • R34

Table 1

Primer sequences used for qRT-PCR"

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

Table 2

shRNA sequences of lentiviruses"

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

Figure 1

Expression level of MIR4697HG and adipogenesis-related genes after adipogenic induction in BMSCs BMSCs, bone marrow mesenchymal stem cells; The other abbreviations as in Table 1.* P<0.05, vs. 0 d; A, the expression level of MIR4697HG after 0, 1, 2, 3, 5, 7, 10 days of adipogenic induction in BMSCs; B, the expression level of PPARγ after 0, 1, 2, 3, 5, 7, 10 days of adipogenic induction in BMSCs; C, the expression level of CEBP/α after 0, 1, 2, 3, 5, 7, 10 days of adipogenic induction in BMSCs; D, the expression level of ADIPQ after 0, 1, 2, 3, 5, 7, 10 days of adipogenic induction in BMSCs."

Figure 2

Examination of lentivirus transfection effect and efficiency GFP, green fluorescent protein;MIR4697HG, MIR4697 host gene.*P<0.05; vs. shNC group; A, the transfection effect was observed by fluorescence microscopy 24 hours after shMIR4697HG-1 group, shMIR4697HG-2 group and shNC group transfection;B, the transfection efficiency of MIR4697HG was examined by qRT-PCR."

Figure 3

Results after adipogenic induction in MIR4697HG-knockdown BMSCs PM, proliferation medium; AM, adipogenic medium; BMSCs, bone marrow mesenchymal stem cells; The other abbreviations as in Table 1; *P<0.05; vs. shNC group; A, qRT-PCR results after adipogenic induction in MIR4697HG-knockdown BMSCs; B, Western blot results after adipogenic induction in MIR4697HG-knockdown BMSCs; C, oil red staining results after adipogenic induction in MIR4697HG-knockdown BMSCs."

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