环状RNA mmu_circ_0000818在地塞米松导致的MC3T3-E1细胞凋亡中的作用及机制

doi:10.3969/j.issn.1006-5725.2025.04.004

Abstract

Abstract:

Objective To screen for differentially expressed apoptosis-related circular RNAs （circRNAs） in osteoblasts from steroid-induced osteonecrosis of the femoral head （SONFH） and to investigate their roles and mechanisms in osteoblast apoptosis. Methods MC3T3-E1 cells were cultured and divided into two groups： Control and DEX-treated. Western blot analysis was employed to evaluate the expression levels of BCL2-Associated X protein （Bax） and B-cell lymphoma 2 （Bcl-2）. Cell apoptosis was assessed using TUNEL staining and flow cytometry. RNA was extracted from both normal and DEX-treated MC3T3-E1 cells， followed by RNA-seq to identify differentially expressed circular RNAs （circRNAs）. The functions and pathways of these circRNAs were analyzed using Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG）. The differentially expressed mmu_ circ_0000818 was selected for further verification at the cellular level. Its chromosomal location and conservation were examined using the UCSC Genome Browser Gateway and circBase. Overexpression plasmids and small interfering RNAs （siRNAs） targeting mmu_circ_0000818 were constructed and transfected into the cells. Subsequently， apoptosis in MC3T3-E1 cells from each group was evaluated by flow cytometry. Results Compared with the control group， the apoptosis rate of MC3T3-E1 cells was significantly increased in the DEX group （P < 0.01）. Differentially expressed circRNAs were identified based on log₂foldchange （≥ 2） and P value （P < 0.05）. Relative to the control group， there were 234 differentially expressed circRNAs in the DEX group， including 138 up-regulated and 96 down-regulated circRNAs. GO and KEGG enrichment analyses of the target genes of these differentially expressed circRNAs revealed significant associations with apoptosis and the PI3K-Akt signaling pathway. qRT-PCR results demonstrated that the expression level of mmu_circ_0000818 was markedly higher in the DEX group compared to the control group （P < 0.01）. Analysis using the UCSC Genome Browser and CircBase indicated that mmu_circ_0000818， located at chromosome 17：78712463-78715086， is formed by the cyclization of exons 6-7 of the Crim1 gene and exhibits high conservation across species. Flow cytometry results indicated that knockdown of mmu_circ_0000818 attenuated DEX-induced apoptosis in MC3T3-E1 cells， while overexpression of mmu_circ_0000818 exacerbated apoptosis. Conclusions CircRNA mmu_circ_0000818 was significantly upregulated in DEX-treated MC3T3-E1 cells， and its downregulation mitigated DEX-induced apoptosis. Consequently， mmu_circ_0000818 may represent a promising therapeutic target for the prevention and treatment of SONFH.

Key words: circRNA mmu_circ_0000818, dexamethasone, MC3T3-E1 cells, RNA sequencing, apoptosis

CLC Number:

R285.5

Huixia YANG,Ning DING,Runqiu MA,Guizhong LI,Yinju HAO,Shengchao MA,Yideng JIANG,Zhigang. BAI. Role and mechanism of circular RNA mmu_circ_0000818 in dexamethasone⁃induced apoptosis of MC3T3⁃E1 cells[J]. The Journal of Practical Medicine, 2025, 41(4): 478-489.

Figures/Tables 8

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References 21

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名称		序列
mmu_circ_0000818	上游引物	5′?GGGGAACTGAACTGCGAAAG?3′
	下游引物	5′?AGGTACAGTCATCTTCCCGC?3′
GAPDH	上游引物	5′?AGGTCGGTGTGAACGGATTTG?3′
	下游引物	5′?GGGGTCGTTGATGGCAACA?3′
si-mmu_circ_0000818-1	正义链	5′?CCGUGUGUGAAGAAACAAAGCTT?3′
	反义链	5′?GCUUUGUUUCUUCACACACGGTT?3′
si-mmu_circ_0000818-2	正义链	5′?UUGCCCCGUGUGUGAAGAAACTT?3′
	反义链	5′?GUUUCUUCACACACGGGGCAATT?3′
si-mmu_circ_0000818-3	正义链	5′?GAAGAAACAAAGCCAGCCUGCTT?3′
	反义链	5′?GCAGGCUGGCUUUGUUUCUUCTT?3′
si-NC	正义链	5′?UUCUCCGAACGUGUCACGUTT?3′
	反义链	5′?ACGUGACACGUUCGGAGAATT?3′

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Role and mechanism of circular RNA mmu_circ_0000818 in dexamethasone⁃induced apoptosis of MC3T3⁃E1 cells

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