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

摘要/Abstract

摘要：

目的筛选激素性股骨头坏死（steroid-induced osteonecrosis of femoral head， SONFH）成骨细胞中差异表达的凋亡相关环状RNA（circular RNA，circRNA），并研究其对成骨细胞凋亡的作用及机制。方法（1）培养小鼠MC3T3-E1细胞，分为Control组和dexamethasone（DEX）组；（2）采用Western blot检测各组细胞BCL2-Associated X的蛋白质（Bax）和B淋巴细胞瘤-2（Bcl-2）蛋白的表达，TUNEL染色和流式细胞术检测细胞凋亡情况；（3）提取正常和DEX处理的MC3T3-E1细胞RNA，进行RNA测序，筛选出差异表达的circRNAs，并通过GO和KEGG分析其功能；（4）筛选出差异表达的mmu_circ_0000818并进行细胞水平验证以及UCSC Genome Browser Gateway和circbase网站分析mmu_circ_0000818在染色体的位置和保守性；（5）构建mmu_circ_0000818过表达质粒及小干扰RNA（siRNA）并转染细胞，流式细胞术检测各组MC3T3-E1细胞凋亡情况。结果（1）与Control组相比，DEX组MC3T3-E1细胞凋亡升高（P < 0.01）；（2）根据log₂foldchange（≥ 2）和P值（P < 0.05）筛选差异表达的circRNAs，与Control组相比，DEX组中共有234个表达差异的circRNAs，其中上调的138个，下调的96个。差异表达circRNAs的靶基因利用GO和KEGG进行富集分析，Apoptosis（凋亡）和PI3K-Akt signaling pathway（磷脂酰肌醇-3激酶/蛋白激酶B信号通路）差异最显著。（3）qRT-PCR结果显示，与Control组相比，DEX组MC3T3-E1细胞中mmu_circ_0000818表达明显升高（P < 0.01），UCSC Genome Browser Gateway和circbase分析发现mmu_circ_0000818主要位于17：78712463-78715086，由Crim1基因第6 ~ 7外显子环化形成并且在不同物种间具有高的保守性。（4）流式细胞术结果提示抑制mmu_circ_0000818可改善DEX引起的MC3T3-E1细胞凋亡，过表达则促进其凋亡。结论 mmu_circ_0000818在DEX处理的MC3T3-E1细胞中显著增高，降低其可抑制DEX引起的细胞凋亡。

关键词: 环状RNA mmu_circ_0000818, 地塞米松, MC3T3-E1细胞, RNA测序, 凋亡

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

中图分类号:

R285.5

杨慧霞,丁宁,马润秋,李桂忠,郝银菊,马胜超,姜怡邓,白志刚. 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[J]. 实用医学杂志, 2025, 41(4): 478-489.

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.

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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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