Mechanism and influence of down-regulation of miR-29a-3p expression on apoptosis in mouse macrophage
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摘要:目的
研究miR-29a-3p下调表达对小鼠巨噬细胞凋亡及相关基因表达的影响，为探讨结核病发病机制、研发新的诊断与治疗方法提供依据。
方法将重组抑制载体pEZX-AM02-miR-29a-3p转染RAW264.7细胞，24、36和48 h荧光显微镜观察转染效率，Real time-PCR法检测miR-29a-3p及凋亡相关基因caspase3、caspase7、caspase8、Bcl-2、Mcl-1和Bax的表达水平，流式细胞术检测RAW264.7细胞的凋亡率。
结果重组抑制载体转染后，细胞内miR-29a-3p表达水平明显下降；而caspase7、caspase8、Bcl-2、Mcl-1和Bax等基因的表达出现不同程度的上调；流式细胞术分析发现随着转染时间延长，细胞凋亡率增加，且36 h凋亡率最高。
结论pEZX -AM02-miR-29a-3p重组载体可抑制小鼠巨噬细胞中miR-29a-3p的表达，通过靶向上调caspase7、caspase8、Bcl-2和Mcl-1等基因的表达促进巨噬细胞的凋亡。
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关键词:
- miR-29a-3p /
- 抑制载体 /
- 巨噬细胞 /
- 细胞凋亡 /
- 靶基因
Abstract:ObjectiveTo study the influence of down-regulation of miR-29a-3p expression on apoptosis in mouse macrophage and expression of apoptosis-associated genes, and provide references for investigating the pathogenesis mechanism of mycobacterium tuberculosis (MTB) and developing new diagnosis and treatment methods.
MethodThe recombinant inhibitor vector pEZX-AM02-miR-29a-3p was transfected into RAW264.7 cells. Transfection efficiency was observed through fluorescence microscopy, the expression levels of miR-29a-3p and apoptosis-associated genes caspase3, caspase7, caspase8, Bcl-2, Mcl-1 and Bax were detected by RT-PCR, and the apoptosis rates of RAW264.7 cells were detected by flow cytometry at 24, 36, 48 h after transfection.
ResultAfter transfected with pEZX-AM02-miR-29a-3p recombinant inhibitor vector, miR-29a-3p expression in RAW264.7 cells decreased significantly, while the target genes caspase7, caspase8, Bcl-2, Mcl-1 and Bax were up-regulated at different levels. The results of flow cytometry indicated that the apoptosis rates increased as the transfection time progressed and reached the peak at 36 h.
ConclusionThe expression of miR-29a-3p in mouse macrophage are inhibited by transfecting with pEZX-AM02-miR-29a-3p, which promotes the apoptosis of macrophage by up-regulating the expression of target genes, suah as caspase7, caspase8, Bcl-2 and Mcl-1.
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Keywords:
- miR-29a-3p /
- inhibitor vector /
- macrophage /
- cell apoptosis /
- target gene
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图 1 miRNA抑制对照以及pEZX-AM02-miR-29a-3p转染RAW264.7细胞
1~3为24 h的抑制载体对照(pEZX-AM02)；4~6为24 h的pEZX-AM02-miR-29a-3p载体；7~9为36 h的抑制载体对照(pEZX-AM02)；10~12为36 h的pEZX-AM02-miR-29a-3p载体；13~15为48 h的抑制载体对照(pEZX-AM02)；16~18为48 h的pEZX-AM02-miR-29a-3p载体
Figure 1. RAW264.7 cells tranfected with miRNA inhibitor control or pEZX-AM02-miR-29a-3p
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图 2 不同处理组凋亡相关基因24、36和48 h的表达
“*”、“**”和“***”分别表示与对照组在0.05、0.01和0.005水平差异显著(Welch法)
Figure 2. The expression of genes related to apoptosis in different groups at 24, 36 and 48 h after transfection
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图 4 巨噬细胞凋亡率
“*”、“**”和“***”分别表示与对照组在0.05、0.01和0.005水平差异显著(Welch 法)
Figure 4. Macrophages apoptosis rate
表 1 基因引物序列
Table 1 Sequences of gene primers
基因 上游引物(5'→3') 下游引物(5'→3') caspase3 TCTGACTGGAAAGCCGAAAC GCAAGCCATCTCCTCATCA caspase7 AAACCCTGTTAGAGAAACCCAA TAAGCAAAGAGGAAGTCGGC caspase8 GCTGCCCTCAAGTTCCTGT GATTGCCTTCCTCCAACATC Bcl-2 GGTGGAGGAACTCTTCAGGG ACATCTCCCTGTTGACGCTC Bax ATGCGTCCACCAAGAAGC CAGTTGAAGTTGCCATCAGC Mcl-1 TGTAAGGACGAAACGGGACT CAAAAGCCAGCAGCACATT 
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