The Regulatory Mechanism of miR-153-3p on Lumbar Degeneration
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摘要:
目的 初步探讨H2O2对于腰椎髓核细胞的影响及miR-153-3p对于腰退行性病变的调节机制。腰椎间盘退变（Lumbar disc degeneration，LDD）是导致下腰痛的主要因素。髓核（nucleus pulposus，NP）细胞异常增殖或过度凋亡是LDD中最明显的细胞和生化变化之一。 方法 分别采用CCK8检测对照组及H2O2组细胞活力，流式检测两组细胞活性氧及线粒体膜电位，另采用qPCR检测上述两组的基质金属蛋白酶3（MMP3）、Nrf2、miR-153-3p、Ⅱ型胶原（COL-Ⅱ）表达含量。 结果 经H2O2处理的腰椎髓核细胞活力下降、线粒体膜电位下降比例较对照组减少；qPCR检测对照组、H2O2组的miR-153-3p、COL-Ⅱ、MMP3、Nrf2表达含量，结果提示对照组、H2O2组Nrf2表达量差异无统计学意义（P > 0.05），H2O2组的miR-153-3p、MMP3表达量较对照组减少，而其COL-Ⅱ表达量较对照组降低且差异有统计学意义（P < 0.05）。Westernblot检测对照组、inhibitor-NC组及nhibitor组的Nrf2表达含量，inhibitor组胞质及胞核中的Nrf2表达量升高，分别与对照组、inhibitor-NC组相比，有统计学差异（P < 0.05）；inhibitor-NC组胞质及胞核中的Nrf2表达量与对照组相比，差异无统计学意义。 结论 抑制miR-153-3p表达，可上调腰椎髓核细胞Nrf2表达水平，提示miR-153-3p参与存进LDD进程的过程可能与其调控Nrf2表达、调节线粒体自噬过程有关。 -
关键词:
- 腰椎退行性病变 /
- 基质金属蛋白酶3 /
- Nrf2 /
- miR-153-3p /
- Ⅱ型胶原
Abstract:objective To investigate the effect of H2O2 on lumbar nucleus pulposus cells and the regulatory mechanism of miR-153-3p on lumbar degeneration. Methods CCK8 was used to detect cell viability in control group and H2O2 group, and flow cytometry was used to detect reactive oxygen species and mitochondrial membrane potential in the two groups, and qPCR was used to detect the expression levels of matrix metalloproteinase 3 （MMP3）, Nrf2, miR-153-3p and type II collagen （COL-Ⅱ） in the above two groups. Results Compared with the control group, the H2O2-treated lumbar nucleus pulposus cells showed reduced viability and mitochondrial membrane potential. The expression levels of miR-153-3p, COL-Ⅱ, MMP3 and Nrf2 in the control group and the H2O2 group were detected by qPCR, and the results showed that there was no significant difference in Nrf2 expression levels in the control group and the H2O2 group （P < 0.05）. The expression levels of miR-153-3p and MMP3 in the H2O2 group were lower than those in the control group, while the expression of COL-Ⅱ was lower than that in the control group and there was a statistical difference （P < 0.05）. Western blot showed that the expression of Nrf2 in the control group, inhibitor-NC group and the inhibitor group was significantly higher than that in the control group and inhibitor-NC group （P < 0.05）. The expression of Nrf2 in the cytoplasm and nucleus of inhibitor-NC group was not significantly different from that of control group （P > 0.05）. Conclusion Inhibition of miR-153-3p expression can up-regulate the expression level of Nrf2 in lumbar nucleus pulposus cells, suggesting that miR-153-3p's involvement in the process of LDD may be related to its regulation of Nrf2 expression and mitochondrial autophagy. -
Key words:
- lumbar degenerative diseases /
- Matrix metalloproteinase 3 /
- Nrf2 /
- miR-153-3p /
- Type Ⅱ collage （COL-Ⅱ）
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图 1 qPCR检测3组miR-153-3p相对表达量图
与inhibitor-NC组相比，**P < 0.01。
Figure 1. Relative expression of miR-153-3p in the three groups detected by qPCR
图 2 Westernblot检测3组的Nrf2相对表达量
*P < 0.05。
Figure 2. Relative expression levels of Nrf2 detected by Western blot in the three groups.
表 1 RT-qPCR 引物序列
Table 1. Rt-qPCR primer sequence
引物名称 引物序列 产物长度（bp） 数据库 种属 厂家 熔解曲线（℃） miR-153-3p-F TTGCATAGTCACAAAAGT 70 MIMAT0000439 人 生工 79 miR-153-3p-R CAGTGCGTGTCGTGGAGT U6-F CTCGCTTCGGCAGCACA 80 NR_004394.1 人 生工 82.5 U6-R AACGCTTCACGAATTTGCGT Nrf2-F GCCAACTACTCCCAGGTTGC 122 NM_006164.5 Homo 人 生工 85.5 Nrf2-R AACGTAGCCGAAGAAACCTCA ACTB-F GTCATTCCAAATATGAGATGCGT 121 NM_001101.5 人 生工 85.5 ACTB-R GCTATCACCTCCCCTGTGTG 
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表 2 CCK8检测对照组、H2O2组细胞活力比较[（
$\bar x \pm s$ ）%]Table 2. Comparison of cell viability between CCK8 control group and H2O2 group [（
$\bar x \pm s $ ）%]组别 细胞活力 t P 对照组 1.00 ± 0.10 8.592 0.000# H2O2组 0.55 ± 0.08
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表 3 流式检测对照组、H2O2组细胞活性氧（
$\bar x \pm s $ ）Table 3. Flow detection of reactive oxygen species in control group and H2O2 group （
$\bar x \pm s $ ）组别 平均荧光强度值 t P 对照组 32423.33 ± 4157.62 −7.555 0.000# H2O2组 62127.83 ± 8686.98*
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表 4 流式检测对照组、H2O2组细胞线粒体膜电位（
$\bar x \pm s $ ）Table 4. Flow cytometry detection of mitochondrial membrane potential in control group and H2O2 group （
$\bar x \pm s $ ）组别 线粒体膜电位下降比例 t P 对照组 16.43 ± 1.30 11.736 0.000* H2O2组 7.98 ± 1.19 注：经正态性检验，两样本符合正态分布，采用两独立样本的t检验。*P < 0.05。
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表 5 qPCR检测对照组、H2O2组相关miRNA或mRNA相对表达量（
$ \bar x \pm s $ ）Table 5. Relative expression levels of miRNA or mRNA in control group and H2O2 group detected by qPCR （
$\bar x \pm s $ ）组别 miR-153-3P COL-Ⅱ MMP3 Nrf2 对照组 1.01 ± 0.18 1.01 ± 0.16 1.01 ± 0.16 1.01 ± 0.15 H2O2组 2.29 ± 0.44* 0.52 ± 0.16* 2.52 ± 0.75* 0.98 ± 0.13 与对照组相比，*P < 0.05。
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表 6 qPCR检测3组的Nrf2、miR-153-3p相对表达量（
$\bar x \pm s $ ）Table 6. Relative expression levels of Nrf2 and miR-153-3p in the three groups detected by QPCR （
$\bar x \pm s $ ）分组 miR-153-3p NRF2 对照组 1.01 ± 0.18 1.01 ± 0.18 miR-153-3p inhibitor-NC组 1.19 ± 0.22* 0.98 ± 0.18 miR-153-3p inhibitor组 0.37 ± 0.07* 2.65 ± 0.41* *P < 0.05。
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表 7 Westernblot检测3组的Nrf2相对表达量（
$\bar x \pm s $ ）Table 7. Relative expression of Nrf2 in the three groups detected by Westernblot （
$\bar x \pm s $ ）分组 胞质 胞核 对照组 1.00 ± 0.17 1.00 ± 0.14 miR-153-3p inhibitor-NC组 1.10 ± 0.13 0.95 ± 0.15 miR-153-3p inhibitor组 1.47 ± 0.23* 2.33 ± 0.41* 与对照组，inhibitor-NC组相比，*P < 0.05。
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