MiR-346 Inhibits Chondrocyte Proliferation in Idiopathic Short Stature by Targeting SMAD3
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摘要:
目的 探讨miR-346在特发性矮小症（idiopathic short stature，ISS）患者中的表达及其对软骨细胞增殖的影响，并阐明其通过靶向调控SMAD家族成员3（SMAD family member 3，SMAD3）发挥作用的分子机制。 方法 采用病例对照研究设计类型，选取2019年12月至2023年12月期间于绵阳市儿童医院就诊的ISS患者作为研究对象（ISS组，n = 38），同时选取同期于该院就诊的身高在同年龄、同性别、同种族人群平均水平±1个标准差以内的儿童（对照组，n = 38）的外周血样本，通过qRT-PCR检测血清中miR - 346和SMAD3的表达水平。体外培养人软骨细胞C28/I2，设置4组实验：miR-NC组（转染阴性对照miR-NC）、miR-346 inhibitor组（转染miR-346抑制剂）、miR-346 inhibitor + si-NC组（共转染miR-346抑制剂和阴性对照si-NC）和miR-346 inhibitor + si-SMAD3组（共转染miR-346 inhibitor和si-SMAD3）。利用CCK-8法和Edu染色检测细胞增殖能力变化。采用流式细胞术评估细胞在不同周期阶段的分布情况。通过蛋白质印迹技术分析细胞周期、成骨分化相关蛋白及SMAD3的表达水平变化。利用生物信息学方法预测miR-346与SMAD3的靶向结合，并通过双荧光素酶报告基因实验对该预测进行验证。 结果 与对照组相比，ISS组血清miR-346表达显著增加，SMAD3表达显著降低（P < 0.05）。与miR-NC组相比，miR-346 inhibitor组和miR-346 inhibitor+si-NC组细胞的miR-346表达水平、G1期和S期细胞比例、p21蛋白表达水平显著降低（P < 0.05），细胞增殖活力、EDU阳性比例、G2期细胞比例、Cyclin D1、CDK4、CDK6、MMP-13、COL10A、RUNX2、OCN和OPN表达水平、SMAD3的mRNA表达及p-SMAD3蛋白水平显著升高（P < 0.05）；与miR-346 inhibitor组和miR-346 inhibitor+si-NC组相比，miR-346 inhibitor+si-SMAD3组细胞的G1期和S期细胞比例、p21蛋白表达水平显著升高（P < 0.05），细胞增殖活力、EDU阳性比例、G2期细胞比例、Cyclin D1、CDK4、CDK6、MMP-13、COL10A、RUNX2、OCN和OPN表达水平、SMAD3的mRNA表达及p-SMAD3蛋白水平显著降低（P < 0.05）。 结论 miR-346在ISS患者中表达上调，可能通过靶向抑制Smad3，进而干扰软骨细胞的增殖与软骨内成骨过程。 Abstract:Objective To investigate the expression of miR-346 in patients with idiopathic short stature （ISS） and its effect on chondrocyte proliferation, and to elucidate the molecular mechanism by which it acts through targeted regulation of SMAD family member 3（SMAD3）. Methods A case-control study design was adopted. Patients with ISS admitted to Mianyang Children's Hospital between December 2019 and December 2023 were enrolled as the ISS group （n = 38）. Meanwhile, children whose height was within ±1 standard deviation of the average level of the population of the same age, gender and ethnicity were recruited as the control group （n = 38）, and their peripheral blood samples were collected. The serum expression levels of miR-346 and SMAD3 were detected by quantitative real-time polymerase chain reaction （qRT-PCR）. Human chondrocytes C28/I2 were cultured in vitro, and four experimental groups were established: miR-NC group （transfected with negative control miR-NC）, miR-346 inhibitor group （transfected with miR-346 inhibitor）, miR-346 inhibitor + si-NC group （co-transfected with miR-346 inhibitor and negative control si-NC）, and miR-346 inhibitor + si-SMAD3 group （co-transfected with miR-346 inhibitor and si-SMAD3）. Changes in Cell proliferation capacity were assessed using the CCK-8 assay and Edu staining. Cell cycle distribution was analyzed by flow cytometry. Protein expression levels of cell cycle phases and osteogenic differentiation-related proteins, as well as SMAD3, were measured by western blotting. The targeting relationship between miR-346 and SMAD3 was predicted using bioinformatics and validated through dual-luciferase reporter gene assays. Results Compared with the control group, the serum expression level of miR - 346 in the ISS group was significantly increased, while the expression level of SMAD3 was significantly decreased （P < 0.05）. Compared with the miR-NC group, the miR-346 expression levels, the proportions of cells in the G1 and S phases, and the p21 protein expression levels in cells of the miR-346 inhibitor group and the miR-346 inhibitor + si-NC group were significantly decreased （P < 0.05）. In contrast, cell proliferation viability, the proportion of EDU-positive cells, the proportion of cells in the G2 phase, and the protein expression levels of Cyclin D1, CDK4, CDK6, MMP-13, COL10A, RUNX2, OCN, and OPN, as well as the mRNA expression of SMAD3 and the p-SMAD3 protein level, were significantly increased （P < 0.05）. Compared with the miR-346 inhibitor group and the miR-346 inhibitor + si-NC group, the proportions of cells in the G1 and S phases and the p21 protein expression level in cells of the miR-346 inhibitor + si-SMAD3 group were significantly increased （P < 0.05）. Conversely, cell proliferation viability, EDU-positive ratio, the proportion of cells in the G2 phase, and the protein expression levels of Cyclin D1, CDK4, CDK6, MMP-13, COL10A, RUNX2, OCN, and OPN, as well as the mRNA expression of SMAD3 and the p-SMAD3 protein level, were significantly decreased （P < 0.05）. Conclusion miR-346 expression is up-regulated in ISS patients and may interfere with chondrocyte proliferation and osteogenic differentiation by targeted inhibition of SMAD3. -
Key words:
- miR-346 /
- SMAD3 /
- Idiopathic short stature /
- Chondrocyte /
- Proliferation /
- Osteogenesis
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图 2 EDU检测各组细胞增殖活性（×400，n = 3）
Figure 2. EDU detection of cell proliferation activity in each group（×400，n = 3）
图 3 各组细胞周期分布和变化情况的比较（n = 3）
A：miR-NC组；B：miR-346 inhibitor组；C：miR-346 inhibitor+si-NC组；D：miR-346 inhibitor+si-SMAD3组。
Figure 3. Comparison of cell cycle distribution and changes among the groups（n = 3）
图 4 各组细胞周期相关蛋白表达水平的比较（n = 3）
A：WB检测各组细胞周期相关蛋白表达；B：Cyclin D1蛋白表达水平的比较；C：CDK4蛋白表达水平的比较；D：CDK6蛋白表达水平的比较；E：p21蛋白表达水平的比较与miR-NC组相比，▽P < 0.05；与miR-346 inhibitor组相比，#P < 0.05；与miR-346 inhibitor+si-NC组相比，△P < 0.05。
Figure 4. Comparison of the cell cycle-related protein expression levels among the groups（n = 3）
图 5 各组细胞软骨内成骨相关蛋白表达水平的比较（n = 3）
A：WB检测各组细胞软骨内成骨相关蛋白表达；B：MMP-13蛋白表达水平的比较；C：COL10A蛋白表达水平的比较；D：RUNX2蛋白表达水平的比较；E：OCN蛋白表达水平的比较；F：OPN蛋白表达水平的比较。与miR-NC组相比，▽P < 0.05；与miR-346 inhibitor组相比，#P < 0.05；与miR-346 inhibitor+si-NC组相比，△P < 0.05。
Figure 5. Comparison of the expression levels of osteogenesis-related proteins in chondrocytes among the groups（n = 3）
图 6 miR-346靶向SMAD3（n = 3）
A：miR-346与SMAD3结合靶点；B：各组细胞SMAD3表达水平的比较。与miR-NC组相比，▽P < 0.05；与miR-346 inhibitor组相比，#P < 0.05；与miR-346 inhibitor+si-NC组相比，△P < 0.05。
Figure 6. miR-346 targets SMAD3 （n = 3）
表 1 引物序列信息
Table 1. Primer sequence information
基因 引物
方向序列 5'→3' miR-346 F ACACTCCAGCTGGGAGGCAGATGCGGA R TGGTGTCGTGGAGTCG U6 F CTCGCTTCGGCAGCACATACT R ACGCTTCACGATTGCGTGT SMAD3 F AGTGGAGCTGACACGGAGAC R AGCGAACTCCTGGTTGTTGA β-actin F CGACCCATCCTACGAGC R TAGGCTGCGTCACTTGC 
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表 2 各组细胞miR-346表达、细胞增殖活力和EDU阳性细胞比例的比较（$ \bar x \pm s $，n = 3）
Table 2. Comparison of miR-346 expression，cell proliferation viability and EDU positive cell ratio in each group （$ \bar x \pm s $，n = 3 ）
组别 miR-346 细胞增殖活力（OD450） EDU阳性比例 0 h 24 h 48 h 72 h miR-NC组 1.00 ± 0.05 0.13 ± 0.03 0.39 ± 0.05 0.78 ± 0.09 1.19 ± 0.10 0.13 ± 0.02 miR-346 inhibitor组 0.25 ± 0.02▽ 0.15 ± 0.02 0.57 ± 0.04▽ 1.23 ± 0.15▽ 1.69 ± 0.13▽ 0.29 ± 0.03▽ miR-346 inhibitor+si-NC组 0.26 ± 0.03▽ 0.15 ± 0.03 0.60 ± 0.05▽ 1.20 ± 0.13▽ 1.72 ± 0.11▽ 0.27 ± 0.04▽ miR-346 inhibitor+si-SMAD3组 0.26 ± 0.02 0.14 ± 0.02 0.44 ± 0.04#△ 0.90 ± 0.08#△ 1.46 ± 0.09#△ 0.18 ± 0.03#△ F 394.738 0.423 14.927 11.004 15.397 17.974 P <0.001* 0.742 0.001* <0.001* <0.001* <0.001* *P < 0.05；与miR-NC组相比，▽P < 0.05；与miR-346 inhibitor组相比，#P < 0.05；与miR-346 inhibitor+si-NC组相比，△P < 0.05。
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表 3 各组细胞周期分布和变化情况的比较（$ \bar x \pm s $，n = 3）
Table 3. Comparison of cell cycle distribution and changes among the group （$ \bar x \pm s $，n = 3）
组别 G1期/（%） S期/（%） G2期/（%） miR-NC组 70.39 ± 3.56 4.59 ± 0.15 26.02 ± 1.13 miR-346 inhibitor组 47.92 ± 2.67▽ 2.20 ± 0.21▽ 50.88 ± 2.46▽ miR-346 inhibitor+si-NC组 47.76 ± 3.02▽ 2.13 ± 0.15▽ 49.11 ± 3.50▽ miR-346 inhibitor+si-SMAD3组 58.07 ± 2.94#△ 4.29 ± 0.17#△ 36.64 ± 1.99#△ F 36.685 177.053 69.051 P <0.001* <0.001* <0.001* *P < 0.05；与miR-NC组相比，▽P < 0.05；与miR-346 inhibitor组相比，#P < 0.05；与miR-346 inhibitor+si-NC组相比，△P < 0.05。
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