miR-10b靶向TGFBR1/SMAD3通路对特发性矮小症的软骨细胞增殖、肥大的影响机制

doi:10.11958/20230659

天津医药 ›› 2024, Vol. 52 ›› Issue (2): 124-128.doi: 10.11958/20230659

miR-10b靶向TGFBR1/SMAD3通路对特发性矮小症的软骨细胞增殖、肥大的影响机制

胡娜(), 李正宇, 叶春风, 吴英, 姚庆, 黄世祥, 李文, 朱海琴^△()

南昌大学第二附属医院儿科（邮编330038）

收稿日期:2023-04-30 修回日期:2023-09-05 出版日期:2024-02-15 发布日期:2024-01-26
通讯作者: ^△ E-mail：904668463@qq.com
作者简介:胡娜（1982），女，主治医师，主要从事儿童生长发育方面研究。E-mail:hunajx@163.com
基金资助:
江西省卫生健康委科技计划项目(20204274)

The mechanism of miR-10b targeting TGFBR1/SMAD3 pathway on chondrocyte proliferation and hypertrophy in idiopathic short stature

HU Na(), LI Zhengyu, YE Chunfeng, WU Ying, YAO Qing, HUANG Shixiang, LI Wen, ZHU Haiqin^△()

Department of Pediatrics, the Second Affiliated Hospital of Nanchang University, Nanchang 330038, China

Received:2023-04-30 Revised:2023-09-05 Published:2024-02-15 Online:2024-01-26
Contact: ^△ E-mail：904668463@qq.com

胡娜, 李正宇, 叶春风, 吴英, 姚庆, 黄世祥, 李文, 朱海琴. miR-10b靶向TGFBR1/SMAD3通路对特发性矮小症的软骨细胞增殖、肥大的影响机制[J]. 天津医药, 2024, 52(2): 124-128.

HU Na, LI Zhengyu, YE Chunfeng, WU Ying, YAO Qing, HUANG Shixiang, LI Wen, ZHU Haiqin. The mechanism of miR-10b targeting TGFBR1/SMAD3 pathway on chondrocyte proliferation and hypertrophy in idiopathic short stature[J]. Tianjin Medical Journal, 2024, 52(2): 124-128.

摘要/Abstract

摘要：

目的研究miR-10b对特发性矮小症（ISS）的影响及作用机制。方法收集ISS患儿（ISS组）和体检健康儿童（健康对照组）各54例，qPCR检验血清miR-10b表达量，分析ISS组患儿血清miR-10b表达与患儿临床资料的关系。采用miR-10b inhibitor、si-TGFBR1及各自阴性对照转染C28/I2细胞，利用CCK-8实验检测C28/I2细胞增殖能力，Western blot检测侏儒相关转录因子2（RUNX2）、X型胶原α1链（COL10A1）、转化生长因子β受体1（TGFBR1）、SMAD3、pSMAD3蛋白表达量。在StarBase数据库筛选miR-10b靶点，利用双萤光素酶报告基因实验验证miR-10b与TGFBR1的靶向关系。结果 ISS组血清miR-10b表达量高于健康对照组，且miR-10b表达越高，患儿的身高、IGF-1、骨特异性碱性磷酸酶的下降越明显（P<0.05）。与NC组相比，miR-10b inhibitor组细胞增殖能力升高，RUNX2、COL10A1、TGFBR1、pSMAD3蛋白表达上调（P<0.05）；StarBase数据库提示miR-10b存在TGFBR1的结合位点，双萤光素酶报告基因实验证实两者结合。与si-NC相比，si-TGFBR1组TGFBR1表达量下调，细胞增殖能力下降（P<0.05）。结论 miR-10b通过靶向TGFBR1/SMAD3通路在特发性矮小症中抑制软骨细胞的增殖、肥大。

关键词: 特发性矮小症, 受体, 转化生长因子βⅠ型, miR-10b, Smad3蛋白质

Abstract:

Objective To investigate the effect and mechanism of microRNA-10b (miR-10b) on idiopathic short stature (ISS). Methods A total of 54 children with ISS and 54 healthy children were collected. The serum expression of miR-10b was detected by RT-qPCR, and the relationship between serum miR-10b expression and clinical data of children with ISS was analyzed. miR-10b inhibitor, si-TGFBR1 and their negative control transfection C28/I2 cells were used. CCK-8 experimental detection was used to detect C28/I2 cell proliferation. Western blot assay was used to detect gnome related transcription factor 2 (RUNX2), collagen type X alpha 1 chain (COL10A1), transforming growth factor beta receptor 1 (TGFBR1), SMAD3 and pSMAD3 protein expression. The target of miR-10b was screened in StarBase database, and the targeting relationship between miR-10b and TGFBR1 was verified by dual luciferase reporter gene assay. Results The serum expression of miR-10b was higher in the ISS group than that of the healthy control group, and the higher the miR-10b expression, the more obvious the decrease of child height, IGF-1 and alkaline phosphatase (P<0.05). Compared with the NC group, the cell proliferation ability and RUNX2, COL10A1, TGFBR1, and pSMAD3 protein expression were up-regulated in the miR-10b inhibitor group (P<0.05). StarBase database suggested that miR-10b had a binding site of TGFBR1, and dual luciferase reporter gene assay confirmed that TGFBR1 interacted with miR-10b (P<0.05). Compared with the si-NC group, the expression of TGFBR1 was down-regulated and the cell proliferation ability was decreased in the si-TGFBR1 group (P<0.05). Conclusion miR-10b inhibits chondrocyte proliferation and hypertrophy in idiopathic short stature by targeting TGFBR1/SMAD3 pathway.

Key words: idiopathic short stature, receptor, transforming growth factor-beta type Ⅰ, miR-10b, Smad3 protein

中图分类号:

R752.8

图/表 7

参考文献 21

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基因名称	引物序列（5′→3′）	产物大小/bp
GAPDH	上游：TGCTGGTGCTGAGTATGTCG	85
GAPDH	下游：GCATGTCAGATCCACAACGG	85
U6	上游：CTCGCTTCGGCAGCACA	105
U6	下游：AACGCTTCACGAATTTGCGT	105
miR-10b	上游：AACGCTTCACGAATTTGCGT	78
miR-10b	下游：GTGCTACCCTGTAGAAC	78
TGFBR1	上游：GTAGCTCTGATGAGTGCAATGAC	120
TGFBR1	下游：CAGATATGGCAACTCCCAGTG	120
SMAD3	上游：AGTGGAGCTGACACGGAGAC	135
SMAD3	下游：AGCGAACTCCTGGTTGTTGA	135

基因名称	引物序列（5′→3′）	产物大小/bp
GAPDH	上游：TGCTGGTGCTGAGTATGTCG	85
GAPDH	下游：GCATGTCAGATCCACAACGG	85
U6	上游：CTCGCTTCGGCAGCACA	105
U6	下游：AACGCTTCACGAATTTGCGT	105
miR-10b	上游：AACGCTTCACGAATTTGCGT	78
miR-10b	下游：GTGCTACCCTGTAGAAC	78
TGFBR1	上游：GTAGCTCTGATGAGTGCAATGAC	120
TGFBR1	下游：CAGATATGGCAACTCCCAGTG	120
SMAD3	上游：AGTGGAGCTGACACGGAGAC	135
SMAD3	下游：AGCGAACTCCTGGTTGTTGA	135

组别		身高/cm		体质量/kg
低表达组		128.6±11.46		23.61±6.15
高表达组		121.1±12.34		21.43±5.23
t		2.314^＊		1.403
组别	BMI/（kg/m²）		IGF-1/（μg/L）		BAP/（U/L）
低表达组	17.31±1.82		241.51±23.56		122.43±22.21
高表达组	16.25±2.06		221.34±32.43		110.39±21.51
t	0.995		2.615^＊		2.023^＊

组别		身高/cm		体质量/kg
低表达组		128.6±11.46		23.61±6.15
高表达组		121.1±12.34		21.43±5.23
t		2.314^＊		1.403
组别	BMI/（kg/m²）		IGF-1/（μg/L）		BAP/（U/L）
低表达组	17.31±1.82		241.51±23.56		122.43±22.21
高表达组	16.25±2.06		221.34±32.43		110.39±21.51
t	0.995		2.615^＊		2.023^＊

组别	miR-10b	细胞增殖活力（OD₄₅₀）
组别	miR-10b	0 h	24 h	48 h	72 h
NC组	1.03±0.12	0.15±0.07	0.41±0.08	0.85±0.12	1.12±0.14
miR-10b inhibitor组	0.23±0.11	0.16±0.05	0.55±0.05	1.21±0.11	1.72±0.12
t	8.512^＊＊	0.201	0.439	3.380^＊	5.636^＊＊

miR-10b靶向TGFBR1/SMAD3通路对特发性矮小症的软骨细胞增殖、肥大的影响机制

The mechanism of miR-10b targeting TGFBR1/SMAD3 pathway on chondrocyte proliferation and hypertrophy in idiopathic short stature

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组别	TGFBR1	细胞增殖活力（OD₄₅₀）
组别	TGFBR1	0 h	24 h	48 h	72 h
si-NC组	0.95±0.08	0.21±0.06	0.59±0.10	0.98±0.11	1.52±0.12
si-TGFB- R1组	0.41±0.12	0.22±0.05	0.45±0.06	0.73±0.13	0.95±0.13
t	6.485^＊	0.222	2.079	2.543	5.580^＊

组别	TGFBR1	细胞增殖活力（OD₄₅₀）
组别	TGFBR1	0 h	24 h	48 h	72 h
si-NC组	0.95±0.08	0.21±0.06	0.59±0.10	0.98±0.11	1.52±0.12
si-TGFB- R1组	0.41±0.12	0.22±0.05	0.45±0.06	0.73±0.13	0.95±0.13
t	6.485^＊	0.222	2.079	2.543	5.580^＊