miR-370-3P通过调节JAK2/STAT5B信号通路抑制人关节软骨细胞增殖

陈蓝妮; 张惠姣; 徐雪姣; 许珂; 朱岷

doi:10.16016/j.1000-5404.202101053

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miR-370-3P通过调节JAK2/STAT5B信号通路抑制人关节软骨细胞增殖

基础医学 | 更新时间：2026-01-09

- miR-370-3P通过调节JAK2/STAT5B信号通路抑制人关节软骨细胞增殖
- MiR-370-3P inhibits proliferation of human articular chondrocytes by regulating JAK2/STAT5B signaling pathway
- 第三军医大学学报 2021年43卷第13期页码：1212-1218
- 作者机构：
  
  400014 重庆，重庆医科大学附属儿童医院内分泌科，国家儿童健康与疾病临床医学研究中心，儿童发育疾病研究教育部重点实验室，儿科学重庆市重点实验室
- 作者简介：
  
  [朱岷，E-mail: zhumin323@aliyun.comZHU Min, E-mail: zhumin323@aliyun.com
- 基金信息：
  
  国家自然科学基金面上项目(81170723)
- DOI：10.16016/j.1000-5404.202101053
  中图分类号： R322.72;R392.28;R394.3
- 收稿：2021-01-06，
  
  修回：2021-04-07，
  
  纸质出版：2021-07-15
- 稿件说明：
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陈蓝妮, 张惠姣, 徐雪姣, 等. miR-370-3P通过调节JAK2/STAT5B信号通路抑制人关节软骨细胞增殖[J]. 第三军医大学学报, 2021,43(13):1212-1218.

Lanni CHEN, Huijiao ZHANG, Xuejiao XU, et al. MiR-370-3P inhibits proliferation of human articular chondrocytes by regulating JAK2/STAT5B signaling pathway[J]. Journal of Third Military Medical University, 2021, 43(13): 1212-1218.
陈蓝妮, 张惠姣, 徐雪姣, 等. miR-370-3P通过调节JAK2/STAT5B信号通路抑制人关节软骨细胞增殖[J]. 第三军医大学学报, 2021,43(13):1212-1218. DOI： 10.16016/j.1000-5404.202101053.

Lanni CHEN, Huijiao ZHANG, Xuejiao XU, et al. MiR-370-3P inhibits proliferation of human articular chondrocytes by regulating JAK2/STAT5B signaling pathway[J]. Journal of Third Military Medical University, 2021, 43(13): 1212-1218. DOI： 10.16016/j.1000-5404.202101053.

摘要

目的

研究非编码单链RNA(micro RNA，miRNA)miR-370-3P是否通过调控JAK2/STAT5B信号通路调节人关节软骨细胞(human chondrocytes- articular，HC-a)的增殖，并探讨其潜在作用机制。

方法

① 通过脂质体转染HC-a细胞构建miR-370-3P过表达/低表达人关节软骨细胞模型，分为5组: miR-370-3P模拟物mimic组(miR-370-3P过表达组)、miR-370-3P遏制物inhibitor组(miR-370-3P低表达组)、正常对照组、mimic空载体对照组(mimic normal control，mNC)、inhibitor空载体对照组(inhibitor normal control，iNC)。②miRNA实时荧光定量PCR检测各组HC-a细胞miR-370-3P的表达。③qRT-PCR检测各组HC-a细胞JAK2、STAT5B mRNA表达。④Western blot检测各组HC-a细胞中JAK2、STAT5B蛋白表达。⑤MTT比色法检测各组HC-a细胞相对增殖情况。⑥双荧光素酶实验: 构建JAK2、STAT5B野生型和突变型双荧光素酶报告基因载体，分别与miR-370-3P模拟物和阴性对照序列共转染293T细胞，检测各组荧光素酶活性。

结果

miRNA实时荧光定量PCR结果显示: 与正常对照组相比，mimic组miR-370-3P表达上调(

0.05)，inhibitor组miR-370-3P表达下调(

0.05)，且空载体对照组和正常对照组之间的差异无统计学意义，表明miR-370-3P过表达/低表达软骨细胞模型构建成功。qRT-PCR结果显示: 相比于正常对照组，JAK2和STAT5B在mimic

组的mRNA相对表达量下调，在inhibitor组JAK2 mRNA和STAT5B mRNA相对表达量上调(

0.05)。Western blot检测结果显示: JAK2和STAT5B蛋白表达在mimic组也有显著下调，在inhibitor组上调。MTT检测结果显示: miR-370-3P的过表达会降低HC-a的存活率(

0.05)，提示miR-370-3P的过表达会抑制人关节软骨细胞(HC-a)的增殖。双荧光素酶实验结果显示: miR-370-3P和JAK2-WT(野生型)、STAT5B-WT(野生型)质粒共转染后，荧光表达较对照组有显著下调(

0.05)，提示miR-370-3P可与JAK2-3'UTR、STAT5B-3'UTR直接结合发挥作用。

结论

miR-370-3P可通过直接靶向调控JAK2/STAT5B信号通路进而抑制人关节软骨细胞(HC-a)的增殖。

Abstract

Objective

To investigate whether the non-coding single chain RNA miR-370-3P regulates the proliferation of human articular chondrocytes (HC-a) through the JAK2/STAT5B signaling pathway and to explore its potential mechanism.

Methods

The over- /under-expression of miR-370-3P models were constructed by transfecting HC-a cells

and then the cells were divided into 5 groups: miR-370-3P mimic group (over-expression group)

miR-370-3P inhibitor group (under-expression group)

mimic normal control group (mNC group)

inhibitor normal control group (iNC group)

and normal control group (NC group). The mRNA levels of miR-370-3P

JAK2 and STAT5B in each group were detected by qRT-PCR

and the protein levels of JAK2 and STAT5B were determined using Western blotting. In addition

MTT assay was adopted to measure the proliferation of HC-a cells in each group

and the binding of miR-370-3P with JAK2 and STAT5B was detected by dual luciferase assay.

Results

As compared with the NC group

the mRNA expression of miR-370-3P was upregulated in the mimic group and down-regulated in the inhibitor group (

0.05)

indicating successful construction of miR-370-3P over-expression/under-expression models. By contrast

the mRNA expression of JAK2 and STAT5B was decreased in the mimic group and increased in the inhibitor group (

0.05)

with no si

gnificant difference between the empty vector control groups (mNC and iNC groups) and the NC group. Western blotting results also confirmed the down-regulation of JAK2 and STAT5B in the mimic group and upregulation in the inhibitor group (

0.05). MTT assay showed that miR-370-3P overexpression reduced the survival rate of HC-a cells (

0.05)

suggesting an inhibitory effect on HC-a cells proliferation. The results of dual luciferase assay displayed that after miR-370-3P was co-transfected with JAK2 (wild type) as well as STAT5B (wild type)

the fluorescence intensity was significantly down-regulated compared with the control group (

0.05)

indicating that miR-370-3P can directly bind to JAK2-3'UTR and STAT5B-3'UTR to function.

Conclusion

miR-370-3P can inhibit the proliferation of HC-a cells by direct regulation of JAK2/STAT5B signaling pathway.

关键词

Keywords

references

HALLETT S A, ONO W, ONO N. Growth plate chondrocytes: skeletal development, growth and beyond[J]. Int J Mol Sci, 2019, 20(23): 6009. DOI:10.3390/ijms20236009.

VANNELLI S, BAFFICO M, BUGANZA R, et al . SHOX deficiency in children with growth impairment: evaluation of known and new auxological and radiological indicators[J ] . Italian J Pediatr, 2020, 46(1): 163. DOI:10.1186/s13052-020-00927-z.

许珂, 徐雪姣, 宋萃, 等. 软骨细胞SHOX基因过/低表达的差异miRNA筛选与验证[J]. 第三军医大学学报, 2018, 40(3): 211-215. DOI:10.16016/j.1000-5404.201709050.

XU K, XU X J, SONG C, et al . Screening and verification of differential miRNAs in chondrocytes with over-expression and low expression of SHOX[J ] . J Third Mil Med Univ, 2018, 40(3): 211-215. DOI:10.16016/j.1000-5404.201709050.

GENONI G, MONZANI A, CASTAGNO M, et al . Improving clinical diagnosis in SHOX deficiency: the importance of growth velocity[J ] . Pediatr Res, 2018, 83(2): 438-444. DOI:10.1038/pr.2017.247.

BUNYAN D J, BAFFICO M, CAPONE L, et al . Duplications upstream and downstream of SHOX identified as novel causes of Leri-Weill dyschondrosteosis or idiopathic short stature[J ] . Am J Med Genet A, 2016, 170A(4): 949-957. DOI:10.1002/ajmg.a.37524.

RAZMARA E, BITARAF A, YOUSEFI H, et al . Non-coding RNAs in cartilage development: an updated review[J ] . Int J Mol Sci, 2019, 20(18): 4475. DOI: 10.3390/ijms20184475.

JOUNG Y H, LIM E J, DARVIN P, et al . MSM enhances GH sig naling via the Jak2/STAT5b pathway in osteoblast-like cells and osteoblast differentiation through the activation of STAT5b in MSCs[J ] . PLoS One, 2012, 7(10): e47477. DOI:10.1371/journal.pone.0047477..

莫奇非, 杨鹏, 苏楠, 等. 成纤维细胞生长因子受体在成骨细胞中的表达及作用研究[J]. 第三军医大学学报, 2019, 41(24): 2386-2392. DOI:10.16016/j.1000-5404.201907008.

MO Q F, YANG P, SU N, et al . Expression of fibroblast growth factor receptor and its role in regulating osteoblast differentiation[J ] . J Third Mil Med Univ, 2019, 41(24): 2386-2392. DOI:10.16016/j.1000-5404.201907008.

BERENDSEN A D, OLSEN B R. Bone development[J]. Bone, 2015, 80: 14-18. DOI:10.1016/j.bone.2015.04.035.

JEE Y H, WANG J, YUE S N, et al . Mir-374-5p, mir-379-5p, and mir-503-5p regulate proliferation and hypertrophic differentiation of growth plate chondrocytes in male rats[J ] . Endocrinology, 2018, 159(3): 1469-1478. DOI:10.1210/en.2017-00780.

YU L, CEN X, XIA K, et al . microRNA expression profiles and the potential competing endogenous RNA networks in NELL-1-induced human adipose-derived stem cell osteogenic differentiation[J ] . J Cell Biochem, 2020, 121(11): 4623-4641. DOI:10.1002/jcb.29695.

JIA B, WANG Z P, SUN X, et al . Long noncoding RNA LINC00707 sponges miR-370-3p to promote osteogenesis of human bone marrow-derived mesenchymal stem cells through upregulating WNT2B[J ] . Stem Cell Res Ther, 2019, 10(1): 67. DOI:10.1186/s13287-019-1161-9.

PERNER F, PERNER C, ERNST T, et al . Roles of JAK2 in aging, inflammation, hematopoiesis and malignant transformation[J ] . Cells, 2019, 8(8): 854. DOI: 10.3390/cells8080854.

JIANG L, ZHAO X H, MAO Y L, et al . Long non-coding RNA RP11-468E2.5 curtails colorectal cancer cell proliferation and stimulates apoptosis via the JAK/STAT signaling pathway by targeting STAT5 and STAT6[J ] . J Exp Clin Cancer Res, 2019, 38(1): 465. DOI:10.1186/s13046-019-1428-0..

SANPAOLO E R, ROTONDO C, CICI D, et al . JAK/STAT pathway and molecular mechanism in bone remodeling[J ] . Mol Biol Rep, 2020, 47(11): 9087-9096. DOI:10.1007/s11033-020-05910-9.

HWA V. Human growth disorders associated with impaired GH action: defects in STAT5B and JAK2[J]. Mol Cell Endocrinol, 2021, 519: 111063. DOI: 10.1016/j.mce.2020.111063.

KLAMMT J, NEUMANN D, GEVERS E F, et al . Dominant-negative STAT5B mutations cause growth hormone insensitivity with short stature and mild immune dysregulation[J ] . Nat Commun, 2018, 9(1): 2105. DOI:10.1038/s41467-018-04521-0.

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