长链非编码RNA KLHL7-DT对人退变髓核细胞增殖和凋亡的影响及其相关机制

doi:10.3760/cma.j.cn101202-20220707-00204

摘要
图/表
参考文献
相关文章 (15)

全文: PDF (3196 KB) HTML (2 KB)
输出: BibTeX | EndNote (RIS)

摘要目的探讨长链非编码RNA KLHL7-DT对人退变髓核细胞增殖和凋亡的影响及其相关机制。方法选取2018年1月—2019年10月南京江北医院骨科脊柱骨折患者手术切除的正常椎间盘标本18例,其中男11例、女7例,年龄为22~46 （38.3±4.3）岁,PfirrmannⅠ级6例、Ⅱ级12例。取椎间盘标本常规分离、培养髓核细胞,使用10 ng/mL IL-1β处理髓核细胞获得退变髓核细胞。将退变髓核细胞分为沉默对照组、KLHL7-DT沉默组、过表达对照组、KLHL7-DT过表达组。4组细胞分别对应转染沉默对照序列、siRNA-KLHL7-DT沉默序列、过表达对照序列、KLHL7-DT过表达序列。取转染后4组退变髓核细胞采用5-乙炔基-2’脱氧尿嘧啶核苷（EdU）法检测细胞增殖情况,流式细胞术检测细胞凋亡情况,Western blot检测聚集蛋白聚糖（Aggrecan）、Ⅱ型胶原（Col Ⅱ）蛋白的表达情况。结果（1）KLHL7-DT过表达组EdU染色阳性细胞/DAPI染色阳性比值（0.147±0.002）低于过表达对照组（0.203±0.007）,而KLHL7-DT沉默组比值（0.428±0.050）高于沉默对照组（0.240±0.032）,差异均有统计学意义（t=14.25、-5.44,P值均<0.05）。（2）KLHL7-DT过表达组细胞凋亡率（19.01%±0.41%）高于过表达对照组（14.38%±0.31%）,KLHL7-DT沉默组细胞凋亡率（16.08%±0.59%）低于沉默对照组（17.42%±0.36%）,差异均有统计学意义（t=15.69、3.36,P值均<0.05）。（3）Western blot结果显示,KLHL7-DT过表达组细胞Aggrecan和Col Ⅱ蛋白的相对表达量（分别为0.34±0.29、0.57±0.11）均低于过表达对照组（1.00±0.22、1.05±0.10）,KLHL7-DT沉默组Aggrecan和Col Ⅱ蛋白的相对表达量（分别为1.77±0.14、1.63±0.12）均高于沉默对照组（1.10±0.18、0.98±0.08）,差异均有统计学意义（t=3.10、5.54、-5.05、-7.66,P值均<0.05）。结论上调KLHL7-DT的表达可抑制退变髓核细胞的增殖,促进退变髓核细胞的凋亡,其机制可能是通过调节细胞外基质Aggrecan、Col Ⅱ蛋白的合成,进而参与椎间盘退变的发展。

	服务

	把本文推荐给朋友
	加入我的书架
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	孙中仪
	田纪伟

关键词 ：椎间盘退行性变, 髓核细胞, 长链非编码RNA, 细胞增殖, 细胞凋亡, 聚集蛋白聚糖, Ⅱ型胶原

Abstract：Objective This study aims to evaluate the effect of long non-coding RNA KLHL7-DT on the proliferation and apoptosis of degenerate nucleus pulposus cells and its related mechanism. Methods A total of 18 normal intervertebral disc specimens were selected from orthopedic spine fracture patients in Nanjing Jiangbei Hospital from January 2018 to October 2019, including 11 males and 7 females, with an average age of 22-46 (38.3±4.3) years old. There were 6 and 12 patients with Pfirrmann grades Ⅰ and Ⅱ, respectively. Nucleus pulposus cells were routinely isolated and cultured from normal intervertebral discs removed by the patient. Then, the degraded nucleus pulposus cells were obtained by treating them with 10 ng/mL IL-1β. The degenerated nucleus pulposus cells were divided into silencing control, KLHL7-DT silencing, overexpression control, and KLHL7-DT overexpression groups. The four groups of cells were corresponding to the transfected silenced control, siRNA-KLHL7-DT silenced, overexpressed control, and KLHL7-DT overexpressed sequences, respectively. Cell proliferation was detected by the 5-acetyne-2 'deoxyuracil nucleoside (EdU) method, cell apoptosis was detected by flow cytometry, and the expression of aggrecan and type Ⅱ collagen (Col Ⅱ) protein was detected by Western blot analysis. Results (1) The EdU staining positive cell/DAPI staining positive ratio in the KLHL7-DT overexpression group (0.147±0.002) was lower than that in the control group (0.203±0.007), and the EdU staining positive cell/DAPI staining positive ratio in the KLHL7-DT silencing group (0.428±0.050) was higher than that in the control group (0.240±0.032). The differences were statistically significant (t=14.02,-5.44, P<0.05). (2) The apoptosis rate of the KLHL7-DT overexpression group (19.01%±0.41%) was higher than that of the overexpression control group (14.38%±0.31%), and the apoptosis rate of the KLHL7-DT silenced group (16.08%±0.59%) was lower than that of the silenced control group (17.42%±0.36%). The differences were statistically significant (t=15.69, 3.36, P<0.05). (3) Western blot analysis results showed that the relative expression levels of aggrecan and Col Ⅱ in the KLHL7-DT overexpression group were 0.34±0.29 and 0.57±0.11, which were lower than 1.00±0.22 and 1.05±0.10 in the overexpression control group, respectively. The relative expression levels of aggrecan and Col Ⅱ in the KLHL7-DT silenced group were 1.77±0.14 and 1.63±0.12, respectively, which were higher than those in the KLHL7-DT silenced group (1.10±0.18 and 0.98±0.08). A statistical significance was observed between the two groups (all P values<0.05). Conclusion The upregulation of the KLHL7-DT expression can inhibit the proliferation and promote the apoptosis of degraded nucleus pulposus cells which may be involved in the development of intervertebral disc degeneration by regulating the synthesis of extracellular matrix aggrecan and Col Ⅱ proteins.

Key words： Intervertebral disc degeneration Nucleus pulposus cells Long non-coding RNA Cell proliferation Apoptosis Aggrecan Type Ⅱ collagen

收稿日期: 2022-07-07

基金资助:江苏省科研培育项目（JKK201902）; 南京市医学科技发展专项基金（YKK20182）

通讯作者: 田纪伟,Email：tjw609@163.com

引用本文:

孙中仪, 田纪伟. 长链非编码RNA KLHL7-DT对人退变髓核细胞增殖和凋亡的影响及其相关机制[J]. 中华解剖与临床杂志, 2023, 28(4): 269-274.
Sun Zhongyi, Tian Jiwei. Expression of long non-coding RNA KLHL7-DT in human intervertebral disc and its effect on proliferation and apoptosis of nucleus pulposus cells and its related mechanism. Chinese Journal of Anatomy and Clinics, 2023, 28(4): 269-274.

链接本文:

http://www.cjac.com.cn/CN/10.3760/cma.j.cn101202-20220707-00204 或 http://www.cjac.com.cn/CN/Y2023/V28/I4/269

[1]	Cazzanelli P, Wuertz-Kozak K.MicroRNAs in intervertebral disc degeneration, apoptosis, inflammation, and mechanobiology[J]. Int J Mol Sci, 2020, 21(10): 3601. DOI: 10.3390/ijms21103601.
[2]	Zhu J, Yu W, Wang Y, et al.lncRNAs: function and mechanism in cartilage development, degeneration, and regeneration[J]. Stem Cell Res Ther, 2019,10(1):344. DOI: 10.1186/s13287-019-1458-8.
[3]	李瑶, 孙中仪, 戴健, 等. 椎间盘退变IL-1β激活NF-κB信号通路增强ADAMTS-4的表达[J].中国矫形外科杂志, 2021, 29(23): 2177-2181. DOI: 10.3977/j.issn.1005-8478.2021.23.14Li Y, Sun ZY, Dai J, et al.IL-1β activates the NF-κB signal pathway to enhance the expression of ADAMTS-4 in intervertebral disc degeneration[J]. Orthopedic J China, 2021, 29(23): 2177-2181. DOI: 10.3977/j.issn.1005-8478.2021.23.14
[4]	Zhang GZ, Deng YJ, Xie QQ, et al.Sirtuins and intervertebral disc degeneration: Roles in inflammation, oxidative stress, and mitochondrial function[J]. Clin Chim Acta, 2020,508:33-42. DOI: 10.1016/j.cca.2020.04.016.
[5]	Brusko GD, Wang MY.Endoscopic lumbar interbody fusion[J]. Neurosurg Clin N Am, 2020,31(1):17-24. DOI: 10.1016/j.nec.2019.08.002.
[6]	Li Z, Chen X, Xu D, et al.Circular RNAs in nucleus pulposus cell function and intervertebral disc degeneration[J]. Cell Prolif, 2019,52(6):e12704. DOI: 10.1111/cpr.12704.
[7]	Tendulkar G, Chen T, Ehnert S, et al.Intervertebral disc nucleus repair: hype or hope?[J]. Int J Mol Sci, 2019, 20(15): 3622. DOI: 10.3390/ijms20153622.
[8]	Li Z, Li X, Chen C, et al.Long non-coding RNAs in nucleus pulposus cell function and intervertebral disc degeneration[J]. Cell Prolif, 2018,51(5):e12483. DOI: 10.1111/cpr.12483.
[9]	于召龙, 王云浩, 黄凯, 等. 长链非编码RNA及其介导椎间盘退变机制的研究进展[J]. 中国矫形外科杂志, 2019, 27(17): 1585-1588. DOI: 10.3977/j.issn.1005-8478.2019.17.10.Yu ZL, Wang YH, Huang K, et al.Research progress of long non-coding RNAs and their mediating effects in the mechanism of intervertebray disc degeneration[J]. Orthopedic J China, 2019, 27(17): 1585-1588. DOI: 10.3977/j.issn.1005-8478.2019.17.10.
[10]	王毅峰, 宋庆鑫, 黄轩, 等. TargetScan筛选退变椎间盘髓核细胞凋亡相关lncRNA的研究[J].中国矫形外科杂志, 2016, 24(7): 643-647. DOI: 10.3977/j.issn.1005-8478.2016.07.14.Wang YF, Song QX, Huang X, et al.TargetScan as a tool in screening of apoptosis related long non-coding RNA in degenerative intervertebral nucleus pulposus cell[J]. Orthopedic J China, 2016, 24(7): 643-647. DOI: 10.3977/j.issn.1005-8478.2016.07.14.
[11]	Sun Z, Tang X, Wang H, et al.LncRNA H19 aggravates intervertebral disc degeneration by promoting the autophagy and apoptosis of nucleus pulposus cells through the miR-139/CXCR4/NF-κB Axis[J]. Stem Cells Dev, 2021,30(14):736-748. DOI: 10.1089/scd.2021.0009.
[12]	Chen X, Li Z, Xu D, et al.LINC01121 induced intervertebral disc degeneration via modulating miR-150-5p/MMP16 axis[J]. J Gene Med, 2020,22(10):e3231. DOI: 10.1002/jgm.3231.
[13]	Cui S, Liu Z, Tang B, et al.LncRNA MAGI2-AS3 is down-regulated in intervertebral disc degeneration and participates in the regulation of FasL expression in nucleus pulposus cells[J]. BMC Musculoskelet Disord, 2020,21(1):149. DOI: 10.1186/s12891-020-3086-y.
[14]	孙中仪, 晏鹏, 王圣杰, 等. 长链非编码RNA在人退变椎间盘组织中的基因表达谱[J]. 中华医学杂志, 2017, 97(33): 2582-2586. DOI: 10.3760/cma.j.issn.0376-2491.2017.33.006.Sun ZY, Yan P, Wang SJ, et al.Gene expression profiles of long non-coding RNAs in human degenerated intervertebral disc tissue[J]. Chinese Medical J, 2017, 97(33): 2582-2586. DOI: 10.3760/cma.j.issn.0376-2491.2017.33.006.
[15]	Pfirrmann CW, Metzdorf A, Zanetti M, et al.Magnetic resonance classification of lumbar intervertebral disc degeneration[J]. Spine (Phila Pa 1976), 2001,26(17):1873-1878. DOI: 10.1097/00007632-200109010-00011.
[16]	王云浩, 孙中仪, 于召龙, 等. Linc01776在椎间盘退行性变中生物学功能的研究[J]. 现代生物医学进展, 2019, 19(18): 3405-3409,3481. DOI: 10.13241/j.cnki.pmb.2019.18.002.Wang YH, Sun ZY, Yu ZL, et al.The biological function analysis of Linc01776 in the degeneration of intervertebral disc[J]. Progress in Modern Biomedicine, 2019, 19(18): 3405-3409,3481. DOI: 10.13241/j.cnki.pmb.2019.18.002.
[17]	Ran R, Liao HY, Wang ZQ, et al.Mechanisms and functions of long noncoding RNAs in intervertebral disc degeneration[J]. Pathol Res Pract, 2022,235:153959. DOI: 10.1016/j.prp.2022.153959.
[18]	Ouyang ZH, Wang WJ, Yan YG, et al.The PI3K/Akt pathway: a critical player in intervertebral disc degeneration[J]. Oncotarget, 2017,8(34):57870-57881. DOI: 10.18632/oncotarget.18628.
[19]	童通,雷涛,申勇.椎间盘退行性变中细胞凋亡的分子机制[J].中国矫形外科杂志, 2017, 25(19): 1788-1792. DOI: 10.3977/j.issn.1005-8478.2017.19.12.Tong T, Lei T, Shen Y.Molecular mechanism of apoptosis in intervertebral disc degeneration[J]. Orthopedic J China, 2017, 25(19): 1788-1792. DOI: 10.3977/j.issn.1005-8478.2017.19.12.
[20]	Tang N, Dong Y, Xiao T, et al.LncRNA TUG1 promotes the intervertebral disc degeneration and nucleus pulposus cell apoptosis though modulating miR-26a/HMGB1 axis and regulating NF-κB activation[J]. Am J Transl Res, 2020,12(9):5449-5464.
[21]	Chen J, Jia YS, Liu GZ, et al.Role of LncRNA TUG1 in intervertebral disc degeneration and nucleus pulposus cells via regulating Wnt/β-catenin signaling pathway[J]. Biochem Biophys Res Commun, 2017,491(3):668-674. DOI: 10.1016/j.bbrc.2017.07.146.
[22]	Koch CD, Lee CM, Apte SS.Aggrecan in cardiovascular development and disease[J]. J Histochem Cytochem, 2020,68(11):C952902.
[23]	王晖, 李政, 张翔, 等. 襁褓体位对大鼠髋关节软骨中二型胶原、蛋白聚糖及金属基质蛋白酶-13表达的影响[J].解剖与临床,2013,18(5):385-389. DOI: 10.3969/j.issn.1671-7163.2013.05.007.
[24]	Ruan Z, Ma H, Li J, et al.The long non-coding RNA NEAT1 contributes to extracellular matrix degradation in degenerative human nucleus pulposus cells[J]. Exp Biol Med (Maywood),2018, 243(7): 595-600. DOI: 10.1186/s40001-021-00481-2.