中国骨质疏松杂志

lncRNA LINC00339调控细胞自噬抑制BMSC成骨的机制研究

Mechanism of regulation of autophagy and inhibition of BMSC osteogenesis by lncRNA LINC00339

DOI：10.3969/j.issn.1006-7108.2024.08.009

英文关键词:LINC00339 bone marrow derived mesenchymal stem cells autophagy osteogenic differentiation

基金项目:新疆维吾尔自治区人民医院院内项目（20220206）

作者	单位
李祖涛蔡昱徐江波孙俊刚车立新*	新疆维吾尔自治区人民医院，新疆乌鲁木齐 830001

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中文摘要:

目的探讨lncRNA LINC00339调控细胞自噬并抑制骨髓间充质干细胞（bone marrow derived mesenchymal stem cells，BMSC)成骨分化的可能机制。方法：以大鼠原代BMSC为研究对象，根据给予的处理因素不同，分为以下4组：敲减LINC00339组、敲减BECN1组、空载对照组、敲减LINC00339+敲减BECN1组。分别应用ELISA检测试剂盒检测细胞上清液中ALP、BGP、PICP；Western blot检测成骨相关指标BMP2、Osterix、Runx2以及自噬相关蛋白BECN1、LC3-Ⅱ/Ⅰ、p62的表达；应用茜素红染色技术检测成骨分化；通过免疫荧光检测LC3斑点。结果与对照组相比， ELISA法检测结果显示siLINC00339组ALP、BGP、PICP显著增高；Western blot结果显示成骨相关蛋白BMP2、Osterix、Runx2和自噬相关蛋白BECN1、LC3-Ⅱ/Ⅰ、p62蛋白表达上调；茜素红染色后细胞视野可见成骨明显增多；免疫荧光观察LC3蛋白明显增多，以上结果差异均有统计学意义（P＜0.05）；与对照组相比，siBECN1组ALP、BGP、PICP显著下降，成骨相关基因BMP2、Osterix、Runx2 和自噬相关蛋白BECN1、LC3-Ⅱ/Ⅰ、p62表达下调，茜素红染色后细胞视野可见成骨明显减少，免疫荧光观察LC3蛋白明显减少，以上结果差异均有统计学意义（P＜0.05）；而双基因操作的siLINC00339+ siBECN1组以上各检测结果分别与siLINC00339组和siBECN1组相比差异均有统计学意义（P＜0.05），但与对照组相比均无统计学意义（P＞0.05）。结论 lncRNA LINC00339可抑制BMSC成骨分化，其机制可能与调控了BECN1参与的细胞自噬现象有关。

英文摘要:

Objective To explore the possible mechanism of lncRNALINC00339 in regulating autophagy and inhibiting osteogenic differentiation by BMSC. Methods The primary BMSCs of rats were studied and divided into four groups based on different treatment, including knockdown LINC00339 group, knockdown BECN1 group, empty control group, and knockdown LINC00339+knockdown BECN1 group. ELISA detection kits were used to detect ALP, BGP, and PICP in cell supernatants. Western blotting was used to detect the expression of osteogenic related markers BMP2, Osterix, Runx2, as well as autophagy related proteins BECN1, LC3-II/I, and p62. Alizarin red staining was used to detect osteogenic differentiation. LC3 spots were detected with immunofluorescence. Results ELISA test results showed a significant increase in ALP, BGP, and PICP in siLINC00339 group. Western blotting results showed that the expression of osteogenic-related proteins BMP2, Osterix, Runx2, and autophagy related proteins BECN1, LC3-II/I, and p62 were up-regulated. After staining with Alizarin red, there was a significant increase in osteogenesis in the cell field of view. Immunofluorescence observation showed a significant increase in LC3 protein, and the differences in the above results were statistically significant (P<0.05). Compared with the control group, siBECN1 group showed a significant decrease in ALP, BGP, and PICP, while the expressions of osteogenic related genes BMP2, Osterix, Runx2, and autophagy related proteins BECN1, LC3-II/I, and p62 were down-regulated. After staining with Alizarin red, the cell field of view showed a significant decrease in osteogenesis, while immunofluorescence showed a significant decrease in LC3 protein. The above results showed statistical significance (P<0.05). The above test results in the siLINC00339+siBECN1 group with dual gene operation showed statistically significant differences compared to those in the siLINC00339 group and siBECN1 group (P<0.05), but there was no statistically significant difference compared to the control group (P>0.05). Conclusion LncRNA LINC00339 inhibits the osteogenic differentiation of BMSC, Its mechanism may be related to the regulation of autophagy by BECN1.

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