miR-1260b regulates osteogenic differentiation of periodontal ligament stem cells by targeting ATF6β

LIU Qiang; LIU Ya-hui; LI Li-kun; GUO Jian-ru

doi:10.11659/jjssx.03E024103

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miR-1260b regulates osteogenic differentiation of periodontal ligament stem cells by targeting ATF6β

Basic Research | 更新时间：2025-09-24

- miR-1260b regulates osteogenic differentiation of periodontal ligament stem cells by targeting ATF6β
- JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY Vol. 34, Issue 9, Pages: 765-771(2025)
- 作者机构：
  
  1.唐山职业技术学院附属医院口腔牙周科，河北唐山 063000
  2.唐山职业技术学院附属医院检验科，河北唐山 063000
  3.唐山职业技术学院附属医院病理科，河北唐山 063000
- 作者简介：
- 基金信息：
- DOI：10.11659/jjssx.03E024103
  CLC： R781.4
- Received：20 March 2024，
  
  Published：25 September 2025
- 稿件说明：
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柳强,刘亚辉,李立坤等.miR-1260b通过靶向ATF6β调控牙周膜干细胞成骨分化的作用研究[J].局解手术学杂志,2025,34(09):765-771.

LIU Qiang,LIU Ya-hui,LI Li-kun,et al.miR-1260b regulates osteogenic differentiation of periodontal ligament stem cells by targeting ATF6β[J].JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY,2025,34(09):765-771.
柳强,刘亚辉,李立坤等.miR-1260b通过靶向ATF6β调控牙周膜干细胞成骨分化的作用研究[J].局解手术学杂志,2025,34(09):765-771. DOI： 10.11659/jjssx.03E024103.

LIU Qiang,LIU Ya-hui,LI Li-kun,et al.miR-1260b regulates osteogenic differentiation of periodontal ligament stem cells by targeting ATF6β[J].JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY,2025,34(09):765-771. DOI： 10.11659/jjssx.03E024103.

摘要

目的

探究miR-1260b靶向活化转录因子6β（ATF6β）在人牙周膜干细胞（hPDLSCs）成骨分化中的作用。

方法

从牙周炎患者拔除的第三磨牙上刮取牙周膜组织，并分离培养hPDLSCs。使用流式细胞术检测CD34、CD45、CD29、CD90、CD105阳性细胞的比例，免疫荧光染色检测vimentin和pan-cytokeratin表达。采用双荧光素酶报告基因实验分析miR-1260b mimic+WT/MUT组（转染miR-1260b mimic和ATF6β-WT/MUT）、mimic-NC组（转染miR-NC和ATF6β-WT/MUT）中ATF6β-WT和ATF6β-MUT的细胞荧光素酶活性。另外将细胞分为miR-1260b mimic组（单独转染miR-1260b mimic）、ATF6β-OE组（单独转染ATF6β过表达载体）、联合组（同时转染miR-1260b mimic和ATF6β过表达载体）及NC组（转染空载体）。对NC组、miR-1260b mimic组、ATF6β-OE组、联合组hPDLSCs进行茜素红S及碱性磷酸酶染色，同时采用qRT-PCR检测各组细胞miR-1260b、ATF6β、Runt相关转录因子2（Runx2）、骨钙素（OCN）、骨桥蛋白（OPN）的mRNA表达水平。采用Western blot检测各组细胞葡萄糖调节蛋白78（GRP78）、C/EBP同源蛋白（CHOP）、蛋白激酶R样内质网激酶（PERK）蛋白表达。

结果

流式细胞术检测显示，分离培养的细胞表面CD29、CD90、CD105呈阳性，CD34、CD45呈阴性；免疫荧光染色显示，细胞vimentin呈阳性，pan-cytokeratin呈阴性，符合hPDLSCs的特征。荧光素酶检测显示，miR-1260b mimic能显著降低ATF6β-WT的荧光素酶活性（

0.05），而对ATF6β-MUT荧光素酶活性无明显影响（

0.05）。茜素红S染色显示，hPDLSCs培养3 d、5 d、7 d染色逐渐加深，提示成骨分化能力增强；随着hPDLSCs成骨分化能力增强，细胞内miR-1260b表达呈逐渐升高趋势，而ATF6β的mRNA及蛋白表达水平则呈逐渐下降趋势（

0.05）。茜素红S染色和碱性磷酸酶染色结果显示，miR-1260b mimic组细胞的阳性面积占比高于NC组（

0.05），ATF6β-OE组和联合组细胞的阳性面积占比则低于NC组（

0.05）。miR-1260b mimic组细胞ATF6β mRNA及蛋白水平低于NC组（

0.05），ATF6β-OE组和联合组细胞ATF6β mRNA及蛋白水平高于NC组（

0.05）。miR-1260b mimic组细胞Runx2、OCN、OPN的mRNA水平显著高于NC组（

0.05）；ATF6β-OE组和联合组细胞Runx2、OCN、OPN的mRNA水平显著低于NC组（

0.05）。miR-1260b mimic组细胞GRP78、CHOP、PERK蛋白表达水平显著低于NC组（

0.05）；ATF6β-OE组和联合组细胞GRP78、CHOP、PERK蛋白表达水平显著高于NC组（

0.05）。

结论

miR-1260b可通过靶向ATF6β抑制hPDLSCs内质网应激水平，促进hPDLSCs成骨分化。

Abstract

Objective

To explore the role of miR-1260b targeting activating transcription factor 6β （ATF6β） in osteogenic differentiation of human periodontal ligament stem cells （hPDLSCs）.

Methods

The periodontal ligament tissue was scraped from the third molar extracted from the patient with periodontitis，and hPDLSCs were isolated and cultured.The proportions of positive cells of CD34，CD45，CD29，CD90 and CD105 were detected by flow cytometry， and the expression of vimentin and pan-cytokeratin were detected by immunofluorescence staining. The cellular luciferase activities of ATF6β-WT and ATF6β-MUT in the miR-1260b mimic+WT/MUT group （transfected with miR-1260b mimic and ATF6β-WT/MUT） and the mimic-NC group （transfected with miR-NC and ATF6β-WT/MUT） were analyzed by the dual luciferase reporter gene assay. In addition，the cells were divided into the miR-1260b mimic group （transfected with miR-1260b mimic alone），the ATF6β-OE group （transfected with the ATF6β overexpression vector alone），the combined group （transfected with miR-1260b mimic and ATF6β overexpression vector simultaneously），and the NC group （transfected with empty vector）. Alizarin red S staining and alkaline phosphatase staining were performed on hPDLSCs in the NC group，the miR-1260b mimic group，the ATF6β-OE group and the combined group. Meanwhile，the mRNA expression levels of miR-1260b，ATF6β，Runt-related transcription factor 2 （Runx2），osteocalcin （OCN），and osteopontin （OPN） in each group of cells were detected by qRT-PCR. The protein expressions of glucose-regulated protein 78 （GRP78），C/EBP homologous protein （CHOP），and protein kinase R-like endoplasmic reticulum kinase （PERK） in each group of cells were detected by Western blot.

Results

The flow cytometry detection showed that CD29，CD90 and CD105 on the surface of the isolated and cultured cells were positive，while CD34 and CD45 were negative. Immunofluorescence staining showed that vimentin in the cells was positive and pan-cytokeratin was negative，which was in line with the characteristics of hPDLSCs. Luciferase assay showed that miR-1260b mimic could significantly reduce the luciferase activity of ATF6β-WT （

0.05），but had no significant effect on the luciferase activity of ATF6β-MUT （

0.05）. After alizarin red S staining，the staining of hPDLSCs gradually deepened after 3 days，5 days and 7 days of culture，suggesting an enhanced osteogenic differentiation ability. With the enhancement of osteogenic differentiation ability of hPDLSCs，the expression of miR-1260b in the cells showed a gradually increasing trend，while the mRNA and protein expression levels of ATF6β showed a gradually decreasing trend （

0.05）. The results of alizarin red S staining and alkaline phosphatase staining showed that the proportions of positive area in the miR-1260b mimic group were higher than those in the NC group （

0.05），while the proportions of positive area in the ATF6β-OE group and the combined group were lower than those in the NC group （

0.05）. The mRNA and protein levels of ATF6β in the miR-1260b mimic group were lower than those in the NC group （

0.05），while the mRNA and protein levels of ATF6β in the ATF6β-OE group and the combined group were higher than those in the NC group （

0.05）. The mRNA levels of Runx2，OCN and OPN in the miR-1260b mimic group were significantly higher than those in the NC group （

0.05）. The mRNA levels of Runx2，OCN and OPN in the ATF6β-OE group and the combined group were significantly lower than those in the NC group （

0.05）.The protein expression levels of GRP78，CHOP and PERK in the miR-1260b mimic group were significantly lower than those in the NC group （

0.05）. The protein expression levels of GRP78，CHOP and PERK in the ATF6β-OE group and the combined group were significantly higher than those in the NC group （

0.05）.

Conclusion

miR-1260b can inhibit endoplasmic reticulum stress level and promote osteogenic differentiation of hPDLSCs by targeting ATF6β.

关键词

Keywords

references

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