Sun Xiang, Cao Junchuan, Han Jiusong, Jia Bo, Wang Jing, Lian Junxiang, Gao Jianwei, Liu Shuguang, Xiao Hui
Department of Oral and Maxillofacial Surgery, Stomatological Hospital, Southern Medical University (Guangdong Provincial Stomatological Hospital), Guangzhou, Guangdong 510280, China.
Department of Stomatology, Chengdu Women's and Children's Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, Sichuan 611731, China.
Biomed Res Int. 2021 Mar 26;2021:5512370. doi: 10.1155/2021/5512370. eCollection 2021.
This study is aimed at investigating the role of long noncoding RNA (lncRNA) RP11-815M8.1 in the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).
RT-PCR was used to detect the expression of lncRNA RP11-815M8.1 before and after osteogenic differentiation of hBMSCs. The lncRNA RP11-815M8.1 in hBMSCs was overexpressed or silenced via lentiviral transfection. The transfection efficiency was detected by RT-PCR, and the proliferation of hBMSCs was determined by CCK-8. After 14 days of osteogenic differentiation of transfected hBMSCs, the expression of osteogenic transcription factors (ALP, OCN, OPN, Runx2, and Osterix) was detected by alizarin red staining and RT-PCR. The mRNAs directly regulated by lncRNA RP11-815M8.1 and targeted miRNAs were analyzed according to the positional relationship between lncRNA and mRNA in the genome and miRanda software.
The expression of lncRNA RP11-815M8.1 enhanced with increasing osteogenic differentiation time of hBMSCs. Two days after the transfection of hBMSCs, lncRNA RP11-815M8.1 expression was significantly increased in the overexpression group and significantly decreased in the knockdown group, compared to control cells. The CCK-8 assay showed that overexpression and knockdown of lncRNA RP11-815M8.1 did not affect the proliferation of hBMSCs. After 14 days of differentiation of hBMSCs, stronger alizarin red staining was observed in the overexpression groups, and the expression of osteogenic transcription factors was increased in the overexpression group compared to the control. In the knockdown group, alizarin red staining and the expression of osteogenic transcription factors were decreased. Bioinformatics analysis showed that lncRNA RP11-815M8.1 was directly associated with one mRNA, 27 interacting miRNAs, and 20 miRNA-targeted mRNAs.
The osteogenic differentiation of hBMSCs can be promoted by lncRNA RP11-815M8.1 in vitro.
本研究旨在探讨长链非编码RNA（lncRNA）RP11 - 815M8.1在人骨髓间充质干细胞（hBMSCs）成骨分化中的作用。
采用RT-PCR检测hBMSCs成骨分化前后lncRNA RP11 - 815M8.1的表达。通过慢病毒转染使hBMSCs中的lncRNA RP11 - 815M8.1过表达或沉默。用RT-PCR检测转染效率，用CCK-8法检测hBMSCs的增殖情况。转染后的hBMSCs成骨分化14天后，通过茜素红染色和RT-PCR检测成骨转录因子（碱性磷酸酶、骨钙素、骨桥蛋白、Runx2和osterix）的表达。根据lncRNA与mRNA在基因组中的位置关系及miRanda软件分析lncRNA RP11 - 815M8.1直接调控的mRNA和靶向的miRNA。
随着hBMSCs成骨分化时间的增加，lncRNA RP11 - 815M8.1的表达增强。hBMSCs转染两天后，与对照细胞相比，过表达组中lncRNA RP11 - 815M8.1表达显著增加，敲低组中显著降低。CCK-8检测显示lncRNA RP11 - 815M8.1的过表达和敲低不影响hBMSCs的增殖。hBMSCs分化14天后，过表达组观察到更强的茜素红染色，与对照组相比，过表达组成骨转录因子的表达增加。在敲低组中，茜素红染色和成骨转录因子的表达降低。生物信息学分析表明lncRNA RP11 - 815M8.1与1个mRNA、27个相互作用的miRNA和20个miRNA靶向的mRNA直接相关。
lncRNA RP11 - 815M8.1在体外可促进hBMSCs的成骨分化。
