Silencing of Long Non-Coding RNA NONHSAT009968 Ameliorates the Staphylococcal Protein A-Inhibited Osteogenic Differentiation in Human Bone Mesenchymal Stem Cells.
OBJECTIVE
Osteomyelitis is defined as an inflammation of the bones and bone marrow. The inflammatory microenvironment attenuates the osteogenic differentiation capacity of stem cells and inhibits osteoblast-mediated bone formation, leading to net bone loss. However, the whole expression profile, function and side effect of long non-coding RNAs (lncRNAs) on osteogenic differentiation of stem cells in an inflammatory microenvironment of osteomyelitis are not known.


METHODS
In the present study, human bone mesenchymal stem cells (hBMSCs) were treated with different concentrations of Staphylococcal protein A (SpA) to trigger an inflammatory microenvironment in vitro to partly duplicate the inflammatory microenvironment of osteomyelitis, which was confirmed using ELISA for detecting the inflammatory cytokines. The complete expression profiles of lncRNAs and mRNA during osteogenic differentiation of hBMSCs in an inflammatory microenvironment triggered by SpA were analyzed using a lncRNA microarray. LncRNA expression levels were verified by quantitative reverse transcription PCR analysis (qRT-PCR). The expression of NONHSAT009968 in hB-MSCs was silenced by infection with lentivirus expressing NONHSAT009968-shRNA. The expression of Runx2, OCN, OPN, COL1A1, and alkaline phosphatase (ALP) activity was detected by western blot. Alizarin red staining and ALP activity detection were carried out.


RESULTS
The results of ELISA showed that SpA treatment induced secretion of inflammatory cytokines IL-1A, IL-6, and TNFA. The results of alizarin red staining and ALP detection showed that SpA treatment suppressed the osteogenic differentiation of hBMSCs. A total of 2033 lncRNAs were found with aberrant expression in SpA-treated hBMSCs compared to controls. Among these lncRNAs, 641 were down-regulated and 1392 were up-regulated. Based on the results of qRT-PCR, lncRNA NONHSAT009968 was chosen for further investigation. The results of alizarin red staining, ALP activity detection, and western blot detection of Runx2, OCN, OPN, COL1A1, and ALP indicated that NONHSAT009968 silencing ameliorates SpA-inhibited osteogenic differentiation in hBMSCs.


CONCLUSIONS
Our present study provides a basis for future analyses of the role of lncRNAs in osteoblastic differentiation in an inflammatory environment triggered by SpA, and lncRNA NONHSAT009968 might be a new target for promoting osteoblast formation.
The Third Military Medical University, Chongqing, China.
Yi
Y
Cui
Sheng
S
Lu
Hongbo
H
Tan
Jun
J
Li
Min
M
Zhu
Yongqing
Y
Xu
D000469:Alkaline Phosphatase / Q000235:genetics / Q000276:immunology; D001842:Bone and Bones / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D002454:Cell Differentiation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000276:immunology; D000090602:Collagen Type I, alpha 1 Chain; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000276:immunology; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007249:Inflammation; D053582:Interleukin-1alpha / Q000235:genetics / Q000276:immunology; D015850:Interleukin-6 / Q000235:genetics / Q000276:immunology; D016086:Lentivirus / Q000235:genetics / Q000276:immunology; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D020411:Oligonucleotide Array Sequence Analysis; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D010012:Osteogenesis / Q000187:drug effects* / Q000235:genetics; D053495:Osteopontin / Q000235:genetics / Q000276:immunology; D062085:RNA, Long Noncoding / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology; D034741:RNA, Small Interfering / Q000235:genetics / Q000276:immunology; D015398:Signal Transduction; D013205:Staphylococcal Protein A / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000276:immunology
D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D050659:Core Binding Factor Alpha 1 Subunit; C508584:IL1A protein, human; C508600:IL6 protein, human; D053582:Interleukin-1alpha; D015850:Interleukin-6; D062085:RNA, Long Noncoding; D034741:RNA, Small Interfering; C493709:RUNX2 protein, human; D013205:Staphylococcal Protein A; D014409:Tumor Necrosis Factor-alpha; D053495:Osteopontin; D000469:Alkaline Phosphatase