Long noncoding RNA lncAIS downregulation in mesenchymal stem cells is implicated in the pathogenesis of adolescent idiopathic scoliosis

摘要

LncAIS is downregulated in the BM-MSCs of AIS patients. a Differentially expressed lncRNAs were analyzed by microarray using BM-MSCs from healthy donors versus AIS patients. BM-MSCs were derived from 5 healthy donors and 12 AIS patients. b Location of lncAIS in human genome. LncAIS locates on human chromosome 1, comprising 4 exons. cLncAIS transcript was analyzed in normal BM-MSCs and AIS BM-MSCs by real-time qPCR. BM-MSCs were derived from 20 healthy donors and 30 AIS patients. Primers were listed in Table S1. Relative gene expression folds were normalized to endogenous β-actin and counted as means ± S.D. **P < 0.01. dLncAIS expression in normal BM-MSCs and AIS BM-MSCs were examined by Northern blot. A 217 nt probe of lncAIS (11–228nt) was labeled for northern blot analysis. RNAs were extracted from indicated cells. 18S RNA was used as a loading control. BM-MSCs were derived from 3 healthy donors and 3 AIS patients. eLncAIS displayed no coding potential by CPAT analysis. XIST transcript served as a non-coding gene control. GAPDH and RUNX2 served as coding gene controls. fLncAIS transcript was cloned into pcDNA4-mychisplasmid and transfected into 293 T cells for 48 h. Expression of Myc-fused protein was analyzed by immunoblotting with anti-Myc antibody. KLF4 was used as a coding protein control. g Fractionation of BM-MSCs followed by qPCR. BM-MSCs were lyzed followed by nuclear and cytoplasmic fractionation and RNA extraction for qRT-PCR analysis. ACTIN RNA and GAPDH RNA served as positive controls for cytoplasmic gene expression. U1 RNA served as a positive control for nuclear gene expression. N: nuclear fraction. C: cytoplasmic fraction. Primers were listed in Table S1. The data are from three independent experiments using BM-MSCs derived from 3 healthy donors. hLncAIS was visualized in BM-MSCs by RNA-FISH assays followed with immunofluorescence staining. Red: lncAIS probe; Green: Actin; nuclei were counterstained by DAPI. Scale bar, 20 μm. Probes Sequences were listed in Table S1. More than 100 typical cells were observed