Long non-coding RNA XIST promotes osteoporosis by inhibiting the differentiation of bone marrow mesenchymal stem cell by sponging miR-29b-3p that suppresses nicotinamide N-methyltransferase

Figures & data

Table 1. The sequences of the PCR primers in this study

Primer	Sequences
XIST	Forward: 5’-GCTCTTCATTGTTCCTATCTGCC-3’
	Reverse: 5’-TGTGTAAGTAAGTCGATAGGAGT-3’
NNMT	Forward: 5’-GAATCAGGCTTCACCTCCAA-3’
	Reverse: 5’-CCCAGGAGATTATGAAACACC-3’
ALP	Forward: 5’-GACCTCCTCGGAAGACACTC-3’
	Reverse: 5’-TGAAGGGCTTCTTGTCTGTG-3’
Runx2	Forward: 5’-TCTTAGAACAAATTCTGCCCTTT-3’
	Reverse: 5’-TGCTTTGGTCTTGAAATCACA-3’
miR-29b-3p	Forward: 5’-CTGCTAGCACCATTTGAAA-3’
	Reverse: 5’-GTGCAGGGTCCGAGGT-3’
GAPDH	Forward: 5’-ACGGATTTGGTCGTATTGGG-3’
	Reverse: 5’-CCTGGAAGATGGTGATGGGATT-3’
U6	Forward: 5’-CTCGCTTCGGCAGCACA −3’
	Reverse: 5’-AACGCTTCACGAATTTGCGT −3’

Figure 1. XIST/miR-29b-3p/NNMT axis may participate in osteoporosis.A. The protein-protein interaction analysis of the top 50 most significantly upregulated genes of GSE35959 data series (adjusted P < 0.05 and |logFC|≥1.5) by string algorithm. B. The intersection between the target miRNAs of XIST, the target miRNAs of NNMT, and the differentially expressed miRNAs in osteoporosis by analyzing GSE91033. The target prediction of XIST and NNMT was conducted using starbase algorithm. The DE-miRNAs of GSE91033 were selected at adjusted P < 0.05 and |logFC|≥1.5

Figure 2. XIST is highly expressed in OP and is low expressed during osteogenic differentiation.(a) XIST expression in the serum of OP patients and non-OP patients. ** P < 0.001. (b) Phenotypic identification of bone marrow mesenchymal stem cells. The shape of BMSCs on the 4th day. (c) The expression levels of osteoblast marker genes ALP, RUNX2 on osteogenesis differentiation. (d) Following cultured in osteogenic induction medium for 14 days, B MSCs exhibited more mineralized nodules according to ARS staining, whereas the control group did not. (e) The expression of XIST during the time of osteogenesis differentiation

** P < 0.001 vs. Control.

Figure 3. Down-regulation of XIST promotes BMSCs osteogenic differentiation, and up-regulation of XIST inhibits BMSCs osteogenic differentiation.(a) The expression of XIST in BMSCs after transfection of sh-XIST #1, sh-XIST 2# or sh-XIST 3#. (b) The expression of XIST in BMSCs after transfection of pcDNA-XIST. (c) The expression of ALP mRNA in BMSCs after transfection of sh-XIST 2#. (d) The expression of ALP mRNA in BMSCs after transfection of pcDNA-XIST. (e) The expression of RUNX2 mRNA in BMSCs after transfection of sh-XIST 2#. (f) The expression of RUNX2 mRNA in BMSCs after transfection of pcDNA-XIST. (g)The expression of RUNX2 and ALP protein in BMSCs after transfection of sh-XIST 2# or pcDNA-XIST. (h) Calcium deposition in BMSCs transfected with sh-XIST 2# or pcDNA-XIST was detected by ARS staining

* P < 0.05, ** P < 0.001 vs. blank.

Figure 4. XIST targets miR-29b-3p.(a) Schematic diagram of predicted binding sites of XIST in 3’-UTR of miR-29b-3p. (b) Determination of the dual-luciferase activity of BMSCs transfected with XIST-MUT or XIST-WT and agomiR-NC or agomiR miR-29b-3p. ** P < 0.001 vs. agomiR-NC. (c) The expression of miR-29b-3p in the serum of OP patients and non-OP patients. ** P < 0.001. (d) Pearson analysis of correlation between miR-29b-3p and XIST in the serum of OP patients. (e) The expression of miR-29b-3p in BMSCs after transfection of sh-XIST 2#

** P < 0.001 vs. blank.

Figure 5. XIST inhibits osteogenic differentiation by inhibiting miR-29b-3p.(a) The expression of miR-29b-3p in BMSCs after transfection of antagomiR miR-29b-3p or sh-XIST 2#. (b) The expression of ALP mRNA in BMSCs after transfection of antagomiR miR-29b-3p or sh-XIST 2#. (c) The expression of RUNX2 mRNA in BMSCs after transfection of antagomiR miR-29b-3p or sh-XIST 2#. (d) The expression of RUNX2 and ALP protein in BMSCs after transfection of antagomiR miR-29b-3p or sh-XIST 2#. (e) Calcium deposition in BMSCs transfected with antagomiR miR-29b-3p or sh-XIST 2# was detected by ARS staining

** P < 0.001 vs. blank; ## P < 0.001 vs. antagomiR-29b-3p.

Figure 6. MiR-29b-3p targeted NNMT.(a) Schematic diagram of predicted binding sites of NNMT in 3’-UTR of miR-29b-3p. (b) Determination of the dual-luciferase activity of BMSCs transfected with XIST-MUT (MUT1, MUT2 and Co-MUT) or NNMT-WT and agomiR-NC or agomiR miR-29b-3p. * P < 0.05, ** P < 0.001 vs. agomiR-NC. (c) The protein expression of NNMT in BMSCs transfected with antagomiR miR-29b-3p or agomiR miR-29b-3p. ** P < 0.001 vs. NC. (d) The mRNA expression of NNMT in BMSCs after transfection of antagomiR miR-29b-3p or sh-XIST 2#. (e) The protein expression of NNMT in BMSCs after transfection of antagomiR miR-29b-3p or sh-XIST 2#

** P < 0.01. ** P < 0.001 vs. blank; ## P < 0.001 vs. sh-XIST #2.

Figure 7. MiR-29b-3p promotes osteogenic differentiation by inhibiting NNMT.(a) The NNMT mRNA expression in BMSCs after transfection of sh-NNMT #1, sh-NNMT 2# or sh-NNMT 3#. (b) The NNMT mRNA expression in BMSCs after transfection of sh-NNMT #1 or antagomiR miR-29b-3p. (c) The expression of ALP mRNA in BMSCs after transfection of antagomiR miR-29b-3p or sh-NNMT #1. (d) The expression of RUNX2 mRNA in BMSCs after transfection of antagomiR miR-29b-3p or sh-NNMT #1. (e) The expression of RUNX2 and ALP protein in BMSCs after transfection of antagomiR miR-29b-3p or sh-NNMT #1. (f) Calcium deposition in BMSCs transfected with antagomiR miR-29b-3p or sh-NNMT #1 was detected by ARS staining

** P < 0.001 vs. blank; ## P < 0.001 vs. sh-NNMT #1.