Promoting effect of long non-coding RNA SNHG1 on osteogenic differentiation of fibroblastic cells from the posterior longitudinal ligament by the microRNA-320b/IFNGR1 network

Figures & data

Table 1. Demographic characteristics of the respondents

Number	Age	Gender	Classification
1	31	Male	II
2	35	Male	I
3	62	Female	I
4	47	Male	II
5	64	Female	II
6	52	Male	II
7	65	Male	I
8	59	Male	II
9	61	Male	II
10	67	Male	I
11	34	Male	II
12	51	Female	I
13	64	Female	I
14	68	Male	II
15	38	Male	II
16	55	Male	II
17	41	Male	I
18	32	Female	I
19	27	Male	II
20	53	Male	II
21	49	Male	I
22	42	Male	I
23	63	Female	I
24	43	Male	I
25	51	Female	I
26	53	Female	I
27	49	Female	II
28	43	Female	II
29	38	Male	I
30	38	Male	I
31	53	Female	II
32	64	Female	I
33	49	Male	I
34	65	Female	I
35	60	Male	I
36	46	Female	I
37	30	Male	II
38	27	Male	II
39	51	Female	I
40	31	Female	II
41	61	Female	II
42	30	Female	II
43	55	Female	II
44	35	Male	I
45	63	Male	I
46	53	Female	II
47	64	Male	I
48	63	Male	I
49	51	Male	I
50	53	Male	I
51	58	Female	I
52	25	Female	I
53	58	Female	II
54	52	Male	I
55	55	Male	I
56	51	Female	I
57	42	Male	II
58	59	Female	II
59	25	Male	II
60	26	Female	I
61	57	Male	II
62	27	Male	I
63	67	Male	II
64	67	Male	II
65	52	Male	II
66	42	Female	II
67	28	Male	I
68	28	Male	II
69	67	Male	0
70	62	Male	0
71	45	Male	0
72	47	Female	0
73	36	Female	0
74	33	Female	0
75	37	Male	0
76	41	Female	0
77	66	Male	0
78	60	Male	0
79	47	Male	0
80	39	Male	0
81	45	Male	0
82	49	Male	0
83	67	Male	0
84	66	Female	0
85	28	Male	0

Table 2. Primer sequences used for cell transfection

Primer	Sequence
SNHG1	Sense: 5'-GGGGTACGTTCTCATTTTTTACTGCTCGTG-3’
	Anti-sense: 5'-CGGGATCCATGTAATCAATCATTTTATTATTTTCATC-3’
si-SNHG1	Sense: 5'-CCAGCAUCUCAUAAUCUAUTT-3’
	Anti-sense: 5'-AUAGAUUAUGAGAUGCUGGAA-3’
miR-320b	Sense: 5'-AAAGCUGGUGAGAGGGAA-3’
	Anti-sense: 5'-UUGCUCUCAACCCAGCUUU-3’
miR-inhibitor	Sense: 5'-UUGCCCUCUCAACCCAGCUUUU-3

Note: SNHG1, small nucleolar RNA host gene 1; si-SNHG1, small interfering SNHG1; miR, microRNA.

Table 3. Primer sequences in RT-qPCR

Primer	Sequence
SNHG1	F: 5'-GTGACTTCGCCTGTGATGGA-3’
	R: 5'-GGCCTCTATCTGTACCTTTATTC-3’
BMP-2	F: 5'-AGCCATTGCACACCTCAC-3’
	R: 5'-CGTGGTACCAAGAGGACAGAGT-3’
OCN	F: 5'-TGAGGACCATCTTCTGCTCA-3’
	R: 5'-TGGACATGAAGGCTTTGTCA-3’
RUNX2	F: 5'-UUGCCCUCUCAACCCAGCUUUU-3
	R: 5'-TGGACATGAAGGCTTTGTCA-3’
miR-320b	F: 5'-CCTGGGAGTATGTCGATCTATTG-3’
	R: 5'-TGGTGTCGTGGAGTCG-3’
IFNGR1	F: 5'-AAGTGGCGTGTTCAAGTGTG-3’
	R: 5'-GGCTTTGGAGATCTGAGCTG-3’
U6	F: 5'-CTCGCTTCGGCAGCACA-3’
	R: 5'-AACGCTTCACGAATTTGCGT-3’
GAPDH	F: 5'-CGGACCAATACGACCAA-3’
	R: 5'-AGCCACATCGCTCAGACACC-3’

Note: RT-qPCR, reverse transcription quantitative polymerase chain reaction; SNHG1, small nucleolar RNA host gene 1; BMP-2, bone morphogenetic protein-2; OCN, osteocalcin; RUNX2, runt-related transcription factor 2; miR, microRNA; IFNGR1, interferon gamma receptor 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Table 4. Antibodies used in western blot analysis and immunohistochemistry

Antibody	Item. No.	Dilution ratio
BMP-2	ab214821	1: 2000
RUNX2	ab192256	1: 5000
OCN	ab13420	1: 2000
JAK2	ab108596	1: 100
P-JAK2	ab32101	1: 100
STAT-1	ab92506	1: 1000
P-STAT-1	ab109461	1: 5000
STAT-2	ab231184	1: 1000
p-STAT-2	ab53132	1: 5000
β-actin	ab179467	1: 5000
Secondary antibody	ab150117	1: 5000

Note: BMP-2, bone morphogenetic protein-2; RUNX2, runt-related transcription factor 2; OCN, osteocalcin; JAK, janus kinase; STAT, signal transducers and activators of transcription.

Figure 1. SNHG1 is highly expressed in patients with OPLL. A, PLL- or OPLL-derived LFCs identified by microscope observation and Vimentin immunofluorescence; B-C, mRNA (b) and protein (c) expression of osteogenesis-related factors BMP-2, RUNX2 and OCN determined by QRT-PCR and western blot analysis, respectively; D-E, the osteogenic properties of LFCs determined by Alizarin Red staining (d) and ALP (e) staining; F, the heatmap for differentially expressed lncRNAs between 4 OPLL tissues and 4 healthy PLL tissues determined by Arraystar Human LncRNA microarray V2.0 (Agilent_033010 Probe Name version); G, SNHG1 expression in 68 pairs of OPLL tissues and 18 pairs of PLL tissues identified by QRT-PCR; H, SNHG1 expression in cells after SNHG1 siRNA and SNHG1 overexpressing vector transfection determined by QRT-PCR as well. Data are expressed as the mean ± SD; in panels B and C, data were analyzed using two-way ANOVA, while data in panel H were analyzed using one-way ANOVA, and Tukey’s multiple comparisons test was used for the pairwise comparisons after ANOVA analysis; *, p < 0.05

Figure 2. Silencing of SNHG1 inhibits the osteogenic differentiation of LFCs. A-B, mRNA (a) and protein (b) expression of osteogenesis-related factors BMP-2, RUNX2 and OCN determined by QRT-PCR and western blot analysis, respectively; C-D, the osteogenic properties of LFCs determined by alizarin red staining (c) and ALP (d) staining. The data are expressed as the mean ± SD. Two-way ANOVA and Tukey’s multiple comparison test was used to determine statistical significance; *, p < 0.05

Figure 3. SNHG1 serves as a ceRNA for miR-320b to mediate IFNGR1 expression. A, subcellular localization of SNHG1 predicted on the lncATLAS DataBase (http://lncatlas.crg.eu/); B, FISH experiments with probes targeting SNHG1 were performed to validate the subcellular localization of SNHG1 in LFCs. The cytoplasm was stained with probes targeting SNHG1 (red stain), and the nuclei were stained with DAPI (blue stain); C, nuclear and cytoplasmic expression of SNHG1 in LFCs determined by QRT-PCR; D, heatmap for the top 30 differentially expressed genes between PLL- and OPLL- LFCs analyzed using the Llimma Rstudio based on GSE5464 microarray, with |LogFC ≥ 4.0| and p < 0.05 as the screening criteria; E, KEGG-based clustering analysis was performed on DAVID 6.8 software, and a total of 43 pathways where the differentially genes clustered were figured out; F, genes clustered on the JAK/STAT signal pathway; G, intersected miRNAs that could bind to SNHG1 and IFNGR1 predicted on StarBase; H, binding relationships between SNHG1 and miR-320b and between miR-320b and IFNGR1 validated by dual luciferase reporter gene assay. The data are expressed as the mean ± SD; two-way ANOVA and Tukey’s multiple comparison test was used to determine statistical significance; *, p < 0.05

Figure 4. Overexpression of miR-320b inhibits the osteogenic differentiation of LFCs. miR-320b mimic or mock were transfected into PLL-derived LFCs with overexpressed SNHG1, and miR-320b inhibitor or inhibitor control were transfected into OPLL-derived LFCs with silenced SNHG1. A, miR-320b expression in cells after transfection measured by QRT-PCR; B-C, mRNA (b) and protein (c) expression of BMP-2, RUNX2 and OCN in cells determined by QRT-PCR and western blot analysis, respectively; D-E, osteogenic properties of LFCs detected by alizarin red staining (d) and ALP staining (e). The data are expressed as the mean ± SD; in panel A, data were analyzed using one-way ANOVA, while data in panels B and C were analyzed using two-way ANOVA, and Tukey’s multiple comparison test was used to determine statistical significance; *, p < 0.05

Figure 5. JAK-STAT signaling pathway activation promotes the osteogenic differentiation of LFCs. A, western blot analysis was performed to measure the phosphorylation of JAK2, STAT1 and STAT3 in LFCs; Then, OPLL-derived Fedratinib, a JAK2-specific agonist, was administrated into the LFCs; Next, B-C, the mRNA (b) and protein (c) expression of BMP2, RUNX2 and OCN was determined by QRT-PCR and western blot analysis, respectively; D-E, osteogenic properties of LFCs were detected by alizarin red staining (d) and ALP staining (e). The data are expressed as the mean ± SD; two-way ANOVA and Tukey’s multiple comparison test was used to determine statistical significance; *, p < 0.05

Figure 6. Silencing of SNHG1 inhibited the osteogenic differentiation of LFCs in vivo. HE staining was performed to observe the scaffold morphology and immunohistochemistry staining was performed to identify the expression of OCN, BMP2 and RUNX2 in the scaffolds seeded with different cell types. The data are expressed as the mean ± SD; one-way ANOVA and Tukey’s multiple comparison test was used to determine statistical significance; *, p < 0.05