LncRNA expression in the spinal cord modulated by minocycline in a mouse model of spared nerve injury

Figures & data

Table 1 The primers of 3 lncRNAs used in this study

lncRNA	Primer	Annealing temperature (°C)	Product length (bp)
NR_015491	F:5′TTGCTGAGCAGACTTGAATGA3′	60	78
	R:5′TTGCCTGAGCACAATCCACA3′
ENSMUST00000174263	F:5′CCCAGCATCAGGGTTTAGGA3′	60	239
	R:5′TCTCAGGGGATTTTCTTCTCTC3′
ENSMUST00000146263	F:5′GAGTCTCCCTGACCTAACCTG3′	60	134
	R:5′TGCCTTCTGTTCTTTGTGTTG3′

Abbreviation: lncRNA, long noncoding RNAs.

Figure 1 The paw withdrawal mechanical thresholds (PWMTs) at baseline and 7 days after modeling are presented as mean ± standard error (n=6). ****P<0.0001 indicates a statistically significant difference when compared to its baseline and Sham group and ^##P<0.001 indicates a statistically significant difference when compared to the V-SNI group.

Abbreviations: MC-SNI, group that had minocycline repeatedly administrated before and after SNI; PWMT, paw withdrawal mechanical threshold; SNI, spared nerve injury; V-SNI, group that repeated vehicle injections before and after SNI.

Figure 2 Scatter plots compare the expression of lncRNAs and mRNAs between the sham versus V-SNI group (A and B), and V-SNI versus MC-SNI group (C and D). The values of X and Y axes in the scatter plot are the averaged normalized values in each group (log2 scaled). The green lines indicate a fold change value of 2.0. The transcripts above the top green line and below the bottom green line indicate more than 2-fold changes between pairs. In total, 113 lncRNAs in B1 and 66 in B2 were listed in heat maps E and F; and 47 mRNAs in B1 and 52 in B2 were listed in heat maps G and H. In the heat maps, each column represents a microarray group, while each row represents a regulated gene. The color scale illustrates the relative expression level of lncRNAs: red denotes rich abundance and green denotes poor abundance. Percentages of each pattern for lncRNAs and mRNAs were shown in donut charts (I and J). Pattern A included genes that were modulated only by SNI, whereas pattern B represented genes that were modulated by both SNI and repeated minocycline administration. B1 - genes upregulated by SNI and then downregulated by minocycline, B2 - genes downregulated by SNI and then upregulated by minocycline, B3 - genes upregulated by SNI and further upregulated by minocycline, and B4 - genes downregulated by SNI and further downregulated by minocycline.

Abbreviations: lncRNA, long noncoding RNA; MC-SNI, group that had minocycline repeatedly administered before and after SNI; SNI, spared nerve injury; V-SNI, group that repeated vehicle injections before and after SNI.

Figure 3 The fold changes of the detected lncRNAs NR_015491, ENSMUST00000174263, and ENSMUST00000146263 using microarray (A, C, and E) and qRT-PCR (B, D, and F) were listed. The statistical analysis for each comparison was performed using ANOVA and Turkey’s correction. *P<0.05, **P<0.01, and ****P<0.0001 indicate a statistically significant difference when compared to the sham group; ^#P<0.05, ^###P<0.001, and ^jpr-10-2503P<0.0001 indicate a significant difference when compared to V-SNI.

Abbreviations: ANOVA, analysis of variance; lncRNA, long noncoding RNA; MC-SNI, group that had minocycline repeatedly administered before and after SNI; qRT-PCR, quantitative real-time polymerase chain reaction; SNI, spared nerve injury; V-SNI, group that repeated vehicle injections before and after SNI.

Figure 4 Distribution of four lncRNA classes. The distribution of intergenic, sense, antisense, and bidirectional lncRNAs is identified. Blue, green, and yellow colors indicate the number of DE lncRNAs with expression pattern B1, B2, or B3 in each class. Pattern B represented genes that were modulated by both SNI and repeated minocycline administration. B1 - genes upregulated by SNI and then downregulated by minocycline, B2 - genes downregulated by SNI and then upregulated by minocycline, and B3 - genes upregulated by SNI and further upregulated by minocycline.

Abbreviations: DE, differentially expressed; lncRNA, long noncoding RNA.

Figure 5 (A) The enriched molecular function annotations for DE mRNAs from sham versus V-SNI groups. (B) Part of the P-value annotation tree was displayed to reveal chemokine activity and its greatly enriched related annotations. (C and D) The enriched molecular function and biological process annotations for DE mRNAs from V-SNI versus MC-SNI. GO results are presented as descending enrichment scores.

Abbreviations: DE, differentially expressed; GO, gene ontology; MC-SNI, group that had minocycline repeatedly administered before and after SNI; SNI, spared nerve injury; V-SNI, group that repeated vehicle injections before and after SNI.

Table 2 Molecular function annotation enrichment results comparing sham versus V-SNI

No.	Term	P-value	Genes
1	Heparin binding	0.001499866	VEGFB, FGFR4, CCL2, NAV2, CCL5, CCL7, ITGAM, ABI3BP, CXCL10, CYR61
2	Chemokine activity	0.001548949	CCL2, CCL19, CCL5, CXCL12, CCL7, CXCL10
3	Protein binding	0.001837527	PRX, SGSH, RHOJ, HSP90AB1, APOBEC3, HPS5, CLSTN3, USH1G, PAX6, RHOQ, ANKRD1, JAG1, CXADR, CD96, NOD1, CD44, RAVER2, ANK3, HOMER3, ATG7, SERPINE1, SPG20, ANO2, PIK3AP1, PILRA, EGFR, DYNC1I1, RBFOX2, SH3PXD2A, LYN, CCDC88C, RXRB, FMR1, ZFP467, SIX3, PDXP, BICD2, PPARGC1B, PDCD1LG2, CARD11, ARVCF, DSG1A, CD36, ZFAND2B, ROR2, H2-AA, VGLL2, FLII, CD226, CD300LD, ABCA7, FGFR4, NKD1, IL1R1, CCL2, RAB3D, PRTN3, CD247, CNTFR, CHEK1, ITGB2, BCL2L2, PAXBP1, CBLL1, TCF7L2, CTLA2B, MOV10, STAT4, GFI1, NFATC2, TCF3, PXT1, LRFN2, HAVCR2, FYB, EIF4ENIF1, CR2, TRNP1, TGFBR2, EN1, DOCK7, AMBRA1, IDO1, HS1BP3, VAV1, PPP1R13L, DVL1, SYNE3, EPHA4, LCE1G, EPS8, CMYA5, CYFIP2, MILR1, CIT, BMPR1B
4	Chemokine receptor binding	0.003686081	CCL19, CCL5, S100A14, CXCL12
5	Cytokine activity	0.004934124	IL17A, CCL2, C1QTNF4, IFNE, IL17F, TNFSF12, INHA, CCL5, CXCL12, CCL7, CXCL10
6	Serine-type peptidase activity	0.008639921	TMPRSS11G, PRTN3, CFB, CTRB1, TYSND1, DPP8, CFI, TMPRSS12, HABP2
7	Transmembrane receptor protein tyrosine kinase activity	0.015247211	EGFR, EPHA4, FGFR4, LTK, ROR2
8	Spectrin binding	0.017147753	DYNC1I1, DMTN, ANK3, USH1G
9	Guanyl-nucleotide exchange factor activity	0.021272918	FGD2, DOCK2, CYTH4, PSD4, DOCK7, VAV1, EIF2B3, RGL1
10	G-protein-coupled receptor binding	0.026730704	GNA14, GNA15, CCL2, DNAJC14, USP20
11	RNA polymerase II core promoter proximal region sequence-specific DNA binding	0.027051751	PAX6, ZFP467, EN1, PAXBP1, FOSB, TCF7L2, FEZF2, SMARCD2, PRDM5, ZFP219, NFATC2, TCF3, NFIA
12	Transcriptional repressor activity, RNA polymerase II core promoter proximal region sequence-specific binding	0.027375039	STAT6, PRDM5, ZFP219, EN1, GFI1, NFATC2, TCF3
13	Integrin binding	0.032537287	EGFR, LYN, ITGA2, CXADR, CD226, CYR61
14	Serine-type endopeptidase activity	0.035490515	TMPRSS11G, F5, PRTN3, CFB, CTRB1, TYSND1, CFI, TMPRSS12, HABP2
15	GTP binding	0.040618394	HSP90AB1, RHOJ, GNA14, GNA15, RAB3D, TUBA3B, RHOQ, RAB40C, NPR2, 1700009N14RIK, RND1, TUBB6, NKIRAS2

Abbreviations: SNI, spared nerve injury; V-SNI, group that repeated vehicle injections before and after SNI.

Table 3 Molecular function annotation enrichment results comparing V-SNI versus MC-SNI

No.	Term	P-value	Genes
1	Actin binding	0.002751885	TNNT3, TRIOBP, SHROOM3, TNNC2, SYNPO2, MYOZ1, MYOZ2, TPM4, FLNA, TNNI2
2	Serine-type endopeptidase activity	0.010409829	MMP8, GM2663, LTF, 2210010C04RIK, MCPT2, TMPRSS12, CTSG
3	Metalloendopeptidase activity	0.022158153	ADAM28, ADAMTS9, ADAM29, MMP8, PMPCB
4	Hydrolase activity	0.041880873	APOBEC2, ADAM28, APOBEC3, CPPED1, PTPRN2, MMP8, PTPN14, PGAM2, MCPT2, MTMR2, NT5C3, PLA2G12A, ACOT11, VPS4B, ATP8B1, PARG, LTF, TMPRSS12, HDAC8, CTSG, PMPCB
5	FATZ binding	0.042538506	APOBEC2, APOBEC3
6	Hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in cyclic amidines	0.042538506	MYOZ1, MYOZ2
7	Guanyl–nucleotide exchange factor activity	0.046446397	FGD2, DENND5A, RAPGEF6, DOCK7, DENND4B
8	Metallopeptidase activity	0.050491587	ADAM28, ADAMTS9, ADAM29, MMP8, PMPCB
9	Telethonin binding	0.050827978	MYOZ1, MYOZ2
10	Catalytic activity	0.056869731	APOBEC2, XDH, APOBEC3, SPTLC2, CKMT2, ABAT, PGAM2, PCYT1B, PMPCB

Abbreviations: MC-SNI, group that had minocycline repeatedly administered before and after SNI; SNI, spared nerve injury; V-SNI, group that repeated vehicle injections before and after SNI.