miR-34a-mediated regulation of XIST in female cells under inflammation

Figures & data

Figure 1 XIST is upregulated in a subset of CRPS patients and inversely correlated with miR-34a.

Notes: (A) Relative expression of XIST in blood from control male (n=6) and female (n=10) donors. (B) Relative expression of miR-34a and XIST in CRPS patients receiving ketamine therapy. Responders n=7, poor responders n=6. (C) Relative expression of XIST in CRPS patients (n=40), control females (n=10) and CRPS patients receiving ketamine therapy, responders n=7, poor responders n=6. Expression levels of XIST normalized to GAPDH. miR-34a expression normalized to U6. One-way ANOVA and Student’s t-test were used for statistical analysis. *p<0.05, data represent mean ± SEM.

Abbreviations: ANOVA, analysis of variance; CRPS, complex regional pain syndrome; SEM, standard error of the mean; XIST, X-inactive-specific transcript.

Figure 2 miR-34a can bind and regulate XIST.

Notes: (A) Relative luminescence after transfecting HEK293 cells with XIST pmirGLO plasmid with 5 kb fragment from 5′XIST cloned downstream of the firefly luciferase gene, and precursor miR-34a or control miRNA (n=5). (B) and (C) Relative expression of Xist/XIST in mouse J774A.1 and human SH-SY5Y cells, respectively, after miR-34a transfection. Expression of Xist/XIST normalized to Gapdh/GAPDH. Student’s t-test used for statistical analysis *p<0.05, ***p<0.001. Data represent mean ± SEM.

Abbreviations: SEM, standard error of the mean; XIST, X-inactive-specific transcript.

Figure 3 miR-34a regulates Xist under inflammation.

Notes: Relative expression of Xist and miR-34a in J774A.1 (mouse macrophage cell line) after LPS treatment (1 μg/mL for 4 h) (n≥3). Expression of Xist normalized to Gapdh. miR-34a expression normalized to U6 (n≥3). One-way ANOVA and Student’s t-test were used for statistical analysis *p<0.05, **p<0.01. Data represent mean ± SEM.
Abbreviations: ANOVA, analysis of variance; LPS, lipopolysaccharide; SEM, standard error of the mean; Xist, X-inactive-specific transcript.

Figure 4 Upregulation of Xist and downregulation of miR-34a in blood samples from CFA model of inflammatory pain.

Notes: (A) CFA-induced mechanical hypersensitivity assessed using von Frey filaments in female C57BL/6 mice (n=3–4). Paw withdrawal threshold dropped significantly by 4 h (acute inflammatory phase) and remained low at 14 days (chronic inflammatory phase). (B) Relative expression of Xist in female C57BL/6 mice injected with CFA or saline into the hind paw (n=3–4). Blood samples collected at 4 h and 14 days were used to determine the expression of Xist during acute and chronic inflammatory phases respectively. (C) Relative expression of miR-34a in blood under acute and chronic inflammation in female C57BL/6 mice injected with CFA or saline (n=3–4). One-way ANOVA and Student’s t-test were used for statistical analysis except for mice mechanical hypersensitivity in which Mann–Whitney U test was used *p<0.05, **p<0.01, ***p<0.001, data represent mean ± SEM.

Abbreviations: ANOVA, analysis of variance; CFA, complete Freund’s adjuvant; SEM, standard error of the mean; Xist, X-inactive-specific transcript.

Figure 5 Upregulation of Yy1 parallels Xist.

Notes: (A) Relative expression of Yy1 in female C57BL/6 mice injected with CFA or saline (n=3–4). (B) Relative expression of YY1 in CRPS patients receiving ketamine therapy, responders n=7, poor responders n=6. Expression of Yy1/YY1 normalized to Gapdh/GAPDH. One-way ANOVA and Student’s t-test were used for statistical analysis *p<0.05, **p<0.01. Data represent mean ± SEM.

Abbreviations: ANOVA, analysis of variance; CFA, complete Freund’s adjuvant; CRPS, complex regional pain syndrome; SEM, standard error of the mean; Xist, X-inactive-specific transcript; YY1, Yin-Yang 1.

Table S1 The demographics of control and CRPS patients

Controls	Sex	Age (years)	Weight (lb)	Height (cm)	BMI
C1	F	42	120	63	21.25
C2	F	37	134	67	20.99
C3	F	40	240	67	37.59
C4	F	24	113	62	20.67
C5	F	34	124	66	20.01
C6	F	39	157	67	24.59
C7	F	29	132	62	24.14
C8	F	47	140	65.5	22.94
C9	F	59	117	60	22.85
C10	F	41	110	65	18.30
C11	M	68	220	76	26.78
C12	M	63	190	71	26.50
C13	M	56	235	78	27.15
C14	M	43	165	70	23.67
C15	M	49	186	75	23.25
C16	M	23	165	67	25.84

Patients	Ketamine response		Age (years)	Weight (lb)	Height (cm)	BMI
P1	NA	F	40	128	65	21.30
P2	NA	F	65	164	67	25.68
P3	NA	F	47	233	65	38.77
P4	NA	F	63	102	58	21.32
P5	NA	F	33	142	62	25.97
P6	NA	F	31	170	62	31.09
P7	NA	F	35	230	66	37.12
P8	NA	F	41	155	67	24.27
P9	NA	F	32	165	64	28.32
P10	NA	F	54	212	71	29.56
P11	NA	F	27	128	62	23.41
P12	NA	F	51	120	62	21.95
P13	NA	F	58	272	64	46.68
P14	NA	F	60	165	69	24.36
P15	NA	F	38	147	60	28.71
P16	NA	F	48	140	65	23.29
P17	NA	F	49	175	66	28.24
P18	NA	F	28	102	61	19.27
P19	NA	F	44	99	64	16.99
P20	NA	F	23	163	61	30.80
P21	NA	F	38	160	64	27.46
P22	NA	F	68	154	63	27.28
P23	NA	F	58	191
P24	NA	F	43	145	68	22.04
P25	NA	F	57	155	63	27.45
P26	NA	F	33	166	68	25.24
P27	NA	F	45	160	61	30.23
P28	Responder	F	51	200	64	34.33
P29	Responder	F	49	298	67	46.67
P30	Responder	F	51	176	68.5	26.37
P31	Responder	F	39	194	67	30.38
P32	Responder	F	43	260	63	46.05
P33	Responder	F	65	165	61	31.17
P34	Responder	F	49	173	63	30.64
P35	Poor responder	F	43	175	63	31.00
P36	Poor responder	F	69	152	61	28.72
P37	Poor responder	F	35	150	66	24.21
P38	Poor responder	F	45	160	61	30.23
P39	Poor responder	F	56	145	63	25.68
P40	Poor responder	F	33	166	68	25.24

Abbbreviation: CRPS, complex regional pain syndrome.