Fig 1: HOTAIR Promotes the DNA Methylation of SSTR1(A) CpG islands in the promoter region of SSTR1 analyzed using MethPrimer. (B) The methylation level of CpG islands in the promoter region of SSTR1 gene in PD cell models as determined by MSP. (C) SSTR1 expression in PD cell models after demethylation treatment as determined by MSP. (D) SSTR1 expression in cells treated with 5-Aza-dc detected as determined by qRT-PCR. (E) SSTR1 expression and the extent of ERK1/2 phosphorylation in PD cell models after demethylation treatment as determined by western blot analysis. (F) The enrichment of DNMT1, DNMT3a, and DNMT3b in the promoter region of SSTR1 in PD cell models as determined by ChIP. (G) The location of HOTAIR in PD cell models detected as determined by FISH (×400). (H) Complementary base pairing in HOTAIR sequence and SSTR1 promoter analyzed using BLAST. (I) The expression of HOTAIR in midbrain substantia nigra pars compacta tissues in MPTP-induced PD mouse model and in PD cell models as determined by qRT-PCR. (J) Correlation of HOTAIR and SSTR1 levels in midbrain substantia nigra pars compacta tissues in MPTP-induced PD mouse model. (K) The luciferase activity of HOTAIR co-transfected with WT-SSTR1 and MUT-SSTR1 into HEK293T cells as determined by the dual luciferase reporter gene assay. (L) The expression of HOTAIR and SSTR1 in cells treated with oe-NC, si-NC, oe-HOTAIR, and si-HOTAIR as determined by qRT-PCR; #p < 0.05 versus si-NC cells. (M) The methylation levels of CpG islands in the SSTR1 promoter region of cells treated with oe-NC, oe-HOTAIR, si-NC, and si-HOTAIR detected as determined by MSP assay. (N) The enrichment of methyltransferases DNMT1, DNMT3a, and DNMT3b in PD cell models as determined by RIP assay. In (B), (F), (I), and (N), *p < 0.05 versus control cells. In (C)–(E), *p < 0.05 versus MPP+ + DMSO cells. In (K) and (L), *p < 0.05 versus oe-NC cells. The measurement data are summarized as mean ± standard deviation, the experiment was repeated 3 times independently, comparisons among multiple groups in (L) were analyzed using one-way analysis of variance and comparisons between two groups in the remaining figures were analyzed using the unpaired t test.
Fig 2: SSTR1 Gene Is Poorly Expressed in PD Mouse Models Induced by MPTP and MN9D Cells Treated with MPP+(A) The coordination of limb movements of mice injected with MPTP as evaluated by the pole climbing test. (B) The coordination of limb movements of mice injected with MPTP as evaluated by the rotarod test. (C) The coordination of limb movements of mice injected with MPTP as evaluated by OFT. (D) The apoptosis of dopaminergic neurons of the substantia nigra pars compacta in MPTP mice as determined by TUNEL and immunofluorescence assay. (E) Gene expression of SSTR1 in PD as indicated by bioinformatics analysis. (F) The expression of SSTR1 in the substantia nigra pars compacta tissue of mice injected with MPTP as determined by qRT-PCR. (G) The viability of MN9D cells in midbrain dopaminergic neurons in response to the treatment with MPP+ as determined by CCK-8 assay. (H) The expression of SSTR1 in MN9D cell model of PD as determined by qRT-PCR; *p < 0.05 versus the control group. The measurement data are summarized as mean ± standard deviation, the experiment was repeated 3 times independently, and comparisons between two groups were analyzed using unpaired t test.
Fig 3: Schematic Diagram Depicting the Molecular Mechanisms by which the HOTAIR/SSTR1/ERK1/2 Axis Influences Dyskinesia in Mice with PDHOTAIR promoted SSTR1 gene methylation to decrease SSTR1 expression via the recruitment of DNMT methyltransferase, and then activated the ERK1/2 axis, leading to accelerated PD progression.
Fig 4: SSTR1 Disrupts the Activation of ERK1/2 Axis in PD In Vitro and In Vivo(A) The protein levels of ERK1/2 and extent of ERK1/2 phosphorylation in response to the treatment with MPTP, MPTP + LV-NC, and MPTP + LV-SSTR1 as determined by western blot analysis; *p < 0.05 versus control mice; #p < 0.05 versus MPTP + LV-NC mice. (B) The efficiency of SSTR1 overexpression and silencing in response to the treatment with oe-SSTR1, si-NC, and si-SSTR1 as determined by qRT-PCR; *p < 0.05 versus oe-NC cells; # p < 0.05 versus the si-NC cells. (C) The activation of ERK1/2 in cells treated with oe-NC, oe-SSTR1, si-NC, and si-SSTR1 as determined by western blot analysis; *p < 0.05 versus oe-NC cells; #p < 0.05 versus si-NC cells. The measurement data are summarized as mean ± standard deviation, the experiment was repeated 3 times independently, and comparisons among multiple groups were analyzed using one-way analysis of variance.
Fig 5: SSTR1 Overexpression Alleviates Dyskinesia In Vivo and Decreases Dopaminergic Neuron Apoptosis In Vitro in PD(A) The expression of SSTR1 in response to the treatment with MPTP, MPTP + LV-NC, and MPTP + LV-SSTR1 as determined by qRT-PCR. (B) The coordination of limb movements of mice treated with MPTP, MPTP + LV-NC, and MPTP + LV-SSTR1 as assessed by the pole climbing test. (C) The coordination of limb movements of mice treated with MPTP, MPTP + LV-NC, and MPTP + LV-SSTR1 as assessed by the rotarod test. (D) The coordination of limb movements of mice treated with MPTP, MPTP + LV-NC, and MPTP + LV-SSTR1 as assessed by OFT. (E) The apoptosis of dopaminergic neurons of the substantia nigra pars compacta in mice treated with MPTP, MPTP + LV-NC, and MPTP + LV-SSTR1 as assessed by TUNEL and immunofluorescence assay. (F) The level of SSTR1 overexpression in MN9D cells treated with MPP+, MPP+ + oe-NC, and MPP+ + oe-SSTR1 as determined by qRT-PCR. (G) The apoptosis of MN9D cells treated with MPP+, MPP+ + oe-NC, and MPP+ + oe-SSTR1 as determined by TUNEL assay. In (A)–(E), *p < 0.05 versus control mice; #p < 0.05 versus MPTP + LV-NC mice. In (F) and (G), *p < 0.05 versus control cells; #p < 0.05 versus MPP+ + oe-NC cells. The measurement data are summarized as mean ± standard deviation, the experiment was repeated 3 times independently, and comparisons among multiple groups were analyzed using one-way analysis of variance.
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