Figures & data
Figure 1. Expression features of LncRNA MIR17HG and miR-153-3p in PD patients.
(a-c): qRT-PCR analyzed the profiles of LncRNA MIR17HG, miR-153-3p and SNCA in PD (N = 40) and in the normal population (N = 20). *** P < 0.001 (vs.Normal group). (d): Detection of IL-1β, IL-6, TNF-a expression by qRT-PCR in PD patients and normal controls. *** P < 0.001 (vs.Normal group). €: Pearson correlation analysis was conducted to determine the relationship between MIR17HG and miR-153-3p, MIR17HG and SNCA, and miR-153-3p and SNCA. R2 = 0.5731, P < 0.0001. R2 = 0.4622, P < 0.0001. R2 = 0.5315, P < 0.0001. (f): Pearson was employed to analyze the relationship between MIR17HG and IL-1β, IL-6, TNF-a and SNCA. R2 = 0.3873, P < 0.0001. R2 = 0.5485, P < 0.0001. R2 = 0.5015, P < 0.0001. (g): Pearson was employed to analyze the relationship between miR-153-3p and IL-1β, IL-6, TNF-a. R2 = 0.3712, P < 0.0001. R2 = 0.5835, P < 0.0001. R2 = 0.7091, P < 0.0001. (h): Pearson analysis of the relationship between SNCA and IL-1β, IL-6, and TNF-a in the plasma of PD patients. R2 = 0.3705, P < 0.0001. R2 = 0.3821, P < 0.0001. R2 = 0.3760, P < 0.0001.
Figure 2. Expression features of LncRNA MIR17HG, SNCA and miR-153-3p in the PD model.
(a). The overall survival rate of mice in PD group and sham group was counted. (b-d): Expression of LncRNA MIR17HG, miR-153-3p and SNCA in the SN region of PD mice was checked using qRT-PCR at weeks 2, 4, 6 and 8 after ventricular injection of 6-OHDA. (e): Protein profiles of SNCA in the SN region of PD mice were evaluated using WB at weeks 2, 4, 6 and 8 after ventricular injection of 6-OHDA. *** P < 0.001 (vs.Sham group). (f-h): The relationships between MIR17HG and miR-153-3p (F), MIR17HG and SNCA (G), and miR-153-3p and SNCA (H) were analyzed using Pearson at week 8 after ventricular injection of 6-OHDA. R2 = 0.7117, P < 0.0001. R2 = 0.7363, P < 0.0001. R2 = 0.7493, P < 0.0001.
Figure 3. Attenuating MIR17HG mitigated neuronal apoptosis in the PD model in vitro and in vivo.
Lentiviral Si-MIR17HG was injected into 6-OHDA-treated mice, and SH-SY5Y cells were transfected with Si-MIR17HG to suppress the MIR17HG expression. (a): qRT-PCR assessed MIR17HG expression in brain tissues of PD mice. (b): FISH assay was applied to monitor the location and level of GFP after GFP-marked si-MIR17HG probe injection. (c). Western blot was performed for detecting GFP. (d-f): The effects of MIR17HG inhibition on the behavioral profile of PD mice were examined using APO-induced rotational behaviors (d), the rotarod bar test (e), and the passive avoidance assay (f). G and H: qRT-PCR and IHC tested the expression of TH mRNA and TH-positive cell mass. (i): WB exhibited alterations in TH protein expression in brain tissues of PD mice. (j): The SNCA expression was gauged by IHC. **P < 0.01, ***P < 0.001 (vs.Sham group), &P < 0.05, &&P < 0.01, &&&P < 0.001 (vs.6-OHDA+Si-NC group). (k): qRT-PCR analyzed the relative expression of MIR17HG in SH-SY5Y cells. (l): The viability of transfected SH-SY5Y cells was monitored by MTT. (m): BrdU staining counted the number of BrdU-positive cells in transfected SH-SY5Y cells. (n): The LDH kit was applied to determine LDH levels in SH-SY5Y cells. (o): Cell apoptosis was tested by FCM. (p and q): WB detection of apoptosis-related protein expression (Bax, Bcl2, and Caspase3) and SNCA expression in SH-SY5Y cells. **P < 0.01, *** P < 0.001 (vs.Con group), && P < 0.01, &&& P < 0.001 (vs.6-OHDA+Si-NC group).
Figure 4. Inhibition of MIR17HG curbed the inflammatory response in microglia in PD models both in vivo and in vitro.
In vivo: (a): Expression of Iba1 positive cells was measured by tissue immunofluorescence. (b): The profiles of IL-1β, IL-6, and TNF-α mRNA in PD mice was gauged by qRT-PCR. (c): MDA and SOD kits to quantify the content of MDA and the activity of SOD in the brain tissue of PD mice. (d). WB assayed COX2, iNOS, pNF-κB, Nrf2, and HO-1 expression in the brain tissue of PD mice. ** P < 0.01, *** P < 0.001 (vs.Sham), && P < 0.01, &&& P < 0.001 (vs.6-OHDA+Si-NC group).In vitro: The CM of 6-OHDA-induced SH-SY5Y cells was harvested and co-cultured with BV2 cells transfected with si-NC and si-MIR17HG for 24 hours. (e): qRT-PCR measured the MIR17HG expression in BV2. F: Il-1 β, IL-6 and TNF-α mRNA profiles were examined by qRT-PCR. G: The expression of IL-1β, IL-6, and TNF-α mRNA in the cells was tested by qRT-PCR. H: WB checked COX2, iNOS, pNF-κB, Nrf2, and HO-1 expression in BV2 cells. *P < 0.05, **P < 0.01, ***P < 0.001 (vs. Blank group), &P < 0.05, &&P < 0.01, &&&P < 0.001 (vs.CM6-OHDA+Si-NC group).
Figure 5. MIR17HG targeted miR-153-3p.
(a): ENCORI and lncBase databases have identified 44 miRNAs with binding sites to MIR17HG. (b): All the 44 miRNAs were analyzed by qRT-PCR in cells. (c): The predicted binding sequence between miR-153-3p and MIR17HG. (d and e): The dual-luciferase reporter assay uncovered the interaction between miR-153-3p and MIR17HG. (f and g): RIP validated the combination of miR-153-3p and MIR17HG. NS P > 0.05 (vs.miR-NC), *** P < 0.001 (vs.miR-NC). (h-j): qRT-PCR verified the expression alterations of miR-153-3p in mouse brain tissues, SH-SY5Y, and BV2 after knocking down MIR17HG. *** P < 0.001 (vs. Sham or Con group), & P < 0.05, && P < 0.01, &&& P < 0.001 (vs.6-OHDA+Si-NC or CM-Si-NC group).
Figure 6. Overexpressing miR-153-3p tarnished neuronal apoptosis and microglial inflammation.
miR-NC and miR-153-3p mimics were transfected in SH-SY5Y cells. (a): Detection of miR-153-3p expression in SH-SY5Y by qRT-PCR. (b): MTT was applied to affirm the influence of miR-153-3p overexpression on neural proliferation. (c): BrdU staining verified cell viability. (d): The LDH kit determined the level of LDH released into the cells. (e): FCM gauged cell apoptosis. (f and g): WB gauged the profiles of Bax, Bcl2, Caspase3 and SNCA. * P < 0.05, ** P < 0.01, *** P < 0.001 (vs.Con group), & P < 0.05, && P < 0.01, &&& P < 0.001 (vs.6-OHDA+miR-NC group). The CM of SH-SY5Y was co-cultured with BV2 cells transfected with miR-NC and miR-153-3p mimics for 24 hours. (h): qRT-PCR was performed to make certain the miR-153-3p expression in BV2. (i): The expression of IL-1β, IL-6 and TNF-A in BV2 cells was monitored by qRT-PCR. (j): MDA and SOD kits were employed to determine the expression of MDA and SOD in BV2 cells. (k)(: WB assayed the expression of COX2, iNOS, pNF-κB, Nrf2, and HO-1 in BV2 cells. ** P < 0.01, *** P < 0.001 (vs. Blank group), & P < 0.05, && P < 0.01, &&& P < 0.01 (vs.CM6-OHDA+miR-NC group).
Figure 7. miR-153-3p targeted SNCA.
(a): Predicted binding sequence between miR-153-3p and SNCA. (b and c): The dual-luciferase reporter assay revealed the interaction between miR-153-3p and SNCA. (d and e): RIP validated the binding association between miR-153-3p and SNCA. *** P < 0.001 (vs. miR-NC), NS P > 0.05 (vs. miR-NC).
Figure 8. Overexpressing SNCA impaired the anti-inflammatory and anti-neuronal apoptotic effects of miR-153-3p.
We transfected 6-OHDA-induced SH-SYSY5 cells with SNCA overexpression plasmids and then handled them with miR-153-3p mimics for 24 hours. (a and b): qRT-PCR tested miR-153-3p and SNCA expression in SH-SY5Y cells. (c): SNCA expression in SH-SY5Y cells was gauged by WB. (d): MTT verified SH-SY5Y cell viability. (e): BrdU assay was employed for counting the BrdU-positive cell number in SH-SY5Y cells. (f): The LDH kit was applied to examine the release of LDH from SH-SYSY cells. (g): Cell apoptosis was estimated by FCM. (h): WB was conducted for evaluating the expression of Bax, C-caspase-3 and Bcl-2 in SH-SY5Y cells. * P < 0.05, ** P < 0.01, *** P < 0.001 (vs. Con group), & P < 0.05, && P < 0.01, &&& P < 0.01 (vs.6-OHDA group). NS P > 0.05, # P < 0.05, ## P < 0.01, ### P < 0.01 (vs.6-OHDA+SNCA group).In addition, we harvested CM from SH-SY5Y cells treated as described above and incubated the CM with BV2 cells for 24 hours. (i and j): qRT-PCR gauged miR-153-3p expression in BV2 cells. (k): SNCA profiles in BV2 cells were assayed by WB. (l): The levels of IL-1β, IL-6, and TNF-α in BV2 cells were compared by qRT-PCR. (m): The oxidative stress kit was employed to assess MDA expression and SOD activity in BV2 cells. (n and o): WB detection of protein expression of COX2, iNOS, pNF-κB, Nrf2, and HO-1 in BV2 cells. * P < 0.05, ** P < 0.01, *** P < 0.001 (vs. Blank group), & P < 0.05, && P < 0.01, &&& P < 0.01 (vs.CM6-OHDA group). # P < 0.05, ## P < 0.01, ### P < 0.01 (vs.CM6-OHDA+SNCA group).
Figure 9. MIR17HG hindered the anti-inflammatory and anti-neuronal apoptotic properties of miR-153-3p.
MIR17HG overexpression plasmids, miR-153-3p mimics, and MIR17HG overexpression plasmids+miR-153-3p mimics were transfected into 6-OHDA-treated SH-SY5Y cells, respectively. (a and b): qRT-PCR tested the SNCA expression in SH-SY5Y cells. (c): MTT verified SH-SY5Y cell viability. (d): BrdU assay was employed for counting the number of positive cells for BrdU in SH-SY5Y cells. (e): Cell apoptosis was monitored by FCM. (f): WB was conducted for evaluating the expression of Bax, C-caspase-3 and Bcl-2 in SH-SY5Y cells. * P < 0.05, ** P < 0.01, *** P < 0.001 (vs.6-OHDA group), & P < 0.05, && P < 0.01, &&& P < 0.01 (vs.6-OHDA+miR-153-3p group). The CM of SH-SY5Y was co-cultured with BV2 cells transfected with MIR17HG overexpression plasmids, miR-153-3p mimics, and MIR17HG overexpression plasmids + miR-153-3p mimics for 24 hours. (g): The profiles of IL-1β, IL-6, and TNF-a were assessed in BV2 cells by qRT-PCR. (h): MDA and SOD kits were adopted to analyze the expression of MDA and SOD in BV2 cells. (i and j): WB assayed COX2, iNOS, pNF-κB, Nrf2, and HO-1 expression in BV2 cells. ** P < 0.01, *** P < 0.001 (vs.CM6-OHDA), & P < 0.05, && P < 0.01, &&& P < 0.001 (vs.CM6-OHDA+miR−153−3p group).
Supplemental material
Supplemental Material
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The data sets used and analyzed during the current study are available from the corresponding author on reasonable request.
