Implications of the SNHG10/miR-665/RASSF5/NF-κB pathway in dihydromyricetin-mediated ischemic stroke protection

Methods

Cell culture

Human neuroblastoma SH-SY5Y cells were purchased from the American Type Culture Collection (Manassas, VA, USA), maintained in DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco), and incubated at 37 °C.

Cells were incubated in glucose-free DMEM (Gibco) and placed in an anaerobic chamber (Model 10; Thermo Fisher Scientific) filled with a gas mixture of 95% N2 and 5% CO2 for 4 h to induce OGD. The cells were then returned to normal culture conditions (HF100 incubator; Heal force, Shanghai, China) in glucose-containing DMEM and 21% O2 for 24 h to allow reoxygenation. Cells not subjected to OGD/R served as the negative control, which were cultured under standard conditions, with DMEM supplemented with 10% fetal bovine serum at 37 °C and 21% O2. For drug treatment, DHM (Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO; Beyotime, Shanghai, China) and added to the cells at concentrations of 5, 10, 20, 50, and 100 µM 1 h before OGD/R and incubated for 24 h. An equal volume of DMSO was added to the cell culture medium as a negative control (NC), and the final concentration was 0.01% for the assay of toxicity to cells.

Cell transfection

The SNHG10 overexpression vector was constructed by cloning full-length SNHG10 into a pcDNA3.1 vector (RiboBio, Guangzhou, China), and the empty pcDNA3.1 vector served as the NC. miR-665 mimic, miR-665 inhibitor, their NCs (mimic NC and inhibitor NC, respectively), and three short interfering RNAs (siRNAs) targeting RASSF5 (si-RASSF5) and its NC (si-NC; scrambled) were purchased from Qiagen (Hilden, Germany) (sequences are shown in Table 1). Transfections were performed using Lipofectamine 2000 reagent (Invitrogen, Thermo Fisher Scientific).

Cell viability assay

The viability of the SH-SY5Y cells was assessed using an MTT assay kit (Solarbio, Beijing, China). Cells (3 × 10³/well) were inoculated into 96-well plates overnight., After treatment with different concentrations of DHM and/or exposure to OGD/R to simulate ischemic injury, a 20 µL aliquot of MTT solution (5 mg/mL) was added to each well. The cells were then incubated at 37 °C for 4 h to allow the formation of formazan crystals. Following incubation, the MTT solution was carefully removed, and 150 µL of DMSO was added to dissolve the crystals. The plate was shaken gently to ensure complete dissolution. Absorbance was measured at 570 nm using a microplate reader (Multiskan FC Microplate Reader; Thermo Fisher Scientific), with cell viability expressed as a percentage relative to untreated control cells.

Nucleocytoplasmic separation and RT-qPCR

RNA was isolated from cells using a Cytoplasmic and Nuclear RNA Purification Kit (Norgen Biotek, Ontario, Canada). Total RNA was extracted using TRIzol reagent (Invitrogen), and RNA purity was assessed by measuring the A260/A280 ratio using a spectrophotometer (Thermo Fisher Scientific), with values between 1.8 and 2.0 indicating high purity. Complementary DNA (1 µg) was synthesized using the PrimeScript RT Reagent Kit (Takara Bio, Shiga, Japan). qPCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on a StepOne Real-Time PCR System (Applied Biosystems). Thermal cycling conditions included an initial denaturation at 95 °C for 30 s, 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. RNA levels of lncRNA SNHG10, RASSF5, and miR-665 were calculated using the 2^−ΔΔCt method with GAPDH or U6 as internal references for normalization (Singaravelan, Sivaperuman & Calambur, 2022) (primer sequences are shown in Table 2). All experiments were repeated three times.

ROS analysis

The ROS levels were measured using a spectrophotometer-based reactive oxygen species (ROS) assay kit (S0033, Beyotime). This assay utilizes the DCFH-DA fluorescent probe (Beyotime), which penetrates the cell membrane and is hydrolyzed intracellularly by esterases to form non-fluorescent DCFH. Cells were incubated with DCFH-DA (10 µM final concentration) in serum-free medium for 30 min at 37 °C in the dark. The oxidation of DCFH-DA by intracellular ROS produced the fluorescent compound DCF, and fluorescence intensity was quantified to indicate ROS levels. The fluorescence intensity of DCF, which correlates with ROS levels, was measured at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, providing quantitative analysis of intracellular ROS production.

TUNEL analysis

The TUNEL assay was performed using the In Situ Cell Death Detection Kit (Beyotime). SH-SY5Y cells were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate (Beyotime) for 8 min at 21 °C. After washing with PBS, sections were incubated with TUNEL reaction mixture at 37 °C for 60 min. Sections were then washed three times with PBS and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) for 5 min to visualize cell nuclei.

Enzyme-linked immunosorbent assay (ELISA)

Levels of inflammatory cytokines (TNF-α, IL-6, IL-1β, and IL-18) and oxidative stress markers (MDA and SOD) in the cell culture supernatant were measured using ELISA kits (Solarbio). SH-SY5Y cells were treated under various experimental conditions, and supernatants were collected after centrifugation at 1,000× g (Centrifuge 5810R; Eppendorf, Hamburg, Germany) for 10 min to remove any cell debris. For each target, 100 µL of the cell culture supernatant was added to individual wells of 96-well ELISA plates pre-coated with specific antibodies for each marker. Plates were incubated at 37 °C for 1 h. After incubation, wells were washed five times with the provided wash buffer to remove unbound components. Subsequently, 100 µL of horseradish peroxidase (HRP)-conjugated secondary antibody was added to each well, followed by a 30-minute incubation at 37 °C. Following further washing, 100 µL of TMB (3,3′,5,5′-tetramethylbenzidine, Solarbio) substrate solution was added to develop color, and plates were incubated in the dark for 15–30 min at room temperature. The reaction was then stopped by adding 50 µL of stop solution (Solarbio), changing the color from blue to yellow. Absorbance was measured at 450 nm using a microplate reader (Multiskan FC Microplate Reader; Thermo Fisher Scientific).

Bioinformatic analysis

Target prediction was performed using the starBase database for lncRNA SNHG10, and starBase and TargetScan databases for miR-665. The likelihood of the outcome predicted by the TargetScan database is extremely high when the context++ score is ≥90. The interaction between miR-665 and predicted targets was further verified using a luciferase reporter assay.

Luciferase assay

The psiCHECK-2 dual luciferase reporter vector (Promega, Madison, WI, USA) was cloned including SNHG10 and 3′-UTR segments of RASSF5 that included miR-665 binding sites. The reporter vector and the miR-665 mimic were co-transfected into SH-SY5Y cells using Lipofectamine 2000 for 48 h. A dual-luciferase reporter assay system (Promega) was used to quantify luciferase activity, according to the manufacturer’s instructions. Firefly luciferase data were normalized to the Renilla luciferase activity.

RNA pulldown

Biotinylated lncRNA SNHG10 (bio-SNHG10), a mutant version of bio-SNHG10 (bio-mut), and a biotin-labeled non-specific control RNA (bio-NC) were synthesized (RiboBio, Guangzhou, China). SH-SY5Y cells were transfected with 50 nM of each biotinylated RNA construct using Lipofectamine 2000 (Invitrogen), and cells were harvested 48 h post-transfection. After cell lysis in RIP lysis buffer supplemented with protease and RNase inhibitors, cell lysates were incubated with streptavidin-coated magnetic beads (Dynabeads M-280 Streptavidin; Invitrogen) at 4 °C for 4 h to allow binding of the biotin-labeled RNA to the beads. The bead-bound complexes were then washed five times with cold wash buffer to remove non-specifically bound proteins and RNAs. After washing, RNA was extracted from the complexes using TRIzol reagent (Invitrogen). The co-precipitated RNAs were then quantified by RT-qPCR (Hu et al., 2023).

Western blot analysis

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protein samples were prepared in radioimmunoprecipitation assay lysis buffer (Solarbio) and transferred to a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA). The membranes were blocked with 5% BSA and incubated with RASSF5 (1:300, ab204117), NF-κB (1:1000, ab32536), phosphor (p) NF-κB (1:1000, ab76302), and GAPDH (1:2500, ab9485) primary antibodies, followed by HRP-conjugated secondary antibodies (1:3000, ab6721). All antibodies were purchased from Abcam (Cambridge, UK). Protein bands were visualized using ECL detection reagent (Thermo Fisher Scientific).

Statistical analysis

All experiments were repeated three times, data are presented as mean ± standard deviation. Statistical analyses were performed using SPSS statistical analysis software (version 22.0, IBM Corp., NY, USA), and significance was determined by one-way analysis of variance (ANOVA) with Tukey’s post hoc test, statistical significance defined as p < 0.05. Graphical representations, including bar charts, were created using GraphPad Prism 8.0 (GraphPad Software, Inc., La Jolla, CA, USA).

The effect of DHM on SH-SY5Y viability

Since 50 and 100 µM DHM (chemical structure in Fig. 1) caused a slight inhibition of cell viability (p < 0.05), we chose 5, 10, and 20 µM as safe concentrations of DHM for subsequent experiments (Fig. 2A). Based on the observed concentration-dependent effects on cell viability, we further investigated the influence of DHM on other cellular processes affected by OGD/R. Our data revealed that DHM ameliorated OGD/R-induced cell viability (Fig. 2B), apoptosis (Fig. 2C). neuroinflammation (increased TNF-α, IL-6, IL-1β, and IL-18) (Figs. 3A–3D), and oxidative stress (increased ROS and MDA, decreased SOD) (Figs. 3E–3G) in a dose-dependent manner. Interestingly, overexpression of lncRNA SNHG10 in the OGD/R response was inhibited by DHM (p < 0.05) (Fig. 4A), suggesting the potential involvement of SNHG10 in the protective mechanism of DHM. Furthermore, cytoplasmic-nuclear separation analysis revealed abundant SNHG10 expression in the cytoplasm, similar to that of the GAPDH positive control, but in contrast to U6 (Fig. 4B).

The functionality of SH-SY5Y cells was improved by DHM via SNHG10

To further investigate the role of SNHG10, an SNHG10 overexpression vector was constructed and transfected into SH-SY5Y cells. The efficacy of this overexpression was confirmed through RT-qPCR analysis (p < 0.05), as shown in Fig. 5A. Upon SNHG10 overexpression, we noted a partial reversal of the DHM-induced protective effects in the cells subjected to OGD/R conditions. Specifically, SNHG10 overexpression led to a reduction in cell viability (Fig. 5B), an increase in apoptosis levels (Fig. 5C), and a significant increase in markers of neuroinflammation, including elevated levels of TNF-α, IL-6, IL-1β, and IL-18 (Figs. 6A–6D). Additionally, oxidative stress markers were adversely affected, with an increase in ROS and MDA levels, coupled with a decrease in SOD activity (Figs. 3E–3G). These observations suggest that SNHG10 may act as a regulatory factor that modulates the protective effects of DHM under OGD/R-induced stress, highlighting its role in influencing cell viability, inflammation, and oxidative stress responses.

SNHG10 targets binding to miR-665

According to the starBase database analysis, SNHG10 has eight predicted downstream miRNAs: miR-2278, miR-6884-5p, miR-485-5p, miR-3196, miR-3180, miR-6816-5p, miR-3180-3p, and miR-665. Among them, miR-665 had the lowest expression in the OGD/R group, displayed a dose-dependent increase following DHM treatment (p < 0.05) (Fig. 7), and was suppressed by SNHG10 overexpression (p < 0.05) (Fig. 8A). MiR-485-5p, which was also downregulated in OGD/R but partially counteracted by DHM, was not significantly altered by SNHG10 overexpression (p < 0.05) (Fig. 8B). Therefore, we selected miR-665 for further mechanistic studies. After validating the efficacy of the miR-665 mimic/inhibitor (Fig. 8C), we found no significant change in the expression of SNHG10 (p > 0.05) (Fig. 8D). Using a dual luciferase assay, it was observed that the luciferase activity of WT-SNHG10 co-transfected with the miR-665 mimic was lower than that of mut-SNHG10 (p < 0.05). However, there was no significant difference in luciferase activity between WT-SNHG10 and mut-SNHG10 cells co-transfected with the miR-665 inhibitor (Fig. 8E). RNA pulldown results showed that miR-665 was enriched in the bio-SNHG10 group compared to the bio-NC and bio-mut groups (Fig. 8F), confirming that miR-665 is a direct target of SNHG10.

RASSF5 is a direct target of miR-665

Through a combined analysis of the TargetScan and starBase databases, we identified 60 genes as potential targets of miR-665, with these genes achieving a context++ score of ≥90 in the TargetScan database (Fig. 9A). By excluding genes that were previously demonstrated to play significant roles in IS or SH-SY5Y cells, we identified six genes whose roles and mechanisms in IS or SH-SY5Y cells remained unverified. These genes included RASSF5, NTMT1, LIX1L, SYNGR2, COPS7B, and ZNF740. We then performed RT-qPCR analysis of these six genes, similar to the miRNA screening approach. The results showed that the expression of RASSF5 and LIX1L was increased by OGD/R treatment and was suppressed by DHM (Figs. 9B–9G). However, RASSF5 expression increased with SNHG10 overexpression (p < 0.05) (Fig. 10A), whereas LIX1L expression did not significantly change (p > 0.05) (Fig. 10B). Therefore, we selected RASSF5 as a potential target of miR-665 for further experiments. RASSF5 expression and protein levels were negatively regulated by miR-665 (Figs. 10C, 10D). TargetScan database predicted the binding site between RASSF5 and miR-665 (Figs. 10E). Luciferase activity of WT-RASSF5 co-transfected with the miR-665 mimic was lower than that of mut-RASSF5 (p < 0.05). However, there was no significant difference in luciferase activity between WT-RASSF5 and mut-RASSF5 cells co-transfected with the miR-665 inhibitor (Fig. 10F). Therefore, RASSF5 is a direct target of miR-665. The mRNA levels of RASSF5 were downregulated in the si-RASSF5-transfected group compared to those in the si-NC group (Fig. 10G), indicating the successful synthesis of si-RASSF5, with si-RASSF5-1 having the best inhibitory effect.

DHM improves SH-SY5Y cells via SNHG10/miR-665/RASSF5 axis

To investigate the interaction between DHM and the SNHG10/miR-665/RASSF5 axis, we co-transfected SNHG10 cells with miR-665 mimic/si-RASSF5. The results showed that DHM ameliorated the OGD/R-mediated decrease in SH-SY5Y cell viability (Fig. 11A), apoptosis (Fig. 11B), neuroinflammation (TNF-α, IL-6, IL-1β, and IL-18) (Figs. 12A–12D), oxidative stress (increased ROS and MDA, decreased SOD) (Figs. 12E–12G), and increased RASSF5 expression (Fig. 13A). Intervention with miR-665 promoted the effect of DHM, and the counteracting effect of SNHG10 on miR-665 was inhibited by si-RASSF5. In addition, DHM alleviated OGD/R-induced elevated RASSF5 protein levels and NF-κB phosphorylation, and the addition of miR-665 mimic enhanced the effect of DHM. si-RASSF5 attenuated the effect of SNHG10 on the miR-665 mimic (Fig. 13B). Thus, DHM protected SH-SY5Y cells by reducing the transcription and translation of RASSF5 and inactivating the NF-κB signaling pathway through the SNHG10/miR-665 axis.