lncRNA SNHG6 regulates neuroinflammation in traumatic brain injury by inhibiting the expression of miR-101-3p

Research population

A cohort of 121 patients with TBI who were treated at Guangzhou Panyu District Hexian Memorial Hospital were included in this study. In addition, 110 healthy volunteers who underwent physical examinations in Guangzhou Panyu District Hexian Memorial Hospital were selected as control group. Venous blood was collected from all people, serum was collected after centrifugation and stored at −80 °C.The inclusion of TBI patients met the diagnostic criteria for TBI [15] and were admitted to our hospital within 24 h after injury. In addition, patients with a clear history of cerebral infarction, cerebral hemorrhage, and transient ischemic attacks, major psychiatric disorders (such as schizophrenia) or neurological disorders, and neurodegenerative diseases were excluded. Each participant signed an informed consent form. The Ethics Committee of Guangzhou Panyu District Hexian Memorial Hospital gave its approval for this study (202200694).

Individuals suffering from TBI were evaluated using the GCS score, which provided a measurement of their neurological status. The assessment of their nervous system function was carried out by observing their responses to movement, verbalizations, and eye-opening responses. According to the GCS score, the subjects were divided into a severe group (n=50, 3–8 points) and a mild group (n=71, 9–15 points).

All TBI patients were followed up for 12 weeks. The outcomes of patients were evaluated by Glasgow Outcome Scale (GOS) score and the patients were categorized into two groups: a good prognosis group (n=79, 4–5 points) and a poor prognosis group (n=42, 1–3 points) [16].

In addition, the basic information of the patients included age, gender, BMI, history of hypertension, history of diabetes, body temperature, respiratory rate, heart rate, and time from trauma to admission were recorded (Table 1).

Cell culture and treatment

The BV2 cell line was procured from the Cell Center of the Chinese Academy of Sciences. BV2 cells were maintained in DMEM (Gibco, MA, USA) supplemented with 10 % FBS (Thermo Fisher Scientific, MA, USA). The cells were incubated in an environment containing 5 % carbon dioxide at 37 °C.

Construction of an OGD model

An in vitro model of OGD was developed to mimic the activation of BV2 microglial cells. In summary, BV2 cells were subjected to three-time washes with a glucose-free DMEM (Gibco, MA, USA). The cells were then cultivated in DMEM devoid of glucose and placed in an incubator containing 1 % oxygen, 5 % carbon dioxide, and 94 % nitrogen. They were then maintained at a temperature of 37 °C for a duration of 1.5 h. The control group was subjected to cultivation in a same culture medium within an incubator that provided a normal oxygen atmosphere and maintained a 5 % carbon dioxide concentration for the same duration.

Cell transfection

The BV2 cells were transfected with a siRNAs targeting SNHG6 (si-SNHG6), siRNA control (si-NC), a miR-101–3p inhibitor and miRNA control (miR-NC) (GenePharma Co., Ltd, Shanghai, China) via Lipofectamine 3000 (Invitrogen, CA, USA).

Extraction of RNA and RT-qPCR

Total RNA was isolated with the TRIzol regent (Invitrogen, CA, USA) and reverse transcribed to cDNA using a reverse transcription kit (Invitrogen, MA, USA). The levels of SNHG6 and miR-101–3p were determined employing RT-qPCR with SYBR Green kit (Invitrogen, MA, USA). qPCR was conducted utilizing the SYBR Green Realtime PCR Master Mix on an ABI 7500 Fast Real-Time PCR System (Toyobo Biologics, Japan). The endogenous controls that were employed were U6 and β-actin. The 2^−ΔΔCt technique was used to calculate the results.

Cell Counting Kit-8 assay

CCK-8 kit was utilized to assess the viability of BV2 cells. In brief, 1 × 10³ BV2 cells were seeded in a 96-well plate per well. The CCK-8 regent (10 μL) (Do Jindo Laboratories, Japan) was subsequently added to each well, after which the plate was placed in an incubator for a 2-h duration. Thereafter, a microplate reader was employed to measure the optical density at a wavelength of 450 nm.

ELISA assay

The concentration of IL-1β, TNF-α, IL-6, IL-4 and IL-10 in cells were determined using an ELISA kit (R&D Systems, Minneapolis, USA). The experiments were carried out in triplicate sets.

Luciferase reporter assay

StarBase was used to predicte the binding sites between SNHG6 and miR-101–3p online. SNHG6 was cloned into pGLO vector to generate a wild-type SNHG6 reporter vector (wt-SNHG6). A mutant-type SNHG6 reporter vector (mut- SNHG6) was created through site-directed mutagenesis. The wt-SNHG6 or mut-SNHG6 vectors were co-transfected with miR-101–3p mimics or miR-101–3p inhibitor into BV2 cells for 48 h, Then the relative luciferase activity was measured. The Dual Luciferase Reporter Assay Kit (Promega) was utilized for the quantification of luciferase activity.

Statistical analysis

The data analysis was done by employing GraphPad Prism 9.0 and SPSS v.26.0. The data were displayed as mean±SD The one-way ANOVA was utilized for comparing means across multiple groups, and the Student’s t-test was used for pairwise comparisons between two distinct groups. The ROC analysis was conducted to assess the diagnostic value of SNHG6 in TBI, and its prognostic value was estimated using the ROC analysis and Cox regression analysis. The statistically significant difference was indicted by p<0.05.

The general information of TBI patients and healthy controls

Age, sex, BMI, history of diabetes, body temperature, respiratory rate, heart rate and history of hypertension did not significantly differ from one another. GCS, GOS, and trauma to hospital admission scores varied significantly across the three groups (Table 1).

The expression of SNHG6 in patients with TBI

TBI patients had increased SNHG6 expression, and those with severe TBI had greater SNHG6 levels than those with moderate TBI (Figure 1A). Moreover, the levels of SNHG6 in the serum of poor prognosis patients were noticeably higher in comparison to those with a good prognosis (Figure 1B).

Figure 1:

The expression of SNHG6 in patients with TBI. ^***p<0.001. Expression of SNHG6 in healthy controls, mild TBI patients, and severe TBI patients (A). Expression of SNHG6 in TBI patients with good and poor prognosis (B).

Clinical value of SNHG6 in TBI

The AUC of the ROC curve for SNHG6 to distinguish patients with severe TBI from those with mild TBI was 0.849, the sensitivity was 74.0 %, and the specificity was 77.5 % (Figure 2A). Furthermore, SNHG could differentiate between TBI patients with a good prognosis and those with poor prognosis. The AUC of ROC curve reached 0.881, with a sensitivity of 78.6 % and a specificity of 82.3 % (Figure 2B).

Figure 2:

Clinical significance of SNHG6. The ROC curve of SNHG6 differentiates patients with mild TBI from those with severe TBI (A). The ROC curve of SNHG6 distinguishes between good and poor prognosis TBI patients (B).

Based on their average SNHG6 levels, two groups of TBI patients were created: those with low expression and those with high expression. The level of SNHG6 was significantly correlated with the time from trauma to hospital admission, GCS score, and GOS score (Table 2). In addition, In TBI patients, SNHG6 was a risk factor for a bad outcome (Table 3).

Relationship between miR-101–3p and SNHG6

In TBI patients, the expression of miR-101–3p was downregulated, and the level was lower in severe patients (Figure 3A). In addition, the level of miR-101–3p was negatively correlated with SNHG6 (Figure 3B). Furthermore, miR-101–3p mimic reduced the luciferase activity of wt-SNHG6, while miR-101–3p inhibitor enhanced the luciferase activity of wt-SNHG6 (Figure 3C).

Figure 3:

Relationship between miR-101–3p and SNHG6. ^***p<0.001. miR-101–3p expression in healthy controls, patients with mild TBI, and patients with severe TBI (A). Correlation between expression levels of SNHG6 and miR-101–3p (B). Dual luciferase reporter gene assay shows the interaction between SNHG6 and miR-101–3p (C).

SNHG6 and miR-101–3p regulated the proliferation and inflammatory response of BV2 cells

The expression of SNHG6 was significantly elevated after the cells were treated with OGD, while miR-101–3p expression was down-regulated in the OGD cell model. Additionally, si-SNHG6 could enhance the expression level of miR-101–3p and miR-101–3p level was inhibited after transfection with miR-101–3p inhibitor (Figure 4A).

Figure 4:

SNHG6 and miR-101–3p regulated the proliferation and inflammatory response of BV2 cells. ^***p<0.001, ^*p<0.05. miR-101–3p inhibitor showed no effect on the expression of SNHG6 but reversed its enhancement by SNHG6 silencing (A). Effect of SNHG6 and miR-101–3p on OGD-induced BV2 cell proliferation (B). Effect of SNHG6 and miR-101–3p on DGR-induced BV2 cell inflammatory response (C–D).

After OGD treatment, the proliferation capacity of BV2 cells decreased, and SNHG6 knockdown improved the proliferation capacity of BV2 cells. However, miR-101–3p inhibitor partially offset the enhancement of the proliferation capacity of BV2 cells induced by si-SNHG6 (Figure 4B). Additionally, the concentration of IL-1β, IL-6, and TNF-αin BV2 cells were elevated after OGD treatment, this increase was reversed by inhibiting the expression of SNHG6, transfection of miR-101–3p inhibitor led to a subsequent rise in these cytokines once more (Figure 4C). On the contrary, the concentration of IL-4 and IL-10 in BV2 cells decreased after OGD treatment. Moreover, SNHG6 knockdown upregulated the levels of IL-4 and IL-10, and miR-101–3p inhibitor reversed this upregulation (Figure 4D).