miR-22-3p relieves the osteoarthritis by targeting to inflammasome in vivo and in vitro

Materials

The fetal bovine serum was purchased from Thermo Fisher Scientific (Waltham, MA, USA). DMEM 1640 medium and phosphate buffered saline (PBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Lipopolysaccharide (LPS) was purchased from Solarbio (Beijing, China). IL-1β ELISA detection kit was purchased from Thermo Fisher (Waltham, MA, USA). NF-κB p65 (1:1,000 dilution), phosphorylated NF-κB p65 (1:500 dilution), IκBα (1:2,000 dilution), phosphorylated IκBα (1:1,000 dilution), and NLRP3 (1:1,500 dilution) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-caspase-1 (1:2,000 dilution) and anti-ASC (1:1,000 dilution) antibodies were purchased from Abcam (Cambridge, UK). Unless otherwise specified, other reagents were purchased from Sigma-Aldrich company (St. Louis, Missouri, USA).

Cell culture

Human cartilage cell line C28/I2 were purchased from ATCC. The cells were cultured in DMEM medium supplemented with 10% FBS at 37 °C humidified incubator with 5% CO₂.

RT-PCR

Total RNAs were extracted from C28/I2 cells or tissue using TRIzol Reagent kit (Invitrogen™, Cat:10296028) following the manufacture’s protocol. The total RNAs were subsequently subjected to cDNA synthesis by using superscript II reverse transcriptase (Invitrogen™, Cat:18064071). RT-PCR was conducted using the following primers: NLRP3 (forward, 5′-CACCTGTTGTGCAATCTGAA -3′; reverse, 5-TCCTGACAACATGC TGATGTGA)-3′, human capsase1 (forward, 5′-TTTCCGCAAGGTTCGATTTTCA-3′; reverse, 5′-TGGGCATCTGCGCTCTACCATC-3′), human IL-1β (forward, 5′-TCCAGGGACAGGATATGGAG-3′; reverse, 5′-TCTTTCAACACGCAGGACAG-3′). miR-22: 5′-GTCGTATCCAGTGCGTTGCGTGGAGTC, GGCAATTGCACTGGATACGACAACTGT-3′. The mRNA levels were normalized to the levels of β-actin mRNA. GAPDH: F: forward, 5′-TGTGTCCGTCGTGGATCTGA-3′, Reverse:5′ -CCTGCTTCACCACCTTCTTGA-3′ [16].

Construction of adeno-associated virus (AAV) vector expressing miR-22-3p

Adeno-associated virus (AAV) is a vector widely used in gene therapy. In the current study, adeno-associated virus (AAV) vector expressing miR-22-3p was constructed by Gene Pharma Co., Ltd.

Establishment of OA model

Male C57BL/6J mice aged 10 weeks were used to establish a mouse OA model [17–19]. After the mice were anesthetized with chloral hydrate (400 mg/kg) in the abdominal cavity, the right knee joints of the mice were disinfected with alcohol, and one centimeter was cut adjacent to the patella to expose the joint cavity, and the anterior meniscus ligament was cut. After flushing the joint cavity with sterile saline, the incision was sutured and the skin was disinfected. The experiments were divided into four groups: Group A, control operation group; Group B, operation group; Group C, AVV-mic-22-3p treatment group; Group D, adeno-associated virus vector control group.

Safranin O-fast green stain

The tissue sections were routinely deparaffinized, and then the sections were soaked in xylene solution for 10 min. After treatment with the ethanol, the sections were stained with fast-green. After washing, the sample was stained with safranin O solution, the sections were then observed using a light microscope.

Intra-articular injection of AAV virus

One week after the establishment of the OA model, the AAV virus vector expressing miR-22-3p or control miRNA was injected into the joint cavity, after miR-22-3p treatment for four weeks, the cartilage tissue of the mouse knee joint was extracted for HE and Safranin O-fast green stain analysis.

Cell viability assay

Cell counting kit-8 was used to test the cell proliferation. In brief, C28/I2 cell (1 × 10⁴ cells/mL) were seeded into 96-well plate for 24 h. Cells were pre-treated with different concentrations of LPS for 12 h, 10 µL CCK-8 solution was added and incubated at 37 °C for 4 h, after which, absorbance at 450 nm wavelength was detected using a microplate reader (Bio-rad).

Western-blot

After treatment with LPS for 12 h, total protein was extracted using RIPA lysis buffer. Protein concentrations were determined using the bicinchoninic acid (BCA) kit. Equal amounts of protein (30 μg/Lane) from each sample were subjected to SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membranes were then treated with 5% skim milk powder at 4 °C overnight. After washing, the PVDF membranes were treated with the primary antibodies (NF-κB, 1:500 dilution; NLRP3, 1:1,000) at 4 °C overnight, followed by incubation with HRP-conjugated secondary antibodies (1:2,000 dilutions) for 1 h at RT. The immunoreactive bands were visualized by ECL detection system.

Fluorescence-activated cell sorting (FACS) analysis

After miR-22-3p or control miRNA transfection for the indicated time points, a single cell suspension was prepared. The cells were fixed, blocked and stained with the indicated antibodies, the flow cytometer was then used to analyze the cell samples.

Analysis of ROS content

C28/I2 cells were cultured in 6-well plates for 48 h. The cells were then stimulated with LPS for 12 h. After three washes, C28/I2 cells were treated with 10 uM DCFH-DA for 0.5 h. After washing, cells were collected and determined by Flow Cytometer (BD). ROS levels were determined by BD CellQuest software.

Statistical analysis

All data are expressed as mean ± SD. Three independent experiments were performed. Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software Inc). p<0.05 was identified as significantly difference.

Effect of LPS on human cartilage cell line C28/I2

We used LPS to treat the chondrocyte line (C28/I2) to establish inflammation model. Firstly, the cell viability of C28/I2 was determined by CCK-8 Kit. Figure 1A showed that the relative viability of C28/I2 cells was obviously decreased after treatment with 5–10 ug of LPS for 12 h. However, LPS (5 ug/mL) did not lead to the NLRP3 up-regulation. However, LPS (10 μg/mL) not only could obviously down-regulate the cell viability, but also elevate the NLRP3 expression. Furthermore, the ROS level was evidently enhanced (it has been demonstrated that ROS leads to the NLRP3 inflammasome activation) (Figure 1B). Additionally, the results form RT-PCR and Flow Cytometry showed that the expression levels of NLRP3, ACS, Caspase-1, and IL-1β were also elevated (Figure 1C). The proportion of cell apoptosis was obviously enhanced in LPS-treated group (Figure 1D). These observations suggests that the LPS-induced arthritis cell model was successfully established. Therefore, LPS (10 μg/mL) was used in the subsequent experiments (in vitro).

Figure 1:

(A) The effect on LPS on the viability of C28/I2 cells. C28/I2 cell (1 × 10⁴ cells/mL) were seeded into 96-well plate for 24 h. Cells were pre-treated with different concentrations of LPS, 10 µL CCK-8 solution was added and incubated at 37 °C for 4 h, after which, absorbance at 450 nm wavelength was detected using a microplate reader. (B) The ROS level was obviously enhanced by LPS treatment. C28/I2 cells were cultured in 6-well plates for 48 h. The cells were then stimulated with LPS for 12 h. After three washes, C28/I2 cells were treated with 10 uM DCFH-DA for 0.5 h. After washing, cells were collected and determined by flow cytometer (BD). ROS levels were determined by BD CellQuest software. (C) NLRP3, ACS, Caspase-1, and IL-1β were up-regulated by LPS treatment. D. The proportion of apoptosis was obviously elevated in LPS group. The asterisk (*) indicates a statistically significantdifference (p-value<0.05).

The expression of MiR-22-3p was down-regulated in LPS-treated C28/I2 cells

RT-PCR was performed to test the expression of MiR-22-3p in LPS-treated C28/I2 cells. Figure 2 showed that the expression level of MiR-22-3p was obviously down-regulated compared to control group.

Figure 2:

(A) The expression of MiR-22-3p was down-regulated in LPS-treated C28/I2 cells. Total RNAswere extracted from C28/I2 cells or tissue using TRIzol Reagent kit (Invitrogen™) following the manufacture’s protocol. The total RNAs were subsequently subjected to cDNA synthesis by using superscript II reverse transcriptase. RT-PCR was performed in the materials and Methods section. The asterisk (*) indicates a statistically significant difference (p-value<0.05).

The effect of up-regulation of miR-22-3p on the expression of NLRP3 and downstream cytokines

Bioinformatics analysis (MirDB: http://www.mirdb.org/, miRWalk: http://mirwalk.umm.uni-heidelberg.de/) showed that the miR-22-3p could target to NLRP3 (Figure 4A), the 3′-UTR of NLRP3 mRNA may has a direct target site of miR-22-3p. Therefore, we studied the effect of over-expression of miR-22-3p on NLRP3. It can be found that miR-22-3p over-expression remarkably blocked the expression of NLRP3, IL-1β and Caspase-1 (Figure 3A). In contrast, inhibition of miR-22-3p enhanced the expression of NLRP3, IL-1β and Caspase-1 (Figure 3B). These observations indicated that up-regulation of miR-22-3p could inhibit inflammatory responses via targeting NLRP3.

Figure 3:

(A) The effect of up-regulation of miR-22-3p on the expression of NLRP3, IL-1β or Caspase-1. Total RNAswere extracted from C28/I2 cells or tissue using TRIzol Reagent kit (Invitrogen™) following the manufacture’s protocol. The total RNAs were subsequently subjected to cDNA synthesis by usingsuperscript II reverse transcriptase. RT-PCR was performed in the Materials and Methods section. (B) Inhibition of miR-22-3p enhanced the expression of NLRP3, IL-1β and Caspase-1.

Figure 4:

(A) The predicted binding sites of miR-22-3p on 3′-UTR of NLRP3 mRNA by MirDB and miRWalk (MirDB: http://www.mirdb.org/, miRWalk: http://mirwalk.umm.uni-heidelberg.de/). (B) miR-22-3p significantly inhibited luciferase activity compared with the mutant binding sites. The asterisk indicates thatthe difference is statistically significant (p-value <0.05).

Identification of NLRP3 mRNA as the target gene of miR-22-3p

Here, we analyzed the relationship between NLRP3 and miR-22-3p. Bioinformatics software analysis (MicroRNA Target Prediction Database, MirDB and miRWalk) has showed that the miR-22-3p could bind to 457–464 of the 3′-UTR of NLRP3 mRNA (Figure 4A). Furthermore, a dual-fluorescent reporter assay system was conducted to confirm the result of bioinformatics analysis. As indicated in Figure 4B, miR-22-3p over-expression remarkably reduced the luciferase levels of the wild-type (WT) 3′-UTR fragment of NLRP3 (but not mutated 3′-UTR fragment of NLRP3), these results suggested that NLRP3 may be a direct target gene of miR-22-3p (Figure 5).

Figure 5:

Effects of miR-22-3p on the NF-κB signaling pathway.

Total protein was extracted using RIPA lysis buffer. Protein concentrations were determined using the bicinchoninic acid (BCA) kit. Equal amounts of protein (30 μg/Lane) from each sample were subjected to SDS-PAGE and transferred onto apolyvinylidene fluoride (PVDF) membrane. The membranes were then treated with 5% skim milk powder at 4 °C overnight. After washing, the PVDF membranes were treated with the primary antibodies (NF-κB, 1:500 dilution; NLRP3, 1:1,000) at 4 °C overnight, followed by incubation with HRP-conjugated secondary antibodies (1:2,000 dilutions) for 1 h at RT. The immunoreactive bands were visualized by ECL detection system.

miR-22-3p over-expression inhibited the activation of NF-κB -associated signaling pathway

Studies has indicated that the activation of NLRP3 is closely related to NF-κB signaling pathway. Therefore, we analyzed the activity of the NF-κB signaling pathway. It can be found that p-p65 and p-IκBα expression was evidently enhanced in inflammatory groups compared to control group. However, miR-22-3p transfection decreased the expression levels of p-p65 and p-IκBα. These results indicate that miR-22-3p suppressing inflammatory injury may be though NF-κB signaling pathway.

miR-22 over-expression relieved osteoarthritis in vivo

A lentiviral vector for the over-expression of miR-22-3p was constructed and injected into the joint cavity of osteoarthritis model mice. After treatment for four weeks, we analyzed the effect of over-expression of miR-22-3p on osteoarthritis (OA). Safranin-fast green stain was performed to observe the morphological changes of articular cartilage. As shown in Figure 6A, compared with the sham operation group (Sham), the surface of the knee joint cartilage in the operation group was significantly destroyed, and the cartilage matrix content was reduced; however, the cartilage damage was significantly reduced in AAV-miR22-3p treatment group, while the adeno-associated virus control group (AAV-miR22-NC) had no significant difference compared to operation group.

Figure 6:

(A) AAV-miR22-3p treatment relieved osteoarthritis in vivo evaluated by Safranin O-fast green staining. The tissue sections were routinely deparaffinized, and then the sections were soaked in xylene solution for 10 min. After treatment with the ethanol, the sections were stained with fast-green. After washing, the sample was stained with safranin O solution, the sections were then observed using a light microscope. (B) The effect of AAV-miR22-3p treatment on OA determined by HE staining. (C) Markin score between AAV-miR-22-3p treatment group and operation group.

Additionally, HE staining of knee joint sections showed that in sham operation group, the surface cells of cartilage tissue were well-arranged, the surface of cartilage tissue was smooth, and the matrix staining was uniform. In the OA group, the cells on the surface of cartilage arranged disorderly, and the matrix staining was uneven. Compared with the operation group, the surface cells of cartilage in the operation group were well-arranged by AAV-miR-22-3p treatment (Figure 6B), there was no significant difference in Markin score between AAV-miR22-NC treatment group and operation group (OA group) (Figure 6C).