By linking SMAD4 and firefly luciferase gene, we constructed a dual luciferase reporter system in E. coli. We then engineered chondrocytes with miR-3074-5p (either as mimics, inhibitor, or NC) and SMAD4 overexpression (WT or Mut) plasmids. Notably, the miR-3074-5p inhibitor alleviates the apoptosis and matrix degradation in chondrocytes triggered by obese adipocyte exosomes.

The following are our Proof-of-Concept experiments to verify our design and proposed implementations.

We searched miRNA databases such as miRanda and Targetscan and found that miR-3074-5p had many potential target genes. Among them, SMAD4 gene is one of the important target genes of miR-3074-5p. The binding sequences between miR-3074-5p and SMAD4 gene were determined from the database and subsequent experiments were designed based on the sequence.

Fig.1 Potential binding sequences between miR-3074-5p and SMAD4 gene.

We obtained linearized vectors by digesting the plasmids with restriction enzymes. The target gene fragments were amplified by PCR. Then we performed a recombination reaction between the linearized vector and the target gene to carry out the insertion of the target gene.

Vector Digestion

For vector digestion, a 50 µL digestion reaction was prepared and the reaction was incubated at 37 ° C for 3 h. The vector restriction products were subjected to agarose gel electrophoresis to recover the target band. The intact plasmids may have different conformations such as superhelix, ring opening, and linearity, which leads to different migration rates and show different sizes of bands in agarose gel electrophoresis. After digestion by a single restriction enzyme, the plasmid displays uniform electrophoretic bands.

Fig.2 Restriction Digest Verification of GV272 Plasmid Construct.
1#: DNA marker.
2#: Enzyme digestion product of the vector plasmid.
3#: Control vector plasmid.

Acquisition of Target Gene Segments

In order to obtain enough target genes, we used PCR to amplify the target gene fragments. The sequences of the primers we used for target gene amplification are listed as follows:

Fig.3 Acquisition of target gene segments.
1#: DNA marker.
2#: Segments of target gene.

Construction of Recombinant Plasmids

The linearized vector and target gene amplification products were used to prepare the reaction system for recombination reaction. The linearized vector and target gene fragments were cyclized in vitro by reaction at 37℃ for 30 min. This was followed by cooling in an ice water bath for 5 min.

We transformed the recombinant plasmid into the competent state of E. coli DH5-alpha cells. We selected positive transformants from the recombinant strain to expand the culture. The recombinant plasmid was then extracted for identification by restriction endonuclease digestion.

Fig.4 Verification of the recombinant plasmid.
1#: Blank control (ddH₂O).
2#: Negative control (empty vector without target gene).
3#: Positive control (GAPDH gene was used as a template).
4#: DNA marker.
5-9#: The recombinant plasmid after transformation.

We co-transfected the miR-3074-5p (Mimics or NC) and SMAD4 overexpression (WT or Mut) plasmid carrying firefly luciferase gene into chondrocytes. The intensity of fluorescein was measured after 24 hours.

Fig.5 Relative fluorescence intensity of chondrocytes in each group.

Compared with experimental groups 1, 3, and 4, the relative fluorescence expression in experimental group 2 was significantly decreased (P< 0.05), indicating that miR-3074-5p can bind to the 3' UTR of the SMAD4 gene and inhibit its expression.

Here we designed a protein display system to detect the effect of miR-3074-5p on MMP 13 levels in chondrocytes. We used three types of cells: 1) chondrocytes transformed with miR-NC as control group; 2) chondrocytes co-cultured with Ad-EVs and transformed with miR-NC; 3) chondrocytes co-cultured with Ad-EVs and transformed with miR-inhibitor. We then extracted the protein components of the cells and detected the changes in protein expression using Western blot.

Fig.6 SMAD4 levels of chondrocytes in each group.

The Western Blot results showed that treatment with obese Ad-EVs decreased the level of SMAD4 in chondrocytes, which was successfully reversed by the miR-3074-5p inhibitor. This demonstrates the inhibitory effect of miR-3074-5p on SMAD4.

Fig.7 Protein levels of chondrocytes in each group.

The Western Blot results showed that treatment with Ad-EVs significantly increased the level of MMP13 and decreased the level of COL2 and SOX9 in chondrocytes, which was successfully reversed by the miR-3074-5p inhibitor. In summary, we found that inhibition of miRNA could alleviate matrix degradation induced by Ad-EVs.

Fig.8 Apoptosis of chondrocytes in each group.

The development of osteoarthritis is accompanied by the apoptosis of chondrocytes. It is reported that SMAD4 plays an important role in cell apoptosis. Here we co-cultured chondrocytes with Ad-EVs and simultaneously transformed with miR-inhibitor as mentioned above, and then collected chondrocytes to detect apoptosis. Flow cytometry showed that the miR-3074-5p inhibitor diminished Ad-EVs-triggered apoptosis in chondrocytes.

Overall, we found that miR-3074-5p targets on SMAD4, playing a key role in the impact of obese Ad-EVs on osteoarthritis.