Part:BBa_K4606005
miR-3074-5p coding sequence
Description
Here we construct the sequences that encoding miR-3074-5p. The gene sequence encoding miRNA is first transcribed into pri-miRNA, which is cleaved and modified by ribonuclease to become the final miRNA. MiRNAs are a class of endogenous non-coding single-stranded small RNAs with a length of approximately 20–22 nucleotides and their expression changes are related to many human diseases.
After inserting this sequence into the vector and expressing it in cells, we could test whether the miR-3074-5p was able to target the SMAD4 gene.
Usage
After inserting this sequence into the vector and expressing it in cells or inhibited its expression by miR-inhibitor, we could detect the effect of miR-3074-5p on the pathological state of chondrocytes. When we combined this sequence with a dual luciferase reporter gene system, we could test whether the miR-3074-5p was able to target the SMAD4 gene.
Experience
Our instructors previously found that miR-3074-5p were significantly increased in obese adipose-derived exosomes by biological sequencing. This sequence in this part was designed to study the effect of miR-3074-5p on osteoarthritis.
We measured the miR-3074-5p levels in chondrocytes after transfection with this sequence. The results of qRT-PCR showed that the plasmid containing the miR-3074-5p coding sequence could successfully express miR-3074-5p 24 hours after transfection into chondrocytes. The miR-3074-5p content in chondrocytes was significantly increased (Figure 1).
Figure 1. The relative miR-3074-5p level in chondrocytes transfected with overexpression (OE) plasmid was significantly higher than that of negative control (NC) plasmid.
Because miR-3074-5p was obtained from obese adipose tissue-derived extracellular vesicles (Ad-EVs). Here, we used Ad-EVs to treat chondrocytes and combined with miR-3074-5p inhibition, and then analyzed the pathological changes of chondrocytes. After chondrocytes were treated with Ad-EVs, the intracellular miR-3074-5p content increased. Inhibition of miR-3074-5p level could protect chondrocytes and reduce their apoptosis rate (Figure 2).
Figure 2. Intracellular miR-3074-5p level of chondrocyte after treatment with Ad-EVs and inhibitor of miR-3074-5p.
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 result demonstrates the inhibitory effect of miR-3074-5p on SMAD4 (Figure 3).
Figure 3. SMAD4 levels 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. Flow cytometry showed that the miR-3074-5p inhibitor diminished Ad-EVs-triggered apoptosis in chondrocytes (Figure 4).
Figure 4. Apoptosis of chondrocyte after treatment with Ad-EVs and inhibitor of miR-3074-5p.
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.
Figure 5. Protein levels of chondrocytes in each group.
Overall, we found that miR-3074-5p plays a key role in the effect of obese Ad-EVs on osteoarthritis.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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