Throughout the engineering design cycles of our project, team Nanjing-NFLS designed and characterized 7 new basic parts and 3 new composite parts. We also provided potential targets for the treatment of osteoarthritis in obese subjects.

Part Number	Type	Description	Length
BBa_K4606001	Basic	ZsGreen	693 bp
BBa_K4606002	Basic	Firefly-luciferase	1650 bp
BBa_K4606003	Basic	SMAD4-wild type	414 bp
BBa_K4606004	Basic	SMAD4-mutant type	414 bp
BBa_K4606005	Basic	miR-3074-5p coding sequence	414 bp
BBa_K4606006	Basic	miR-3074-5p	22 bp
BBa_K4606007	Basic	Vector-AmpR	3493 bp
BBa_K4606010	Composite	Vector-/ZsGreen-miR-3074-5p-AmpR	4477 bp
BBa_K4606011	Composite	Vector-Luciferase-SMAD4(WT)-AmpR	5563 bp
BBa_K4606012	Composite	Vector-Luciferase-SMAD4(MUT)-AmpR	5563 bp

Table 1 Parts Overview

Stage 1: Design—miR-3074-5p and Its Target Gene SMAD4

MiRNA is a kind of single-stranded noncoding RNA consisting of approximately 22 nucleotides, which can interfere with the expression of genes by binding to the mRNA of target genes. From previous studies on adipose tissue in obesity (see description page), we found that miR-3074-5p may play a key role in the effect of obesity on osteoarthritis.

Currently, researchers need a way to precisely identify the miRNA targets, prior to doing experimental approaches that allow a better functional characterization of miRNAs in biological processes and can thus predict their effects. Computational prediction tools provide a rapid method to identify putative miRNA targets.

Many bioinformatic tools have been developed to predict miRNA:mRNA interaction. In 2003, Bartel′s group developed TargetScan, becoming the first algorithm used to predict miRNA targets in vertebrates ⁽¹⁾. At the same time, the miRanda algorithm was also developed by Enright et al. in 2003 and designed to find potential target sites for miRNAs in the genomic sequence ⁽²⁾. The miRanda algorithm was included in a miRNA web-based tool ⁽³⁾.

By consulting the above gene database, we found that miR-3074-5p could theoretically target SMAD4, a key factor in OA. In chondrocytes, SMAD4 can affect their morphology and enhance their migration. SMAD4 expression has been reported to be downregulated in articular cartilage of osteoarthritis patients, and activation of the SMAD4 pathway could alleviate the progression of diabetes-related osteoarthritis. However, whether miR-3074-5p can inhibit SMAD4 expression is still unknown. Here, we designed a dual luciferase assay to explore the biological binding between miR-3074-5p and SMAD4 gene.

Fig.1 Structure of SMAD4

Fig.2 The target gene SMAD4 of miR-3074-5p by using Targetscan

Stage 2: Build——Plasmids Construction

We incorporated the sequences of the target gene and the luciferase gene into the GV272 vector. Then the vector plasmid was transfected into E. coli DH5-alpha cells for purification and amplification. In this way, SMAD4 plasmids (wild-type (WT) or mutant (Mut)) with the luciferase gene were obtained.

Fig.3 Schematic of Recombinant Plasmid

Fig.4 Schematic of a dual luciferase assay to test the biological binding between miR-3074-5p and SMAD 4, Created with Biorender

Stage 3: Test——Dual Luciferase Assay

We transfected the luciferase gene-bonded SMAD4 plasmid (WT or Mut) and miR-3074-5p plasmid (Mimics or NC) into chondrocytes. After 24 hours, the fluorescence intensity of chondrocytes was detected to determine whether miR-3074-5p could inhibit SMAD4 expression. We identified the plasmid construction by restrictive endonuclease digestion and agarose gel electrophoresis. The effect of miR-3074-5p on SMAD4 expression was determined by comparing the fluorescence intensity of fluorescein in different groups of chondrocytes.

Fig.5 Schematic of Recombinant plasmid to overexpress the target gene

Fig.6 Relative fluorescence intensity of chondrocytes in SMAD4 WT and Mutant group.

Stage 4: Learn

Through this cycle, we learned how to use TargetScan and miRanda software to theoretically predict target gene. When co-expression is verified, it is necessary to investigate the physical interaction between the miRNA in question and the candidate mRNA localized within the target mRNA. The reporter gene assay is the current gold standard procedure to prove a direct miRNA-mRNA interaction. In this procedure, the 3'-UTR region of the gene of interest, containing the predicted mRNA sequence, is cloned immediately downstream of the luciferase gene or another reporter gene in a plasmid. Subsequently, the plasmid must be subcloned under the control of a ubiquitous promoter. Cells containing the plasmid can be co-transfected with miRNA mimics or miRNA inhibitors in order to perform gain-/loss-of-function experiments. The rationale for performing luciferase assay is based on the evidence that though the mRNA is a real target of miRNA under examination, miRNA mimics, as well as miRNA inhibitors, are able to alter endogenous miRNA concentrations and probably lead to changes at protein levels.

Stage 1: Design—miR-3074-5p Inhibitor

Given the important role of SMAD4 in cartilage homeostasis, it is reasonable to speculate that miR-3074-5p could influence the development of OA by acting on SMAD4. We need use engineering tool to inhibit microRNA in obese adipocyte exosomes.

Locked-nucleic acid oligonucleotides are synthetic, modified antisense RNAs. When introduced into cells, these single-stranded molecules perfectly bind to endogenous miRNAs, preventing hybridization with its cellular mRNA targets and thus decreasing miRNA activity. Similarly, constructs known as 'sponge' inhibitors produce RNA sequences containing several sites specific for miRNA in order to sequester endogenous miRNAs and subsequently inhibit their regulatory capacity ^(4-7).

In this method, appropriate controls need to be employed to determine transfection efficiency. For example, the transfection of scrambled miRNA sequences should prove the specificity of a miRNA-mRNA interaction, and the use of an empty vector should demonstrate the efficiency of the transfection.

Based on the above result, we designed a specific miRNA inhibitor of miR-3074-5p for next experiment to examine whether inhibiting miR-3074-5p can rescue SMAD 4 expression and alleviated the changes of chondrocytes induced by obese adipose tissue-derived exosomes.

Fig.7 Schematic of miR-3074-5p inhibitor

Stage 2: Build—miR-3074-5p Inhibitor Construction

We successfully constructed miR-3074-5p inhibitor in this experiment cycle. Inhibitor is a small, synthetic, single-stranded RNA molecules designed to specifically bind to and inhibit mature miRNAs from functioning.

miR-3074-5p inhibitor can efficiently recover SMAD4 expression in chondrocyte. miR-3074-5p can be potential drug to anti-Obesity and protect our joint from the harm of obesity adipocyte exosomes.

Fig.8 Schematic of miR-3074-5p inhibition in chondrocytes

Stage 3: Test——miR-3074-5p Inhibitor Transfected to Chondrocytes

For this cycle, we obtained obese adipose tissue-derived exosomes (Ad-EVs) from a cooperating instructor (Professor Zhao Yue). We further investigated whether miR-3074-5p was involved in Ad-EVs-mediated pathological changes in chondrocyte by using a specific miRNA inhibitor of miR-3074-5p. We then treated chondrocytes with Ad-EVs and inhibitor of miR-3074-5p and examined the pathological changes of chondrocytes like metabolic marker Collagen 2, SOX 9 and MMP 13. To further examine the pathological effects on chondrocytes, we used flow cytometry to detect their apoptosis.

Fig.9 SMAD4 levels of chondrocytes in each group.

Fig.10 Metabolic marker Collagen 2, SOX 9 and MMP 13 protein levels of chondrocytes in each group.

Stage 4 Learn

From this cycle, we learned if a given mRNA is a genuine miRNA target, significant changes in protein levels should be observed due to their interaction. Increased miRNA activity, resulting from the transfection of miRNA mimic into cells expressing the target protein, should lead to decrease target protein expression. Conversely, reduced miRNA activity resulting from the use of a miRNA inhibitor during cell transfection should lead to an increase in target protein expression. We can use miRNA mimic or miRNA inhibitor to achieve our experimental goal.

Reference

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