This year, the Nanjing-NFLS team utilized the anomalous variations of miR-3074-5p in obese adipocyte exosomes (Ad-EVs) to investigate its role in the regulation of osteoarthritis. By targeting the SMAD4 gene in chondrocytes, miR-3074-5p disrupts chondrocyte homeostasis. A crucial discovery was that inhibiting miR-3074-5p could attenuate the chondrocyte injury induced by obese Ad-EVs.

The rising prevalence of osteoarthritis and obesity in modern society cannot be ignored. Obesity not only doubles the lifetime risk of osteoarthritis but also exacerbates it in both weight-bearing and non-weight-bearing joints. Given that osteoarthritis is a leading cause of disability among older adults, the urgency to find novel therapeutic targets for osteoarthritis related to obesity has never been more pressing.

We realized that obese conditions increase the secretion of exosomes from adipose tissues. These exosomes, especially their component of Micro-RNAs like miR-3074-5p, play pivotal roles in inter-organ communication and joint functions. Using the TargetScan software and a luciferase reporter plasmid, we verified that SMAD4, vital for bone and cartilage development, is a direct target of miR-3074-5p. In chondrocytes, SMAD4 not only impacts their morphology but also promotes their migration. Importantly, decreased SMAD4 expression has been observed in the articular cartilage of osteoarthritis patients. Thus, targeting miR-3074-5p's effect on SMAD4 could pave the way for potential osteoarthritis treatments and anti-Obesity.

To corroborate our findings, we constructed target gene plasmids and performed luciferase assays. The results demonstrated that miR-3074-5p indeed suppresses SMAD4 expression. Additionally, our western blot and apoptosis assays showed that inhibiting miR-3074-5p alleviates the harm posed by obese adipocyte exosomes to chondrocytes.

In our project, we used the firefly lusiferase gene from http://parts.igem.org/Part:BBa_I712019, and spliced the firefly luciferase gene behind the target gene segment. By co-transfecting them into cells and detecting the luciferase activity, we can speculate whether the genes under test are successfully expressed. We contributed an addtion document under the http://parts.igem.org/Part:BBa_I712019.

We compiled protocols for the micro-RNA target gene searching by using TargetScan software and created a troubleshooting document (View detail in Partnership page) to assist future teams who may choose to use a dual luciferase reporter experiment. We also designed and added new documentation to basic Parts and composite parts on that Part's registry page(Part). All protocols and results were carefully documented in our wiki and the Registry. The prevention and treatment of osteoarthritis and obesity have always been popular iGEM topics, and we hope our contributions could be of some reference for future teams!

Fig.1 Schematic engineering design in our project created with Biorender