JAM-A Regulates PTEN To Inhibit Epithelial Cell Proliferation Through CeRNA Mechanism

During wound healing,epidermal cells play a key role and their status profoundly affects the quality of wound healing.And the proliferative activity of epidermal cells is one of the most important mechanisms that affect wound healing.In the process of proliferation of epidermal cell,many molecules and signaling pathways participate in the regulation of its proliferation function.In recent years,junctional adhesion molecule A has gradually become a research hotspot.JAM-A is a transmembrane protein,belonging to the family of junctional adhesion molecules.Its inside PDZ domain stucture could bind to the intracellular PDZ-domain-containing molecules and tansduct signals after homodimerization.At the same time,JAM-A itself is an adhesion molecule,which is closely related to the adhesion and migration function of cells.It is highly expressed on the membrane surface of various cells,such as epidermal cells,endothelial cells,inflammatory cells,and mesenchymal stem cells.It participates in the epithelial cell barrier formation and regulation of immune homeostasis,inflammation and angiogenesis.According to the latest research,in addition to regulating cell adhesion,migration and signal transduction,JAM-A can positively regulate the proliferative activity of tumor cells in myeloma,breast cancer,lung cancer,testicular cancer,head and neck squamous cell carcinoma and etc..In malignant tumors,the expression of JAM-A was significantly increased,showing a positive correlation with the proliferation,invasion,and chemotaxis of cancer cells.In a preliminary experiment,we found that after JAM-A expression was disrupted,the epidermal cell proliferation rate was significantly higher than that of JAM-A overexpressed epidermal cells.Obviously,this is inconsistent with the function of JAM-A in those tumor tissues.Whether JAM-A regulates proliferative activity in normal epidermal cells is positive or not? What’s more,in these tumor-related studies,the functions exhibited by JAM-A are mostly exerted by proteins,and few scholars pay attention to its long 3600 bp 3′UTR region,which contains a large number of non-coding RNA binding sites.Does JAM-A still play a major role in the regulation of proliferative activity in normal epidermal cells as a protein?Since microRNAs mainly exert post-transcriptional repression through binding to the 3′UTR region of mRNA,we suspect that JAM-A regulating proliferative activity in normal epidermal cells may also achieve it through microRNAs.These microRNAs,including miR-21,miR-31,miR-203,miR-205,and miR-210,are involved in the regulation of epidermal cell proliferation after skin wounded.However,there is no literature reported that microRNA regulates cell proliferation by binding to the 3′UTR region of JAM-A.It suggests that we may discover new miRNAs that bind to the JAM-A 3’UTR region.Therefore,based on the research progress of JAM-A,microRNA and cell proliferation and combined with our preliminary experimental results,we intend to discover whether JAM-A exerts its positive regulation role in epidermal cells’ proliferative activities through non-coding RNAs contrary to that in turmor cells.We intend to further screen differentially expressed non-coding RNAs by high-throughput gene sequencing,and combine bioinformatics analysis with literature resources to find a specific non-coding RNA targeted to JAM-A 3’UTR region.Then we’ll explore the underlying mechanisms whether JAM-A regulates the downstream molecules through non-coding RNA,by which negatively regulates epidermal cell proliferation.This will help deepen the understanding of the wound healing process and lay a foundation for the subsequent exploration of mechanisms for promoting healing and searching for potential clinical therapeutic targeted drugs in diabetic foot ulcer,stress ulcers and other chronic non-healing wounds.JAM-A affects epidermal cell proliferation activity.Overexpression of JAM-A inhibits proliferation of epidermal cells.According to our previous study,JAM-A plays a role in epidermal cell proliferation and migration,which is consistent with the reported literatures.To further clarify the effect of JAM-A on proliferation activity in epidermal cells,we constructed a human immortalized epidermal cell HaCaT that overexpresses JAM-A and knocks down JAM-A,using lentiviral vectors.The CCK-8 and ki67 experiment verified that the proliferation of epidermal cells after overexpression of JAM-A was slowed down,and the proliferation of epidermal cells after JAM-A knock-down was accelerated.JAM-A regulates the proliferation of epidermal cells through binding to miR-221/222 cluster with its 3’UTR region.Previous literature has reported that JAM-A plays a role in the migration and proliferation of tumor cells through protein-protein interactions usually.At the same time,we found that there are a lot of microRNA binding sites in the 3’UTR region of the JAM-A mRNA by searching the Gene database on the PubMed website.Therefore,we wanted to explore the molecular mechanism of JAM-A regulating the proliferation of epidermal cells from the microRNA perspective.To verify this idea,we constructed epithelial cells that overexpressed the CDS region and overexpressed the 3’UTR region on the basis of interfering with JAM-A epidermal cells.After RNA extraction and PCR,we found JAM-A regulate cell proliferation activity through its 3’ UTR.Then we designed the Dicer enzyme knock-out experiment and demonstrated that JAM-A exerts its function of regulating epidermal cell proliferation by binding its 3’UTR region to microRNA.Based on above findings,we conducted RNA-seq high-throughput sequencing utilizing over-expressed JAM-A and interfered with JAM-A epidermal cells.Combined with bioinformatics analysis and literature searches,we screened(1)significantly down-regulated miRNAs in the overexpression group,(2)significantly up-regulated miRNA in the knock-down group,(3)an intracellular abundance of about 2000 TPM,(4)microRNAs that targeted to genes associated with cell proliferation.By RT-PCR,we found the expression of miR-221/222 was up-regulated after interfering with JAM-A,but miR-221/222 expression was down-regulated after overexpression of JAM-A 3’UTR region.Through in vitro cell experiments,we also demonstrated that the miR-221/222 mimicry promotes epidermal cell proliferation and miR-221/222 inhibitor suppresses epidermal cell proliferation.Based on this,we identified the miR-221/222 cluster as our study subject.The miR-221/222 cluster exerts post-transcriptional inhibition by simultaneously binding to JAM-A and PTEN.Currently,studies have shown that one of the main action mechanism of microRNAs is recognition and binding with the 3’UTR region of the target gene mRNA,resulting in degradation of target gene mRNA or interference with its normal expression,post-transcriptionally inhibiting its functions.Therefore,to further elucidate the molecular mechanism by which miRC plays a role in inhibiting proliferation of epidermal cells,we intended to find downstream target molecules those are functionally compatible and those could be directly recognized and binded.Through the prediction of miRC target molecules(microRNA.org,targetscan and miRbase)and searches of PubMed and Web of Science literature databases,we have pinpointed PTEN as a potential downstream target molecule for miR-221/222.And our hypothesis has been verified through dual luciferase reporter genes experiments.In addition,the outcomes further demonstrated that miR-221/222 can simultaneously target JAM-A and PTEN.Next,through cell and molecular experiments,we further verified that miR-221-3p and miR-222-5p mimics could negatively regulate the expression of JAM-A and PTEN,and their inhibitors could positively regulate the expression of JAM-A and PTEN.Later on,through rescue experiments,we found that overexpression of miR-221-3p,miR-222-5p can reduce the expression of PTEN after overexpression of JAM-A 3’UTR,furtherly proving that JAM-A mRNA protects PTEN from the degradation of miR-221-3p and miR-222-5p.JAM-A 3′UTR mutant had no effect on PTEN and no inhibitory effect on epidermal cell proliferation.Finally,we explored specific binding sites for the miR-221/222 cluster in the JAM-A 3’UTR region.Since miR-221 has three binding sites in the JAM-3’UTR region and two binding sites in PTEN,miR-221-3p is selected as the key research target.Based on the 3’UTR region sequence of JAM-A and the three binding sites of miR-221-3p,we constructed mutants for each site and whole mutant plasmids.The results of the dual luciferase reporter assay verified that miR221-3p could be synchronized with all three binding sites of JAM-A 3’UTR regions.Therefore,in the following experiments,all mutants were used as experimental subjects.RT-PCR and CCK-8 experiments confirmed that the JAM-A 3′UTR mutants had no regulatory effect on PTEN,and had no inhibitory effect on epidermal cell proliferation.In summary,in this study,it was clarified that JAM-A participates in the negative regulation of epidermal cell proliferation activity,which is dependent on the 3′UTR region of its mRNA,rather than the traditional direct interaction between protein and protein.The miR-221/222 cluster was screened,and the downstream target PTEN of miR-221/222 was further screened.In the subsequent mechanism study,we clarified that miR-221/222 can simultaneously bind to JAM-A and PTEN,clarify the specific binding site of miR-221/222,and outline the clear network of JAM-A,miR-221/222 clusters and PTEN.This research has conducted a beneficial exploration of understanding the regulation mechanism of epidermal cell proliferation activity from the microRNA perspective,deepens the understanding of the regulatory mechanism of epidermal cell proliferation in the process of wound healing,and is beneficial to the development of new drugs targeted to abnormal wound healing in the future.