MiR-142-5p Directly Mediates CDK5 Regulation Of Inflammatory Response In Acquired Middle Ear Cholesteatoma

BackgroundThe European Association for Neuro-Otology(EAONO)and the Japanese Otological Society(JOS)published the "Definition,Classification and Staging of Middle Ear Cholesteatoma" at the 10th International Cholesteatoma and Middle Ear Surgery Conference in 2016.Cholesteatomas,which is a mass composed by the squamous epithelial cells and debris in the connective tissue in the tympanic cavity(or)papilla,usually outbreak with or without inflammatory response,and its pathophysiology is not yet clear.Cholesteatoma can be classified into three categories:congenital cholesteatoma,acquired cholesteatoma,and cholesteatoma undifferentiated.Acquired cholesteatoma has not yet occurred at birth,and can come from the invaginated pocket of the loose and(or)tension part and invade the basement membrane through basal cells.Acquired middle ear cholesteatoma(AMEC)usually manifests as otorrhea,the combination of pain and earache,tinnitus,and conductive or sensorineural hearing impairment.Given its its serious intracranial and extracranial complications,such as peripheral facial paralysis,purulent meningitis,middle ear cholesteatoma should be actively treated.There is still a high risk of recurrence after surgery,so the treatment of AMEC needs further research.MicroRNA(miRNA)serves as a highly conserved non-coding RNA,which with a length of 18-36bp.At present,it is known that there are more than 20,000 miRNA molecules in animals,plants and viruses.They were first discovered in vertebrates and passed 3’-untranslated mRNA The region(3’-UTR)combines with post-transcriptional modification and regulation to affect the expression of protein-coding genes.MiRNA plays an irreplaceable role in the physiological processes of early development,cell proliferation,apoptosis,and inflammatory immune response.MiR-142-5p,as a miRNA related to inflammation,can mediate immune regulation.MiR-142-5p and miRNA-3p are two mature forms of miRNA-142.Among them,miRNA-142-5p plays multiple roles in tumorigenesis,tumor invasion,anti-tumor immunity,immune disorders and inflammatory response.A large number of studies have shown that miRNA-142-5p is involved in cancer progression in non-small cell lung cancer,breast cancer,ovarian cancer and renal clear cell carcinoma.The study on autoimmune Hashimoto’s thyroiditis found that there is significant differential expression of miR-142-5p in the control group and the diseased group.Other studies have shown that miR-142-5p,which is overexpressed under fever and inflammation,can target multiple immune genes and cytokines and the pathogenesis of chronic inflammation.In addition,miR-142-5p is used as a biomarker in the classification of pelvic inflammatory disease in animal experiments.Cell cycle dependent protein kinase 5(CDK5)is a member of the CDK5 family.Compared with most members of the CDK family,it has a similar structure and different functions.Classical cyclin-dependent kinase is an important factor in cell cycle regulation.It plays a role by combining with cyclin to form a heterodimer,where CDK is the catalytic subunit,cyclin is the regulatory subunit,and the cyclin-CDKs complex can catalyzes the phosphorylation of different substrates,and realizes the regulation of the cell cycle.Cdks has three important functional domains.The first functional domain is the binding site of ATP and the active part of the enzyme;the second functional domain is the binding site of the regulatory subunit(Cyclin);the third functional domain is the binding site of P13sucl(P13sucl can inhibit kinase activity and prevent cells Enter or exit phase M).In different CDK molecular structures,they all contain a similar kinase domain.This region has a conserved sequence,namely PSTAIRE(PSTAIRE),which mediates the binding of kinases to cyclin.CDK5,a downstream gene of miR-142-5p,is a special member of the CDK family compared with classic cyclins-dependent protein kinases owing to its unique regulatory pathway and typical effects.CDK5 plays a significant role in synapse production,biological information transmission,nerve cell migration and differentiation,nerve cell apoptosis,etc..In addition,it can play an irreplaceable role in many diseases physiological processes by targeting more than 30 different substrates.A study showed that CDK5 regulates dendritic cell maturation in chronic rhinosinusitis with nasal polyps.In neuro system,CDK5 inhibitory peptide could reduce neuro inflammation.However,no reports have been published on the role of CDK5 in AMEC.ObjectiveIn the study,we explored the regulation of miR-142-5p on the inflammatory response of acquired middle ear cholesteatoma by mediating CDK5,clarifying the relationship between miR-142-5p and CDK5,and regulating miR-142-5p and CDK5 on inflammatory factors secretion.Methods1.The sample of acquired middle ear cholesteatoma and controled external auditory canal skin were treated with 4%formalin.The different expression of CDK5 between acquired middle ear cholesteatoma group and control group was determined by immunohistochemistry and immunohistofluorescence.PCR and Western Blot was used to further verify.The expression levels of miR-142-5p and inflammatory factors in acquired middle ear cholesteatoma were tested by PCR.2.Pearson’s correlation analysis was used to clarify the correlation between miR-142-5p and CDK5.HaCat cells were stimulated with LPS in different time,RNA was extracted to verify the correlation between the two.3.CDK5 and inflammatory factors expression was comfirmed by Pearson’s correlation analysis.HaCat cells were treated with LPS with time prolongation,PCR was used to detect the expression of CDK5 and inflammatory factors.4.We used dual luciferase reporter gene to confirm whether miR-142-5p directly target CDK5.Si-CDK5,miR-142-5p mimic and miR-142-5p inhibitor were transfected into HaCat cells to detect the expression changes of miR-142-5p and CDK5 at the gene and protein level.5.After transfection of si-CDK5 and miR-42-5p inhibitor in HaCat cells,the expression of inflammatory factors were detected by Elisa.Results1.Increased expression of CDK5 in acquired middle ear cholesteatoma epithelium.2.The expression of miR-142-5p is negatively correlated with CDK5.3.The secretion of inflammatory factors is related to the expression of CDK5 in acquired middle ear cholestoma.4.miR-142-5p inhibits CDK5 expression through direct targeting CDK5.5.miR-142-5p mediates CDK5’s regulation of inflammatory factor expression.ConclusionIn this study,we found that miR142-5p was decreased in AMEC compared with that in healthy controls,showing a negative correlation with CDK5.We also confirmed that miR142-5p directly targets CDK5 to affect its expression and that of inflammatory factors.For the first time,we reported that miR142-5p plays a role in acquired middle ear cholesteatoma.