The Function And Molecular Mechanism Of MED27 In Melanoma Development

Objective: The human mediator complex(MED)composed of 28 elements.As a component of these 28 elements,MED27 is an enzyme ubiquitously expressed in humans and universally required in transcriptional initiation.MED27 is found to interact with thyroid hormone receptor(TR)to facilitate TR function by conjunction with other transcriptional factors and co-factors.In addition,the newly findings demonstrated its involvement in HIV-1 transcription.Knockdown of MED27 protein,combined with the knockdown of other MED proteins,significantly impaired viral replication without affecting cell viability.However,to date,unlike other members of MED family,the functional role of MED27 in human diseases is still rarely reported,and especially its function in respect to tumorigenesis and development is poorly uncharacterized.Using si RNA library screening,we has identified a serial of new proteins implicated in melanoma tumor progression.Here,we chose one of these proteins,MED27,as the main study object and provided an evaluation of its activity on melanoma cell growth and apoptosis.Moreover,we explored its potential molecular mechanisms associated with melanoma survival and its clinical significance in melanoma.Our results have shown that knockdown of MED27 leads to reduced cell proliferation,increased cell cycle repression and apoptosis in melanoma,and such proliferation-inhibiting effects of MED27 knockdown is mediated by inactivating PI3K/AKT,MEK/Erk pathway and elevating Bcl-2/Cyt C/Caspase 3 signaling pathway.Our researches not only demonstrated the role of MED27 in melanoma progress at first time,but also provided a potential candidate therapeutic target for melanoma treatment.Methods:(1)We used si RNA library screen technology to test and identify potential target genes that affect melanoma cells.(2)We applied genetransfer technology to specifically activate or inhibit MED27 expression in vivo and in vitro study to study thetumor cell growth,cell proliferation,migration,cycle,apoptosis,and other biological function.(3)We used RT-PCR,Westernblot and immunohistochemistry techniques to detect the expression levels of MED27 in normal skin cells and melanoma tumor cells.We also evaluated the expression levels of MED27 in melanoma patients and analyzed clinical significance,such as diagnostic,prognostic value,recurrence and therapeutic targets.(4)We used co-immunoprecipitation to detect the relation between transcriptional MED27 and transcriptional co-activator p300 as well as the effect of their binding on i NOS expression.(5)We used animal model to validate the MED27 impact on tumor growth and expression of i NOS.Results:(1)We identified the MED27 that can affect the growth of melanoma cells using si RNA library screen.(2)MED27 in melanoma cells and tumor tissues was highly expressed.(3)MED27 promoted the proliferation of melanoma cells(4)PI3K/AKT and MAPK/ERKsignaling pathway was involved in the proliferative regulation of melanoma cells mediated by MED27.(5)MED27 knockdown induced cell cycle arrest and inhibition of melanoma cell migration.(6)MED27 knockdown enhanced apoptosis of melanoma cells through up-regulating Bax/Cyto C/caspase3 signaling pathway.(7)Regulation of MED27 on i NOS expression through affecting NF-?B signaling pathway.(8)MED27 interacted with NF-? B and p300 in melanoma cells.(9)MED27knockdown inhibited melanoma progression in animal model with xenografts.Conclusions: In this study,using si RNA library screen,we successfully identified the transcription factor MED27 from melanoma cells.The analysis of clinical data showed that the MED27 expression in melanoma was positively related with the survival of patients.In vitro,the knockdown of MED27 could regulate the melanoma proliferation induced cell cycle arrest and promoted cell apoptosis.At the same time,our study verified that MED27 regulated i NOS expression.In summary,our study provided a new target for melanom.