Arginine Methyltransferase PRMT5 Regulates The Methylation Of Mxi1 To Promote Radioresistance In Lung Cancer

Objective: Lung cancer still accounts for the most cancer death worldwide with an average 5-year survival rate of about 19%,of which non-small cell lung cancer(NSCLC)represents ～85%.Radiotherapy is considered to be a radical treatment for patients with locally advanced unresectable lung cancer.However,radioresistance is the leading cause of local recurrence and distant metastasis in lung cancer.Therefore,it is necessary to identify novel biomarkers for radioresistance and explore potential radiosensitizers in lung cancer.PRMT5(Protein Arginine Methyltransferase-5)is a type II protein arginine methyltransferase that regulates a variety of cell life activities,including RNA metabolism,genes epigenetic regulation and DNA damage repair,but its role in lung cancer radioresistance is still unknown.The purpose of this study is to explore the role and mechanism of PRMT5 in radioresistance,as well as the radiosensitization effect of PRMT5 specific inhibitor EPZ015666 in lung cancer.Methods:(1)Co-IP and GST pulldown assays were carried out to verify the interaction between PRMT5 and Mxi1.(2)Mxi1 deletion mutants were constructed to map the domains of Mxi1 responsible for binding to PRMT5.(3)In vivo and in vitro methylation assays were performed to detect Mxi1 methylation.(4)Co-IP assays were utilized to detect the interaction between Mxi1 and β-Trcp.(5)In vivo ubiquitination and CHX assays were conducted to assess the effect of PRMT5 on Mxi1 protein stability.(6)Neutral comet assay,Rad51 foci formation assay and clonogenic cell survival assay were conducted to evaluate the radiosensitivity of lung cancer cells in vitro.(7)BALB/c nude mice were injected subcutaneously with Sh Control or Sh PRMT5 A549 cells to determine whether PRMT5 enhanced the radiosensitivity of lung cancer in vivo.(8)In vivo experiments were performed using BALB/c nude mice to assess the therapeutic potential of PRMT5 pharmacological blockade using EPZ015666.(9)Rescue experiments were utilized to identify whether the biological functions of PRMT5 were depended on Mxi1.Results:(1)PRMT5 physically interacts with Mxi1 in vivo and in vitro.(2)The BHLH-LZ domain of Mxi1 binds to PRMT5.(3)PRMT5 methylates Mxi1 in vivo and in vitro.(4)PRMT5 promotes the binding between Mxi1 with β-Trcp.(5)Genetical or pharmacological blockade of PRMT5 decreases Mxi1 poly-ubiquitination and extends the half-life of Mxi1 and greatly increases Mxi1 protein levels in lung cancer cells.(6)Genetical or pharmacological blockade of PRMT5 significantly increases the length and DNA content in the comet tails and impaires the formation of Rad51 foci as well as enhances cell radiosensitivity to irradiation in vitro.(7)Loss of PRMT5 enhances the radiosensitivity in lung cancer in vivo.(8)Pharmacological blockade of PRMT5 with EPZ015666 overcomes lung cancer radioresistance in vivo.(9)PRMT5 exerts its biological effects through methylating and degrading Mxi1 in lung cancer cells.Conclusions: Our work identifies PRMT5 as a type II arginine methyltransferase that binds to and methylates Mxi1 and induces the ubiquitination and destruction of Mxi1,resulting in lung cancer radioresistance in vitro and in vivo.We also reveal that genetical and pharmacological inhibition of PRMT5 impairs radioresistance in a Mxi1-dependent manner in lung cancer.These data not only shed light on the molecular mechanism underlying how Mxi1 is regulated by PRMT5 but also imply that PRMT5 is a potential drug target for lung cancer radiotherapy.