MED13L Integrates Mediator-regulated Epigenetic Control Into Lung Cancer Radiosensitivity

Background:According to the GLOBOCAN estimates of cancer incidence and mortality,lung cancer is the most frequently diagnosed cancer and the leading cause of cancer death in 2018.Lung cancer presents a complex disease,which differs by histologic types,i.e.small-cell lung cancer(SCLC)(about 20%of incident cases)and non-small-cell lung cancer(NSCLC)(about 80%of all cases).Radiotherapy is one indispensable therapeutics in the definitive management for NSCLC.However,radioresistance is an important factor that limits the efficacy of radiotherapy for NSCLC patients.RTOG0617 Phase III randomized clinical trial indicated that patients with locally advanced NSCLC who received high-dose radiotherapy failed to have survival benefits compared with those receiving standard-dose radiotherapy.These results indicate that it might be practical to identify new druggable targets to improve the radiosensitivity of NSCLC cells,but not to simply boost radiation dose.Mediator is a large,highly conserved protein complex and functions as a key transcriptional co-activator in eukaryotes.Mediator has two structural modules(CDK8 kinase module and core Mediator)and is composed of 30 subunits.Mediator could interact with chromatin regulators involved in histone modification,transcription factors(TFs)at enhancers,as well as the Pol II transcription machinery bound at promoters.Cancer cells often arise as a consequence of dysregulation of gene expression.Therefore,Mediator dysfunction is implicated in malignancies.MED13L is one subunit of Mediator,little is known about its role in cancer cells.MicroRNAs(miRNAs)regulate the vast majority of biological events by suppressing the expression of target genes,and their dysregulation plays a crucial part in cancers.Depending on their target genes,miRNAs have been reported to function as either oncogenes or tumor suppressors in lung cancer.In addition,the importance of miRNAs in radiosensitivity has begun to emerge.However,the molecular mechanistic detail of miRNAs regulating NSCLC development and radiosensitivity,remains yet to be fully,systematically characterized.Purpose:In this study,we aimed to systematically evaluate the radiation-induced miRNAs in lung cancer cells and identify their target genes which are involved in regulating NSCLC radiosensitivity.Methods and Materials:1.Human miRNA profilling for evaluating the expression of miRNAs in NSCLC cell lines before and after X-ray radiation was performed using the human miRNA OneArray microarrays(Phalanx,HmiOA7.1).Dysregulated miRNAs were then verified by qRT-PCR.2.The successfully validated miRNAs were examined in paired NSCLC and normal tissues(n=46,Shandong cohort).The association between miR-4497 and the prognosis of NSCLC patients with locally advanced disease treated with radiotherapy(n=108,Jiangsu cohort)was also analyzed.3.We evaluated the role of miR-4497 in NSCLC using cell proliferation assay,colony formation assay,wound healing assay and transwell assay.4.We addressed the combined impacts of miR-4497 and X-ray radiation on colony formation,apoptosis and cell cycle control of NSCLC cells.5.We predicted the potential target genes of miR-4497 by integrating results from different algorithms including TargetScan,MiRDB and TargetMiner.qRT-PCR and dual luciferase reporter gene assays were conducted to validate the candidate MED13L and SHOX genes.6.We examined MED13L mRNA and protein expression in NSCLC cells transfected with the miR-4497 mimics or siRNAs of MED13L.To evaluate expression correlations between miR-4497 and MED13L,we detected MED13L expression in NSCLC and normal tissue samples.7.Different MED13L expression in NSCLC tissues and normal lung tissues(n=46)was analyzed.The association between MED13L expression and the prognosis of NSCLC patients with locally advanced disease treated with radiotherapy was examined.8.We evaluated the role of MED13L in NSCLC using cell proliferation assay,colony formation assay,apoptosis assay,cell cycle assay,wound healing assay and transwell assay.9.We evaluated the in vivo role of miR-4497 or MED13L shRNAs,alone and in combination with radiotherapy(RT),using NSCLC xenografts.10.We examined how MED 13L impacts the structure integrity of Mediator by IP assays.To investigate if MED13L could recruit P300 via MED 12 to Mediator,we performed IP assays using NSCLC cells.11.We performed H3K27ac ChIP-seq to investigate chromatin histone-modification changes of NSCLC PC9 cells with ectopic miR-4497 or silencing of MED13L(sil3L-1 and si13L-2).12.We did integrated analyses of candidate genes with significant changes of H3K27ac modification in the miR-4497,si13L-l and si13L-2 groups.We then performed KEGG pathway analyses with these candidate genes.To evaluate the impacts of MED13L-regulated H3K27ac on expression of these genes,we detected their expression in NSCLC cells using qRT-PCR.13.We explored if P300 could directly bind to regulatory regions around the PRKCA TSS(transcriptional start site)with H3K27ac ChIP-seq peaks using ChIP-qPCR assay.14.PRKCA expression in NSCLC tissues and normal lung tissues were detected.The association between PRKCA expression and patients' prognosis was analyzed.We evaluated the role of PRKCA in NSCLC using cell proliferation and colony formation assays.15.We examined the in vivo role of PRKCA,alone and in combination with RT,using NSCLC xenografts.Results:1.In this study,we found that miR-4497 was significantly up-regulated following radiation in A549 and H1299 NSCLC cell lines(P<0.05).miR-4497 expression is significantly down-regulated in NSCLC tissues comparing to normal tissues(P<0.001).Intriguingly,the elevated miR-4497 expression was evidently associated with longer progression free survival(PFS)and overall survival(OS)of NSCLC patients with locally advanced disease treated with radiotherapy(both Log-rank P<0.001).Overexpression of miR-4497 could significantly suppress proliferation,clonogenicity,migration and invasion capability of PC9,A549 and H1299 NSCLC cells.Conversely,miR-4497 inhibitors could promote the malignant phenotypes of NSCLC cells.In addition,ectopic miR-4497 sensitizes NSCLC cells to radiotherapy in a dose dependent manner.Importantly,as radiation does,miR-4497 could significantly induce apoptosis and G2/M cell cycle arrest of NSCLC cells.2.We predicted the potential candidate target genes of miR-4497 by integrating results from different algorithms including TargetScan,MiRDB and TargetMiner.qRT-PCR and dual luciferase reporter gene assays were conducted to validate the candidate genes,MED13L and SHOX.We found that MED13L is the target gene of miR-4497 in NSCLC.Ectopic miR-4497 could significantly inhibit MED13L mRNA and protein expression in NSCLC cells.In addition,significantly negative expression correlations between miR-4497 and MED13L were observed in NSCLC and normal tissue samples from different patient cohorts.There was an evidently elevated MED13L expression in NSCLC tissues compared to normal lung samples(P<0.001).High MED13L expression was associated with poor prognosis with stage III NSCLC patients treated with concurrent chemoradiotherapy.We then explored the biological significance of MED13L in NSCLC cells.Knockdown of MED13L can remarkably suppress the malignant phenotypes of NSCLC cells.3.Consistent with in vitro data,growth of xenografts with stably ectopic miR-4497 or stably depleted MED13L was significantly suppressed compared to the controls(all P<0.01).After the tumors stably expressing miR-4497 or stably knocking-down MED13L were treated with radiation,xenografts grew much slower and showed a significant decrease in tumor volume compared to control tumors(all P<0.001).4.IP assays indicated that MED13L is a vital linker protein between the core Mediator and the CDK8 kinase module.P300 can be specifically immunoprecipitated by MED13L in NSCLC cells.ChIP-seq data showed that the H3K27ac signals were much weaker in NSCLC cells with ectopic miR-4497 expression or silenced MED13L compared to the control cells.PRKCA,SOS2,AKT3,PAK4,and ARAF in the ErbB signaling pathway showed significantly dysregulated H3K27ac modification around their TSS in PC9 cells after ectopic miR-4497 expression or silencing of MED13L.miR-4497 and silencing of MED13L can significantly suppress expression of PRKCA and ARAF in NSCLC cell lines.Compared to normal lung tissue samples,mRNA of PRKCA but not ARAF was significantly upregulated in the NSCLC samples.Importantly,ChIP-qPCR assays indicate that P300 can directly bind to chromatin regions with H3K27ac ChIP-seq peaks around the PRKCA TSS.However,ectopic miR-4497 and silencing of MED13L leads to reduced recruitment of P300.5.There was an evidently elevated PRKCA expression in NSCLC tissues compared to normal lung samples(P<0.001).High PRKCA expression was associated with poor prognosis with stage ? NSCLC patients treated with concurrent chemoradiotherapy.Inhibition of PRKCA expression sensitized NSCLC to radiotherapy in vitro and in vivo.Conclusions:We identified a novel model that integrates Mediator-regulated epigenetic control into NSCLC radiosensitivity.The fact that radiation can trigger disassemble of the Mediator complex via silencing of MED13L by miR-4497 adds a new layer of gene expression regulation of NSCLC cells receiving radiotherapy.Results linking PRKCA to NSCLC radiosensitivity through a novel epigenetic mechanism may enable the rational targeting of PRKCA to unlock the therapeutic potential of NSCLC in the clinic.